The Pennsylvania State University VITAMIN A METABOLISM IN ...

The Pennsylvania State University

The Graduate School

College of Health and Human Development

VITAMIN A METABOLISM IN THE NEONATAL LUNG:

STUDIES IN A RAT MODEL

A Dissertation in

Nutrition

by

Lili Wu

2011 Lili Wu

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

December 2011

1

The dissertation of Lili Wu was reviewed and approved* by the following:

A. Catharine Ross

Professor of Nutritional Sciences

Occupant of Dorothy Foehr Huck Chair

Dissertation Advisor

Chair of Committee

Okhee Han

Assistant Professor of Nutritional Sciences

Katarzyna Kordas

Assistant Professor of Nutritional Sciences

Pamela J. Mitchell

Associate Professor of Biology and Molecular

Jeffery M. Dodds

Attending Veterinarian for Laboratory Animals

Gordon L. Jensen

Professor of Nutritional Sciences

Head of the Department of Nutritional Sciences

*Signatures are on file in the Graduate School

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ABSTRACT

Vitamin A (VA) is an essential nutrient for differentiation and maturation of the

lungs. Direct evidence has been presented that keratinizing squamous metaplasia of the

bronchopulmonary tree can be caused by VA deprivation in the lungs of VA deficient

animals and this morphologic change can be reversed by refeeding the animals with VA.

Other biochemical and molecular genetic evidence revealed that specific retinoids-

binding proteins and nuclear retinoic acid receptors are contained in the lungs and

retinoids can affect lung cells differentiation by influencing lung gene expression. In rats,

significant storage of VA in the lungs starts in late gestation just before the onset of

alveologenesis and surfactant synthesis, and then quickly is depleted during late

pregnancy and postnatal life as the lungs are still developing, suggesting a high and acute

demand of VA for postnatal lung development. However, the VA status in the lung of

human and other mammals is known to be low at birth and postnatal lungs are very

sensitive to dietary VA deprivation. VA deficiency associated with premature infants or

infants with very-low-body-weight (VLBW) can interrupt normal development and

compromise the respiratory function of the lung, thereby putting this population at high

risk to develop various respiratory diseases. Therefore, it is important to improve lung

VA status at the early time of the postnatal life.

Although supplementation with VA to neonates is an effective way to prevent VA

deficiency, it shows limited effectiveness in improving lung VA status. Previously we

have shown that retinol combined with retinoic acid (RA), a biologically active

iv

metabolite of VA (VARA), is able to increase lung retinyl ester (RE) formation

synergistically and RA redirects more of the VA given as a supplement into the neonatal

lung. Our work investigating the molecular mechanism of VARA synergy revealed that

RA is able to affect lung VA metabolism by upregulating several important retinoid

homeostatic genes: LRAT, lecithin:retinol acyltransferase, an enzyme converting retinol

to its storage form; CYP26, a cytochrome P450, an enzyme metabolizing RA to inactive

polar metabolites; and STRA6, stimulated by retinoic acid gene 6, a transmembrane

receptor for the retinol-RBP complex that mediate cellular retinol uptake. However, these

findings are based on a single dose study, and the activity of RA on gene induction

appears to be transient.

In the present study, we tested the effects of repeated supplementation with

VARA in increasing lung RE contents. We also examined whether inflammation state

and reduced RA concentration could affect the capability of RA in promoting RE

formation. At the same time, we speculated the spatial expression pattern of LRAT,

CYP26B1 and STRA6 to further understand VA metabolism in the lungs of the neonates.

We carried out several studies to 1) investigate how multiple treatments of RA during the

period of lung septation affect RE accumulation and the expression pattern of lung

retinoid homeostatic genes, or genes required for normal lung function; 2) compare a

reduced amount of RA in the VARA dose to test the potential of RA in elevating lung

RE; 3) examine how lipopolysaccharide (LPS)-induced inflammation state affects VA

homeostasis in neonatal lung; 4) determine the localization of retinoid homeostasis

proteins in the lung. The results of our studies have shown that repeated treatments of

v

VARA dramatically increase neonatal lung RE store in a cumulative and synergistic way.

Diluted RA in VARA still promotes higher RE formation in neonatal lung more than VA

alone after a single dose, but not after multiple doses. LPS-induced inflammation doesn’t

significantly impact lung RE formation promoted by RA. The localization study

suggested the expression of LRAT in lipofibroblasts, STRA6 in endothelial cells, and

CYP26B1 in bronchiolar epithelium.

Overall, these studies have shown the great ability of RA in promoting lung RE

formation, even when given in much diluted concentration. Compared with a single dose,

multiple treatments of VARA produced a cumulative effect on RE storage. The

synergistic effect of VARA was not significantly affected by inflammation. These results

together with our findings of the localization of retinoid homeostatic proteins provide a

better understanding of retinoid uptake, accumulation and metabolism in the neonatal

lung. Our findings also suggest a promising therapeutic approach in clinical use for a

rapid restoration of lung VA in preterm or VLBW infants to promote normal lung

maturation and prevent these infants from developing respiratory diseases.

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TABLE OF CONTENTS

LIST OF ABBREVIATIONS......................................................................................x

LIST OF FIGURES .....................................................................................................xiii

ACKNOWLEDGEMENTS.........................................................................................xvi

Chapter 1 LITERATURE REVIEW...........................................................................1

1.1 LUNG PROBLEMS IN PRETERM NEONATES .......................................1

1.2 LUNG STRUCTURE AND DEVELOPMENT ...........................................2

1.2.1 Lung structure and function..................................................................2

1.2.2 Lung development ................................................................................6

1.2.3 Lung cells .............................................................................................8

1.3 GENERAL INTRODUCTION TO VITAMIN A.........................................9

1.3.1 Functions and properties of vitamin A .................................................9

1.3.2 Vitamin A deficiency and toxicity .......................................................12

1.4 VITAMIN A METABOLISM ......................................................................13

1.4.1 Transport and metabolism of retinoids.................................................13

1.4.2 Retinoid homeostatic proteins ..............................................................19

1.5 THE REGULATORY MECHANISM OF RETINOIDS..............................22

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1.6 RETINOIDS IN LUNG DEVELOPMENT AND FUNCTION ...................24

1.6.1 Retinoids in lung morphogenesis .........................................................24

1.6.2 Retinoids in alveolar septation .............................................................25

1.6.3 Retinoids in lung tissue repair ..............................................................27

1.6.4 Retinoid metabolism in the lung...........................................................30

Chapter 2 MULTIPLE TREATMENT STUDY.........................................................32

2.1 ABSTRACT ..................................................................................................32

2.2 INTRODUCTION.........................................................................................33

2.3 HYPOTHESIS AND AIMS..........................................................................35

2.4 MATERIALS AND METHODS ..................................................................36

2.5 RESULTS......................................................................................................42

2.6 DISCUSSION................................................................................................52

Chapter 3 LPS-INDUCED INFLAMMATION STUDY...........................................56

3.1 ABSTRACT ..................................................................................................56

3.2 INTRODUCTION.........................................................................................57



3.5 RESULTS......................................................................................................66

3.6 DISCUSSION................................................................................................77

Chapter 4 ACIDIC RETINOIDS DILUTION STUDY..............................................81

viii

4.1 ABSTRACT ..................................................................................................81

4.2 INTRODUCTION.........................................................................................82



4.5 RESULTS......................................................................................................89

4.6 DISCUSSION................................................................................................95

Chapter 5 LOCALIZATION OF LUNG RETINOID HOMEOSTATIC

PROTEIN .............................................................................................................98

5.1 ABSTRACT ..................................................................................................98

5.2 INTRODUCTION.........................................................................................99



5.5 RESULTS......................................................................................................106

5.6 DISCUSSION................................................................................................114

Chapter 6 DISCUSSION ............................................................................................117

6.1 VITAMIN A SUPPLEMENTATION DURING LUNG SEPTATION

PERIOD.........................................................................................................117

6.2 MOLECULAR MECHANISM OF RETINOL UPTAKE INTO THE

LUNG............................................................................................................121

6.3 THE POTENTIAL OF RA TO PROMOTE LUNG RE FORMATION ......122

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6.4 THE RELATIONSHIP BETWEEN INFLAMMATION AND

RETINOID METABOLISM IN NEONATAL LUNGS ..............................124

6.5 SPATIAL DISTRIBUTION OF THE RETINOID HOMEOSTATIC

PROTEINS IN NEONATAL LUNGS..........................................................125

6.6 FUTURE DIRECTIONS...............................................................................129

REFERENCES ............................................................................................................132

x

LIST OF ABBREVIATIONS

9cRA: 9-cis-retinoic acid

ADH: alcohol dehydrogenase

ADRP: adipocyte differentiation-related protein,

ALDH: aldehyde dehydrogenase

at-RA: all-trans-retinoic acid

ANOVA: analysis of variance

BPD: bronchopulmonary dysplasia

CCL2: chemokine (C-C motif) ligand 2

CRABP: cellular RA-binding protein

CRBP: cellular retinol-binding protein

CRP: c-creative protein

CYP: cytochrome P450

DAPI: 4’,6’-diamidino-2-phenylindole

ECM: extracellular matrix

h: hour(s)

HPLC: high performance liquid chromatography

IGF2R: insulin-like growth factor II receptor

IHC: immunohistochemistry

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IL-6: interleukin-6

ISH: in situ hybridization

IU: international unit

LPL: lipoprotein lipase

LPS: lipopolysaccharide

LRAT: lecithin:retinol acyltransferase

M6P: mannose-6-phosphate

PECAM: platelet endothelial cell adhesion molecule

RA: retinoic acid

RAE: Retinol Activity Equivalents, a term referring to vitamin A activity

RALDH: Retinaldehyde dehydrogenase

RAR: retinoic acid receptor

RARE: retinoic acid response element

RBP: retinol binding protein

RDH: retinol dehydrogenase

RDS: respiratory distress syndrome

RE: retinyl ester

rt-PCR: real-time polymerase chain reaction

RXR: retinoic X receptor

xii

RXRE: retinoid X-response elements

ROH: retinol

RXR: retinoid X receptor

SPA1: surfactant protein A1

STRA6: stimulated by retinoic acid gene 6

TTR: transthyretin

VA: vitamin A

VAD: vitamin A deficiency

VAS: vitamin A sufficiency

VEGF: vascular endothelial growth factors

VARA: vitamin A combined with retinoic acid

VLBW: very-low-body weight

VLDL: very-low-density lipoprotein

xiii

LIST OF FIGURES

Figure 1.1 Cross-section of alveolar wall and major cell types. . ................................5

Figure 1.2 Common natural and synthetic retinoids....................................................11

Figure 1.3 Schematic overview for the transport and uptake of dietrary retinoids

within the body. ....................................................................................................15

Figure 1.4 Uptake of retinoids into extrahepatic tissues..............................................17

Figure 1.5 Regulatory role of RA in retinoid metabolism. ..........................................21

Figure 1.6 Cellular retinoid metabolism and signaling pathway. ................................23

Figure 2.1 Animal experimental design.......................................................................39

Figure 2.2 Lung RE concentration after a single treatment or multiple treatments.....44

Figure 2.3 Expression level of Lung LRAT gene after treatments..............................47

Figure 2.4 Expression level of lung CYP26B1 gene after treatments. ........................48

Figure 2.5 Expression level of lung STRA6 genes after treatments............................49

Figure 2.6 Expression level of lung functional and structural genes after

treatments..............................................................................................................51


xiv

Figure 3.2 Lung RE concentration after a single treatment with oil or VARA in

the presence or absence of LPS induced inflammation state................................68

Figure 3.3 Expression level of lung IL6 gene 6 h or 18 h after LPS administration. ..70

Figure 3.4 Expression level of lung CCL2 gene 6 h or 18 h after LPS

administration. ......................................................................................................71

Figure 3.5 Plasma C Reactive Proteins (CRP) level 6 h or 18 h after LPS

administration. ......................................................................................................72

Figure 3.6 Expression level of lung LRAT gene 6 h after VARA treatment. .............74

Figure 3.7 Expression level of lung CYP26B1 gene 6 h after VARA treatment.. ......75

Figure 3.8 Expression level of lung STRA6 gene 6 h after VARA treatment.............76


Figure 4.2 Lung RE concentration after a single treatment with VA alone, VARA

10%, VARA 5%, VARA 2%, VARA1% and VARA0.5%..................................92

Figure 4.3 Lung RE concentration after multiple treatments with VA alone,

VARA10%, VARA2% and VARA1%.................................................................93


VAAm10%, VAAm2% and VAAm1%. ............................................................94

Figure 5.1 Localization of LRAT protein in 14d old rat lungs....................................109

xv

Figure 5.2 Localization of STRA6 protein in 14d old rat lungs. .................................110

Figure 5.3 Localization of CYP26B1 protein in 14d old rat lungs..............................113

Figure 6.1 Model of VA metabolism in neonatal rat lung...........................................128

xvi

ACKNOWLEDGEMENTS

First and foremost, I would like to express my sincere gratitude to my supervisor Dr. A.

Catharine Ross, whose guidance, encouragement, patience and support helped me in all

the time of research and enabled me to complete my project and writing of this thesis.

I am grateful to my committee members: Dr. Okhee Han, Dr. Katarzyna Kordas, Dr.

Pamela J. Mitchell, and Dr. Jeffery M. Dodds for sharing their expertise and providing

invaluable insights during my research and writing.

I am also thankful to my colleagues: Dr. Qiuyan Chen and Dr. Reza Zolfaghari for their

great encouragement and advice on my graduate research; Yao Zhang, Libo Tan,

Katherine Restori, Amanda Wray, Dr. Kyoko Goto, Dr. Nan-qian Li, and Madeline Stull

for their friendship and assistance in my daily work and life.

I would like to thank the Department of Nutritional Sciences, which likes a big and warm

family to students. For many years, I have got continued help from our department staff,

especially Judy Jones.

Last, but not least, I offer my gratitude and blessings to my husband, Weimin Wu and my

family for their constant support and love. Without your support, I could never have gone

this far and completed my graduate work.

1

Chapter 1

LITERATURE REVIEW

Every year, around half a million infants are born prematurely in the United States. The

human fetal lung normally is not clinically mature until after approximately 35 wk

gestation. Although many premature infants appear healthy, it has been observed that

they may suffer from interrupted lung development, underdeveloped lungs at birth, and

immature immune systems. Due to the physiological and morphological immaturity, the

lung of premature infants is functional insufficient and these infants are at increased risk

of developing various pulmonary diseases during the early postnatal life, such as

respiratory distress syndrome (RDS) and subsequent ventilatory management-caused

bronchopulmonary dysplasia (BPD). The respiratory problems are the leading cause of

the morbidity in preterm neonates. Therefore, premature infant lung development is one

of the largest issues in clinical therapy for premature infants.

1.1 LUNG PROBLEMS IN PRETERM NEONATES

2

The lungs of mammals are among of the largest organs in the body and are the major part

of the respiratory system. They are cone-shaped and have a spongy and soft texture

because of the composition of millions of alveoli, the functional unit of the lung. The

principal function of the lungs is to bring oxygen (O2) into the body, and to remove

carbon dioxide (CO2) out of the body.

In the body of mammals, the distal end of trachea divides into two bronchi which lead

into the left and right lungs, respectively. Each bronchi then starts a series of branches,

called the respiratory tree. It divides into secondary bronchi, tertiary bronchi, and after

multiple divisions, reaches to the level of bronchioles. While the process of branching

continues, bronchioles branch into the terminal bronchioles, the respiratory bronchioles,

alveolar ducts, and finally, give rise to the clusters of alveolar sacs. Each alveolar sac is

tightly wrapped by a dense network of capillaries. In human, the structure of alveoli

increases the surface of the lung to 80-140 m2, depending on body size.

According to the function and properties of each part, the lungs are divided into two

primary zones - the conducting zone and respiratory zone. The conducting zone is

composed of the trachea, bronchi, bronchioles, and terminal bronchioles. In the trachea

1.2 LUNG STRUCTURE AND DEVELOPMENT

1.2.1 Lung structure and function

3

and the upper levels of bronchi, hyaline cartilage is present in a C-ring shape around the

wall of the airway. As the bronchi divide into smaller and smaller passageways, the

amount of hyaline cartilage decreases and the cartilage ring gradually becomes irregular,

discontinued, and finally disappeared and replaced by smooth muscle in the smallest

bronchioles. As the cartilage decreases and the smooth muscle increases, the mucous

membrane also undergoes a transition from ciliated pseudostratified columnar epithelium

in the upper respiratory tract to a simple cuboidal epithelium in the terminal bronchioles.

Throughout the mucosa, goblet cells are present to provide continued mucus secretion.

The conducting zone mainly functions as passageways that deliver inspired air to the

respiratory zone and expired air to the outside environment. It also acts to warm and

humidify the inhaled air and to provide an immunological defense against intruding

organisms.

The respiratory zone is the site of gas exchange. It is made up of respiratory bronchioles,

alveolar ducts and alveoli which only have a thin wall which is primarily made up of

simple squamous epithelial cells. Each alveolar sac is surrounded by a capillary network.

The thin layer of type I epithelial cells, the basal membrane, and the thin layer of

capillary endothelial cells constitute the air-blood barrier (also named as respiratory

membrane) which permits a rapid diffusion of oxygen and carbon dioxide. The structure

of alveolar dramatically increases the surface area for contact with blood vessels. The

alveolar epithelium is simply comprised of type I alveolar cells, type II alveolar cells,

endothelial cells, macrophages and fibroblast (Fig. 1.1). Type I alveolar cells are simple

squamous cells that account for most of the surface wall, although there are just about

4

half as many as type II cells. Type I cells are large and thin, while type II alveolar cells

are small and round, and compose only about 10 % of the alveolar wall. The type II cell

is responsible for the production and secretion of surfactant, which reduces surface

tension and prevents the collapse of alveoli. When type I cell is damaged, the type II cell

is able to differentiate into a type I cell to replace it. Macrophages, derived from blood

monocytes, are also present in the alveolar space to phagocytize the invading bacteria.

5

Figure 1.1 Cross-section of alveolar wall and major cell types. This figure is from

internet resource and is modified by L. Wu.

6

In humans, the process of lung development can be subdivided into five distinct phases:

embryonic phase, pseudoglandular phase, canalicular phase, saccular phase and alveolar

phase (1).

Embryonic phase/ early branching phase

This phase of lung development takes place during weeks 4 and 5 of gestation. The lung

bud originates from endodermal epithelium and grows out from the lower pharynx. It

then subdivides into two main bronchi and begins dichotomous branching into smaller

segmental bronchi. Smooth muscle, airway cartilage, blood and lymph vessels start to

develop.

Pseudoglandular phase

In this stage (4-17 weeks of gestation), the lung resembles an exocrine gland, hence the

name. The major conducting airway continues to develop into a bronchial tree, paralleled

by the formation of a vascular bed. Under the influence of adjacent mesenchyme, the

primitive airway epithelial cells start to differentiate into ciliated cells, goblet cells and

mucous glands, while mesenchymal cells have begun to form cartilage and smooth

muscle cells (2). Type II alveolar cells appear in respiratory bronchioles and start to

produce amniotic fluid.

1.2.2 Lung development

7

Canalicular phase

In this stage (17-26 weeks of gestation), terminal airspaces expand to form primitive

alveoli. Surfactant protein is detectable by 24 weeks of gestation. During this period,

respiratory bronchioli appear, interstitial tissue decreases, the cuboidal epithelium starts

to differentiate into type I and type II alveolar cells, and the blood capillaries proliferate

around the alveoli to form air-blood barrier for limited gas exchange (3). Thus, fetus is

able to survive at around the mid-late canalicular stage (4).

Saccular phase

In the saccular stage (24-36 weeks of gestation), the distal airspaces continue to enlarge,

accompanied with a continued reduction of interstitial tissues. The air sacs are mainly

lined with flattened type I epithelial cells and rounded type II epithelial cells. The

epithelial layer becomes thinner. As a result of the structural change, the gas-exchange

function is enhanced. The primary septa between two alveolar sacculi are still thick and

contain a double capillary network. During this time, surfactant containing laminar

bodies lining in type II pneumocytes can be found. However, the lung of rodents is in this

phase at birth, which is equal to human fetal lung at 28 wks.

Alveolar phase/ septation period

The last stage, alveolar stage, begins approximately 36 week of gestation to term and

continues for at least 3 year of postnatal life. At birth, the lung is still structurally

immature, only 15-20% of alveoli in the adult lung are formed, indicating most of the

alveoli are formed within the postnatal period (5, 6). In rodents (rats and mice), this stage

8

occurs exclusively between day 4 and 14 after birth (7). The alveolar stage is mainly

characterized by repetitive subdivisions of terminal sacs into definitive alveoli by the

growth of secondary septa. In the process of subdivision, secondary septa extend from

primary alveolar wall and subdivide the terminal sacs into multiple smaller and thinner

definitive alveoli. Development of these septae occurs through deposition of new

basement membrane, outgrowth of epithelial cells and myofibroblasts at the tips of

septae, and elastin deposition (5). The epithelial cells lining the alveolar wall decrease in

number and become thinner and flatter. Accompanying the aiveolar septation is the

microvascular maturation. The capillary vessels come into close contact with the alveoli

and the basement membranes underlying the capillary endothelial cells and alveolar

epithelial cells fuse with each other, forming a very thin air-blood barrier. In contrast to

the double capillary in primary septa, the secondary septa contain only a single layer of

capillary. The continued subdivisions of alveoli greatly increases the lung surface area for

gas exchange, thus, the process is also known as alveolarization (8).

The mature lung is a heterogeneous tissue comprised of about 40 morphologically

distinct cell types (9). This cell population including cells in the epithelium, cells in the

connective tissue, blood vessels and nervous tissue, such as squamous epithelial, ciliated

columnar cells, mucus-secreting goblet cells, and clara cells lining in the upper airways,

type I and type II alveolar epithelial cells lining in alveoli; fibroblasts, myofibroblasts,

and smooth muscle cells (SMC) in the lung interstitium, endothelial cells in blood

1.2.3 Lung cells

9

vessels, macrophages and lymphocytes, migrating between circulation system and tissue,

etc. These cells serve various roles in lung development and functions.

Vitamin A (VA) is a group of compounds that play an important role in many functions.

The major functional activities of VA include 1) promoting vision (10), 2) participating

in protein synthesis and cell differentiation in epithelial tissues and skin (11, 12), 3)

supporting reproduction, development and growth (13), and 4) maintaining the integrity

of immunity (14, 15). The group of VA compounds is referred to as retinoids and is

comprised of a large number of natural and synthetic compounds (Fig. 1.2). The natural

compounds include retinol and its metabolites: RE, retinal, retinoic acid (RA) and some

water-soluble metabolites. The basic structure of a retinoid is composed of a substituted

cyclohexenyl ring, a tetraene side chain and a functional group at the end of the side

chain. Retinol (also named vitamin A) has a hydroxyl group at its terminal end. When

this group is esterified with a fatty acid, retinol becomes RE, the form in which VA is

stored. The hydroxyl group also undergoes oxidation to produce an aldehyde (retinal),

which may be further oxidized to a carboxylic acid (retinoic acid). Among these

compounds, RA is the most active metabolite of vitamin A, which interacts with RA

nuclear receptors and subsequently modulates proliferation of epithelial cells, pattern

1.3 GENERAL INTRODUCTION TO VITAMIN A

1.3.1 Functions and properties of vitamin A

10

formation in developing tissues, morphogenesis in the lung, and cellular differentiation.

In addition to these naturally occurring retinoids, a large number of artificial analogs have

been synthesized and used in studies of retinoid signaling within the cell. For example,

Am580, a stable RA analog and a selective agonist of retinoic acid receptor-α (RARα),

belongs to the retinoid family as well (16). The property of metabolism resistance of

Am580 is due to the two methyl groups at the C4 position which prevent the access of

RA metabolism enzyme, thus protect Am580 from being rapidly catabolized.

11

Figure 1.2 Common natural and synthetic retinoids. (a) All-trans-retinol; (b) All-

trans-retinoic acid; (c) Retinyl ester; (d) Retinal; (e) 9-cis-retinoic acid; (f) 13-cis-

retinoic acid; (g) Am580, (an RA analog).

12

Vitamin A deficiency is a major public health problem among young children and, to a

lesser extent, pregnant and breastfeeding women, in most developing countries. It is a

major cause of mortality and severe morbidity in children (17). It is estimated that 140–

250 million children under five years of age are affected by VA deficiency worldwide

(18). Vitamin A deficiency can be caused by inadequate intake, fat malabsorption, or

liver disorders. Deficiency can result in impaired immunity, skin keratinization,

metaplasia, poor growth and typical ocular abnormalities (e.g., xerophthalmia, night

blindness), etc. (19). VA deficiency increases the risk of infection, diarrhea and

developing respiratory disease in children (20). The traditional means of prevention are

supplementation with high-dose vitamin A, for example, infants < 6 mo can be given a

one-time dose of 50,000 IU, and those aged 6 to 12 mo can be given a one-time dose of

30,000 RAE (100,000 IU). For pregnant or breastfeeding women, therapeutic doses

should not exceed 10,000 IU /day to avoid possible damage to the fetus or infant.

On the other hand, just as a deficiency of VA affects all body systems, so does an

overabundance. The manifestations of VA toxicity include headache, vomiting, diplopia,

alopecia, dryness of the mucous membranes, bone and joint pain, liver damage and coma

(21). In very young children treated with 50,000 to 100,000 IU vitamin A, the most

frequently observed symptoms are a temporary bulging of the fontanel and vomiting (22,

23). Vitamin A also has teratogenic properties. High intakes of VA by pregnant women

1.3.2 Vitamin A deficiency and toxicity

13

may increase the incidence of teratogenic effects in the developing fetus (24), and

experiments in pregnant animals have demonstrated that excess VA given to the mothers

can result in increased birth defects in their young (25, 26) Thus, maintaining an adequate

but not excessive amount of VA in the body is vitally important.

Dietary VA is obtained mainly in the form of RE and β-carotene. When ingested, VA is

first converted to retinol in the lumen of the intestine, then re-esterified to RE in the

enterocytes, packaged into newly formed chylomicrons for delivery and stored in the

liver as RE (27). As the center of VA storage, the liver stores approximately 50-80% of

the body’s total VA. To transport VA to extrahepatic tissues, stored RE is mobilized to

produce free retinol which then binds to a liver synthesized transport protein, retinol-

binding protein (RBP). Once bound by RBP, the retinol-RBP complex is secreted into the

bloodstream for transport to peripheral tissues (28, 29). Release of retinol-RBP complex

is determined by the rate of RBP synthesis. Meanwhile, the secretion of retinol-RBP is

also highly regulated by VA status, such that VA deficiency blocks retinol-RBP secretion

(29, 30). When it circulates in the bloodstream, the retinol-RBP complex binds to a serum

carrier, transthyretin (TTR), which is believed to prevent elimination of the relatively

small RBP molecule through the kidney and thereby maintain normal levels of retinol in

1.4 VITAMIN A METABOLISM

1.4.1 Transport and metabolism of retinoids

14

the circulation (29, 31, 32) (Fig. 1.3). It has been thought that retinol enters cell through

passive transport or through a specific cell receptor. Recently, a cell surface protein,

STRA6, has been identified as an RBP receptor, which mediates cellular uptake of retinol

(33) (Fig. 1.3).

Although most of the retinoid compounds found in the circulation are in the form of

retinol bound to RBP, there are small amount of RA and RE circulating in the blood

stream and these are taken up by unknown mechanism (Fig. 1.4) (34). Dietary RA, or RA

produced by metabolism of dietary RE in the small intestine, can be absorbed via the

portal system and then circulate in the plasma bound to albumin (28, 35). A study

investigating the uptake and metabolism of all-trans-[3H]retinoic acid by human foreskin

keratinocytes suggested that the binding of RA to albumin protects RA from conversion

to polar metabolites, and controls delivery of RA from the aqueous extracellular

environment to the cell surface (36). It has been reported that RA does not require a cell

surface receptor for uptake because it is able to traverse the cellular membrane and enter

the cell efficiently (37, 38). However, studies by Kang et al. (39) revealed that a

membrane receptor, mannose-6-phosphate/insulin-like growth factor II receptor

(M6P/IGF2R) might be involved in mediating RA-response pathway and cellular activity

in cells. Moreover, the liver and other tissues do not store RA, and the pool of RA turns

over rapidly (40).

15

Figure 1.3 Schematic overview for the transport and uptake of dietary retinoids

within the body. This figure is based on reference (136). Dietary retinoids, primarily in

the form of retinyl esters, are converted to retinol prior to uptake into the enterocyte.

Within the intestine, retinol undergoes re-esterification and the new RE is incorporated

along with dietary lipids into chylomicrons for transport through the circulation system to

the liver, where excess retinol is stored in the form of RE. To transport VA to

extrahepatic tissues, stored RE is mobilized to produce free retinol, which binds to RBP,

and then it is released into the bloodstream. In the blood, the retinol-RBP complex binds

to a serum protein, transthyretin for transport to target organs. The transmembrane RBP

receptor, STRA6, facilitates retinol uptake into cells and after retinol is taken up into the

tissue, it binds to CRBP and is esterified to form RE, or oxidized to form RA to exert its

biological activities within cells. (RE: retinyl ester; LPL: lipoprotein lipase; R: retinol;

16

RA: retinoic acid; RBP: retinol binding protein; TTR: transthyretin; Chylo: chylomicron;

CRBP: cellular retinol-binding protein; CRABP: cellular RA-binding protein; RAR:

retinoic acid receptor; RXR: retinoid X receptor)

17

Figure 1.4 Uptake of retinoids into extrahepatic tissues. This figure is based on

reference (34). Retinoids in the circulation are present in several forms, including retinol

bound to RBP, RA bound to albumin, and RE contained in lipoproteins (primarily

chylomicrons). The transmembrane protein STRA6, which is identified as a RBP

receptor, can mediate retinol uptake into cells. The mechanisms that mediate cellular

uptake of RE and RA are not fully understood. However, a possible mechanism for RE

uptake in certain tissues has been established that RE is hydrolyzed to retinol by LPL

before it is taken up by tissues and cells.

18

It has been established that ~75% of dietary RE is taken up by the liver, while ~25% RE

is taken up by extrahepatic tissues, including adipose tissue, skeletal muscle, heart, lungs

and kidneys (41, 42). A model of mice lacking RBP indicated that although the VA

stored in the liver cannot be mobilized, RBP-deficient mice are still able to maintain a

normal phenotype (43). A further investigation on RBP-/- mice revealed that a high

concentration of RE in the circulating chylomicron/VLDL meets tissue retinoid

requirements, thus compensating for the absence of retinol-RBP (44). The physiological

importance of postprandial RE is unclear in wild-type animals, but it might be an

important factor in maintaining VA homeostasis in some specific tissues, such as the

lung, which might require a direct delivery of retinoids to accumulate VA stores (45).

There is little known about the cellular uptake of RE as well. It has been hypothesized

that lipoprotein lipase (LPL) plays a role in facilitating cellular uptake of RE, because

LPL is able to catalyze the hydrolysis of chylomicron RE (46). However, LPL-facilitated

chylomicron RE uptake is only observed in skeletal muscle, heart, and adipose tissue,

while other tissues, such as the kidneys and lungs, take up RE independent of LPL

manipulations. This suggests that more than one mechanism exists to mediate the uptake

of chylomicron RE in the extra-hepatic tissues.

Within the cytoplasm of tissue cells, retinol and its oxidized form RA bind with cellular

retinol-binding proteins (CRBP) and cellular retinoic acid-binding proteins (CRABP)

(47), which are believed to regulate the biological action of retinol and RA (48) (Fig. 1,4;

1.6). By binding with these retinoid-binding proteins, the concentration of free cellular

retinoid is limited, and the bound retinoid is directed to specific enzymes for metabolic

19

processing (49). Retinol bound to CRBP is directed to the enzyme lecithin: retinol

acyltransferase (LRAT) for esterification and thus is turned into the storage form RE (50,

51). CRBP-bound retinol is also a substrate for members of the alcohol dehydrogenase

family (ADH) (52), which catalyzes oxidization of retinol to retinal. Retinal is further

irreversibly oxidized to RA by aldehyde dehydrogenase (ALDH) and cytochrome P450

enzyme families (52, 53). CRABP has the similar function of mediating intracellular RA

concentration and directing RA to the enzymes, which metabolizes RA into polar inactive

metabolites, such as 4-oxo-RA, 4-OH-RA, 18-OH-RA and 5,18-epoxy-RA, etc (51, 54).

Thus, the biological effects of excess RA are limited.

Along the pathway of VA metabolism, two important enzymes play prominent roles in

regulating retinoid homeostasis (55). One is LRAT which catalyzes the esterification of

retinol; the other is a cytochrome P450, CYP26, which mediates oxidation of RA (Fig.

1.5). Both of these enzymes are tightly regulated by RA in a tissue-specific manner,

especially in the liver and lung (56). Also, the level of these enzymes is affected by VA

status, for example, VA deficiency down-regulates the expression level of LRAT (57)

and CYP26 (58, 59) to maintain the level of free retinol, while VA sufficiency increase

LRAT (56) and CYP26 (60) level to prevent retinol excess. This is because the level of

RA signal that regulates gene expression is determined by the status of VA. The response

1.4.2 Retinoid homeostatic proteins

20

of LRAT and CYP26 to the RA signal provides a self-regulatory mechanism that

regulates retinol homeostasis to avoid both VA deficiency and toxicity.

Another protein that may contribute to retinoid homeostasis is STRA6, a cell surface

receptor for RBP (Fig. 1.4). This transmembrane protein shows a high affinity for RBP

and mediates cellular uptake of retinol (33). Clinical cases suggest that mutations in the

STRA6 gene result in Matthew-Wood syndrome, which is characterized by multisystem

malformations that include lung hypoplasia (61). When STRA6 and LRAT are co-

expressed, the cells take up retinol more efficiently, suggesting a driving force for retinol

uptake resulting from the conversion of retinol into RE by LRAT (33, 62). Several in

vitro and in vitro studies have shown that STRA6 expression can be upregulated by RA

in several cell lines (63-65), and supplementation with retinoids is able to elevate STRA6

expression in mouse embryos or in the lungs of neonatal rats (66, 67), indicating the

regulation of STRA6 by RA. However, it is not clear whether STRA6 mediated retinol

uptake is the only mechanism facilitating retinoid uptake. As discussed before, the

chylomicron derived RE could be another important source of retinoid for some extra-

hepatic tissues.

21

Adapted from Ross, A.C. J Nutr, 1993

Figure 1.5 Regulatory role of RA in retinoid metabolism. This figure is based on

reference (68). The retinoid homeostasis is tightly regulated by RA through a gene

regulation mechanism. Expression of the LRAT and CYP26B1 mRNA are induced by

RA, or by dietary vitamin A, and are downregulated by VA depletion. STRA6 expression

may also be upregulated in response to RA for facilitating retinol uptake into the cell.

22

Vitamin A is a fat-soluble vitamin that acts like a hormone in the body (49). It functions

as a gene regulator by binding to a series of nuclear receptor proteins that belong to the

steroid/thyroid hormone receptor superfamily (69, 70) (Fig. 1.6). These retinoid receptors

are classified into two families, the RA receptors (RARs), and the retinoid X receptors

(RXRs), each containing three isoforms (α, β, and γ) (70, 71). Upon RA binding, the

RARs are activated, form homodimers of RAR/RAR, or heterodimers of RAR/RXR with

RXR, which then interacts with specific regions of DNA termed retinoid responsive

elements (RARE or RXRE) to activate or repress the transcription of downstream target

genes (70). It is believed that both all-trans and 9-cis RA bind to RAR, but all-trans RA

bind to RAR specifically, whereas 9-cis RA binds to RXR with high affinity (72). Via

this mechanism, RA is able to affect transcription of many genes. Several artificial

retinoids with specific RAR or RXR binding selectivity have been synthesized to be used

in studies of retinoid signaling within cells. For example, Am580, a retinobenzoic acid

analog of RA, which activates RARα selectively and shows resistance to metabolism (16,

73), has been used in our previous study to test the interaction of VA and acidic retinoids

on lung RE formation (67). Due to the prolonged regulating activity on retinoid

homeostatic genes, Am580 can alter neonatal lung retinoid metabolism dramatically (67).

1.5 THE REGULATORY MECHANISM OF RETINOIDS

23

Figure 1.6 Cellular retinoid metabolism and signaling pathway. This figure is based

on references (56, 68, 74). Upon entering the cell, retinol is bound by CRBP, and directed

to LRAT for esterification or directed to alcohol dehydrogenase (ADH), which catalyzes

oxidation of retinol to retinal. Retinoic acid taken into the cell or synthesized from retinal

is bound by CRABP. CRABP-RA is either delivered to CYP26 for further oxidation or

transferred to nuclear. Once in the nuclear, RA activates RAR which forms a heterodimer

with RXR, or forms a homodimer, and then interacts with specific regions of RARE or

RXRE to activate or repress the transcription of downstream target genes.

24

1.6 RETINOIDS IN LUNG DEVELOPMENT AND FUNCTION

The earliest evidence of VA’s importance in the lung can be dated back decades. In

1917, McCollum, who discovered VA, first observed that animals fed VA deficient diet

frequently suffered from prevalent bronchitis (75). Several years later, Bloch, a

pediatrician, supplemented food rich in VA to VA deficient (VAD) infants and young

children, and found the symptoms of bronchopneumonia caused by VAD could be

rescued by the VA rich diet (76). In 1933, Wolbach and Howe (77) observed that the

mucous-secreting epithelium in the trachea and the bronchi of VAD rats was replaced by

stratified squamous keratinizing epithelium, which further confirmed that the respiratory

system is a target for retinol action The morphological alteration in the airway of VAD

rats can be recovered when the dietary retinol is restored. These findings suggest that

retinoids play an important role in regulating differentiation of respiratory epithelium and

this speculation was demonstrated by a number of studies (78-81). As a biologically

active metabolite of retinoid, RA has shown stronger action on lung development than

retinol, although RA is not stored in tissues and is eliminated rapidly (53, 82).

RA signaling is detectable in the lung as early as the very beginning of organogenesis

(83). Absence of RA signaling in the early time of embryonic stage results in impaired

primary lung bud formation and branching morphogenesis (84). During the early stages

1.6.1 Retinoids in lung morphogenesis

25

of lung development, the major RA synthesizing enzyme retinaldehyde dehydrogenase 2

(RALDH2) and RA nuclear receptors (RARs, RXRs) are expressed in a specific pattern

in fetal lung. Mice with compound mutants of the RA receptors showed dramatic

abnormalities in lung phenotype (85-87). Disruption of the mouse RALDH2 gene also

generated severe defects similar to those described in vitamin A-deprivation studies (88).

In the process of airway branching, RA activity needs to be down-regulated to allow the

normal growth of distal epithelial bud. If up-regulated, the branching is inhibited and

lung structure resembles proximal airways (89, 90). When RA signaling is blocked by a

pan-RAR antagonist, the lung branching is increased (90, 91). These findings suggest the

involvement of RA in morphogenesis and in early embryonic development.

Lung septation stage occurs from later gestation until the first few postnatal years in

human. Studies in neonatal rat lung indicate that an acute and significant accumulation

and utilization of RE occurs exclusively during alveolar stage (92, 93). Concurrently,

there is an increase in active forms of retinol and RA in the lung fibroblasts, indicating an

increased demand of retinoids for normal postnatal development of the lung (94).

Associated with dramatic changes in the retinoid homeostasis is the upregulation of

cellular retinoids binding proteins (CRBP, CRABP), retinoid synthesizing enzymes

(ALDH-1, RALDH-2) and specific RA receptors (RARs) during perinatal development

(47, 94-96). After birth, the levels and binding activity of CRABP to RA rise strikingly in

1.6.2 Retinoids in alveolar septation

26

neonatal rat lung and then declines to low level until the septation process is completed

(47, 97). These findings provide evidence that RA is indeed required for alveologenesis.

It seems likely that the effects of VA on lung development, differentiation and

maintenance through the regulation of many genes that related to lung development,

including those involved in patterning, matrix proteins and certain growth factors, etc

(98), and the action of VA is through its interaction with its nuclear receptors which in

turn modulate the transcription of target genes. Studies investigating the relationship

between retinoids and lung development have shown that deletion of RA receptors RAR-

γ and RXR-α, the key nuclear receptors through which RA induces the formation of

alveoli (97, 99, 100), leads to marked failure of septation that associated with reduced

alveolar numbers and elastin production (101). In contrast to RAR-γ and RXR-α, RAR-β

is an endogenous inhibitor of septation during but not after the period of septation, since

RAR-β activation could block septation while its deletion result in early onset of

septation in mice lung (102). RA also has shown its activity in upregulating expression of

lung elastin gene in lung fibroblast (103) and administration of RA to the neonatal rats

resulted in partial recovery of the septation process and formation of alveoli (97, 99).

Inhibition of the RA synthetic enzyme, ALDH, disrupts tropoelastin mRNA and

decreases elastin levels (103).

Premature infants, associated with very low body weight (VLBW), tend to have VA

deficiency, probably because of a shortened period of transplacental VA supply resulting

from premature birth (104-106). The earlier a child is born before due date, the lower his

serum-retinol levels are (107), and this population often has a high risk to develop

27

various pulmonary diseases. In several clinical trials, VA supplements showed beneficial

effects in elevating VA status and preventing BPD in VLBW infants (108, 109).

In clinical therapy, dexamethasone, a typical synthetic glucocorticosteroid hormone, is

commonly used in the postnatal treatment of premature and VLBW infants with BPD to

prevent inflammation and stimulate the lung maturation and the production of surfactant.

Although a short-term improvement in lung function is seen in many infants, side effects

of dexamethasone have been reported in multiple organs in preterm infants (110). Animal

studies have shown that daily dexamethasone treatment to neonatal rats during the

postnatal period resulted in irreversibly impairment in saccule septation (111, 112). The

possible reasons that glucocorticosteroids inhibit the lung septation might due to its

inhibition in DNA synthesis and cell division while the process of the lung septation

needs septa formation, elongation, capillaries and fibroblasts filling (113-115). This

thought is strengthened because that serum glucocorticosteroid concentration was found

to be low during the period of septation (116), and starts to increase as septation ends (7,

117), which suggests the increased concentration of the hormone initiates the end of

septation. Interestingly, it was reported that postnatal dexamethasone treatment is able to

increase plasma VA and RBP significantly in newborn infants (118), which suggest that

dexamethasone could stimulate liver retinol mobilization and retinol-RBP release. in

response to dexamethasone.

1.6.3 Retinoids in lung tissue repair

28

In addition to dexamethasone treatment, preterm infants often receive treatments of

oxygen supplementation and mechanical ventilation support after birth since their lungs

are functional inefficient. But these treatments carry many potential complications

including airway injury, alveolar damage, and ventilator-associated inflammation because

the lung is exposed to hyperoxia. Hypoxia is known to be a major stimulator of VEGF

expression (119, 120) because it can activate the transcription factors hypoxia-inducible

factor 1 (HIF-1) and HIF-2 (121, 122), which subsequently enhance transcription of the

gene. Elevated VEGF levels in hypoxic tissues are thought to induce angiogenesis by

which more nutrients and oxygen can be delivered to the hypoxic cells (123). Therefore,

exposure of the developing lung to high levels of oxygen during the postnatal period may

downregulate VEGF expression, which in turn inhibit the formation of capillaries in the

lung (124, 125) and interferes with the process of septation (126, 127). Moreover,

hyperoxia may deprive VA storage in the lung, which is known as one of the effective

antioxidants, because oxidative stress caused by hyperoxia can result in more oxidation

of retinol.

A number of studies have demonstrated the great potential of RA in lung tissue repair. In

different neonatal rodent models, known for impaired alveologenesis, like postnatal

hyperoxia exposure or dexamethasone treatment, exogenous at-RA treatment can

partially rescue failed septation and stimulate alveolar formation in neonatal rat lung and

(97, 99, 128). In adult rat or mouse models, the similar effects of RA also have been

observed in elastase or dexamethasone-impaired lung tissue (95, 100, 129, 130). It is well

known that RA functions as a gene transcription regulator through interacting with their

29

specific nuclear receptors (69), and the VEGF genes have been reported as RA

responsive genes (131). Since VEGF family of growth factors plays an essential role in

angiogenesis (132), alveolar regeneration induced by RA may be an important novel

therapeutic approach to the treatment of respiratory diseases characterized by a reduced

gas-exchanging surface area such as BPD and emphysema.

The molecular signals that induce alveolarization are not clearly understood, but RA

signaling in lipofibroblasts appears to play a key role (133). Lipofibroblasts not only

serve as retinoid storage cells of the lung, similar to the stellate cell in the liver (134), but

also contain many components of retinoid signaling pathway including receptors and

binding proteins (94), and can produce endogenous RA (135). Furthermore,

lipofibroblasts are a major source of lung ECM proteins, such as collagen and elastin

(133), and they can synthesize elastin in response to RA (103). Collagen and elastin are

two of the most important components of the ECM that give structural support to resident

cells and contribute to the elasticity of the lung, respectively.

The interactions between ECM and cells not only regulate the development of the normal

lung, but it also plays an essential role in repair and formation of new ECM after lung

injury. Lipofibroblasts are located at the base of new septa and adjacent to type II

alveolar cells (7, 136, 137), which are involved in surfactant synthesis and secretion and

can be considered as progenitor of type I cells. Meanwhile, lipofibroblasts are progenitors

of myofibroblasts, which appear at the tips of newly formed septa and are required for

alveolar septation (137, 138). Based on these findings, lipofibroblasts might serve as a

30

proximate endogenous source of retinoid signals and a signaling center for mediating

alveolus formation during lung septation or after lung injury.

The molecular mechanism of retinol uptake by the lung tissue is still not fully

understood. A receptor-mediated cellular uptake of retinol is a possible mechanism

involved in this process. Recent discovery of a membrane receptor for RBP, STRA6,

supports this hypothesis (69, 139). Another mechanism of retinol uptake into the lung

may exist in that chylomicrons or chylomicron remnants carrying RE could be taken up

by the low-density lipoprotein or chylomicron remnant receptor (33).

It is believed that retinoids are stored as RE in lipid interstitial cells (also known as

lipofibroblasts) (140), which are characterized by their lipid droplets, located in the

proximal portion of alveolar septum, proliferate at the tip of the septum to cause the

eruption of septa into the alveolar sacs (134, 141). Abundant evidence suggest that lipid

interstitial cells play an essential role in normal lung development and injury repair via

epithelial-mesenchymal interactions (137, 142), involved in the synthesis of collagen and

elastin in extracellular matrix (133, 143-147). It also provides lipid substrate for the

synthesis of surfactant phospholipid in type II alveolar epithelial cell (133). During the

postnatal period, cellular retinoic acid binding protein (CRABP) increases in whole lung

tissue and lung lipofibroblasts of rats. Also, increases of RARβ and RARγ were observed

in lipofibroblasts from late gestational period to early postnatal period (148). Recently, it

1.6.4 Retinoid metabolism in the lung

31

was demonstrated that lipid interstitial cells are able to produce and secrete at-RA at the

sites where the secondary septum projects, thereby providing endogenous at-RA for

alveolar formation (47, 94). Based on the observations above, signaling between the

lipofibroblasts and type II cells might be crucial for normal alveologenesis.

32

Chapter 2

MULTIPLE TREATMENT STUDY

Previously we have shown that an oral dose of VA combined with acidic retinoids

synergistically increases retinol uptake and RE formation in neonatal rat lung,

concomitant with the upregulation of expression of several important retinoid

homeostatic genes: LRAT (lecithin:retinol acyltransferase), CYP26B1 (a cytochrome

P450), and STRA6 (stimulated by retinoic acid gene 6). In the present study we

compared the response to VA dose alone, or VA combined with acidic retinoids (RA or

Am580) in two timing protocols: a single early dosing (d 4) vs. multiple dosing

throughout the period of lung septation (d 4, 7, 11, 14). We also tested the influence of

dose administration on lung structural and functional genes. The results have shown that

multiple dosing resulted in a higher, cumulative increase in lung RE content for all

treatments. However, the increase of gene upregulation mediated by acidic retinoids after

the single and multiple dosing did not differ between the two treatment protocols. Lung

structural and functional genes remained nearly constant with both treatment and timing.

In conclusion, multiple treatments of VA and acidic retinoids in combination during the

septation period greatly increased neonatal lung RE content in a synergistic and

cumulative manner. Repeated but transient induction of retinoid homeostatic genes by

2.1 ABSTRACT

33

acidic retinoids at the time of each dosing may explain the observed cumulative

synergistic enhancement of RE formation.

Lung VA storage starts in late gestation and then VA is quickly depleted during the

perinatal and postnatal periods (92, 94). It is believed that the depletion of lung VA

storage is due to the high demands of retinoids for normal lung development and

maturation (149). Preterm infants who have low VA status at birth are more susceptible

to various respiratory diseases, such as BPD (106, 150, 151). Several clinical trials have

demonstrated that VA supplementation not only improves VA status but also reduce the

risk of chronic lung disease in preterm infants who often have very low birth weight at

birth (109, 152-155). In weaning rats fed a VA deprived diet, lung epithelium undergoes

striking morphological alterations, such as keratinizing metaplasia, an increased size of

rat lung airspaces, and reduced collagen and elastin in the parenchyma (149, 156, 157) .

The administration of RA to postnatal rats or mice could prevent the dexamethasone

caused low gas-exchange surface area and low alveolar number, and rescue failed

alveolar septation (97, 99). RA also enhanced alveolar septation in emphysematous rats

(100) and adult rats after pneumonectomy (158).

Given that RA is a crucial regulator of lung development, maturation and maintenance of

normal functions, studies on improving VA status in neonatal lung have been of great

interest. Shenai et al. showed that maternal VA supplementation resulted in an obvious

2.2 INTRODUCTION

34

increase in lung RE contents in the fetuses and offspring of rats (159). Previously, we

reported that providing a combination of VA and RA (10:1 molar ratio) directly to the

neonatal rats is much more effective (~4 fold) in increasing lung RE than providing the

same amounts of VA or RA separately (160, 161). Metabolic studies showed that VARA

directs more delivery of [3H]retinol, used to trace the uptake of newly absorbed retinol, to

the lung (67, 161). However, the synergistic effect of VARA was only observed in the

lungs, as RE in the liver was increased equally by VARA and an equal dose of VA only,

without RA (160, 161).

Our latest investigation on the molecular mechanism of VARA synergy suggested that

the VARA promotes RE formation in the lung by upregulating the expression of LRAT,

CYP26B1 and STRA6 mRNAs, thus, enhances uptake and storage of VA in the but

transient activity of RA on retinoid homeostatic genes (67), and this finding leads us to

the next hypotheses that repeated treatments with RA would result in much stronger and

sustained activity on lung RE formation.

In the present study, we supplemented neonatal rats with multiple doses of retinoids from

postnatal day 4 to day 14, the period of septation, to compare effects of multiple dosing

with a single dosing on RE formation. We then determined whether long-term treatment

with RA can raise expression of LRAT, CYP26B1 and STRA6 during the postnatal

period. This study also evaluated the effect of multiple treatments on the expression

pattern of several lung structural and functional genes. Overall, this study was designed

to test the effects of long-term supplementation of VARA on promoting lung RE storage

and its influences on normal lung development in postnatal life.

35

Hypothesis 1: Multiple treatments with VARA or VAAm580 throughout the postnatal

period of lung septation will significantly increase RE content in neonatal lung in a

cumulative manner. Here, we used Am580 to investigate the regulatory activity of RA

throughout the supplemented period. The repeated action from supplementation with RA

will enhance the expression of LRAT, CYP26B1 and STRA6 mRNA to higher levels

compared with the effect of a single treatment.

Hypothesis 2: Treatment with Am580 will produce a stronger induction in expression of

retinoid homeostatic genes compared with RA, thus contributing to a higher RE

formation in the neonatal lung.

Hypothesis 3: Sustained activity of acidic retinoids from multiple treatments will also

regulate the expression pattern of several lung structural and functional genes during

postnatal period.

Aim 1: To investigate how the multiple treatments of RA or Am580 during the period of

lung septation affect retinyl esters (RE) accumulation in the neonatal rat lung.

In this study, we treated neonatal rats with a single dose of VA and acidic retinoids

combination on day 4 or day14, and multiple doses through day 4 to day 14, respectively.

2.3 HYPOTHESIS AND AIMS

36

Then we determined lung total retinol contents by HPLC to determine the synergistic

effect of multiple doses on lung RE formation.

Aim 2: To determine how the retinoid homeostatic genes respond to repeated treatments

with VARA or VAAm580.

In this study, we conducted real-time polymerase chain reaction (rt-PCR) to quantify the

mRNA levels of LRAT, CYP26B1 and STRA6 genes in the lung of neonatal rats to

compare the effects of single or multiple dose of RA.

Aim 3: To determine how multiple treatments of VARA or VAAm580 will affect lung

structural and functional genes.

We examined expression levels of several lung structural and functional genes including

surfactant proteins, collagen, β-laminin, fibronection, elastin mRNA as well as vascular

endothelial growth factors (VEGF), after treatments, to test their responses to multiple

treatments of RA.

Animal procedures were approved by the Institutional Animal Use and Care Committee,

Pennsylvania State University. We conducted three studies (Fig.2.1): single early dosing

2.4 MATERIALS AND METHODS

Animals and experimental design

37

(d 4), single late dosing (d14), and multiple dosing throughout the period of lung

septation (d 4, 7, 11, 14). In each of these studies, neonatal Sprague-Dawley rats were

randomly divided into 6 groups (n=4-5/group) that received treatments of oil (control),

VA alone, RA alone, VA combined with RA (VARA), or VA combined with Am580

(VAAm). Neonatal pups were delivered and nourished by mother rats fed a VA adequate

diet. Since the pups were from several litters, sexes were evenly distributed to each

group. The average body weight of each group was close to each other. Before each

treatment, the pups were weighed and the dose was adjusted to 0.4 µl/g bodyweight.

We purchased VA, in the form of all-trans-retinyl palmitate and all-trans-RA (at-RA)

from Sigma-Aldrich (St. Louis, MO). Am580 was contributed by H. Kagechika,

University of Tokyo. Dosage selection for VA was based on the amount shown in human

newborns to reduce morbidity and mortality (50,000 IU/2.5KG) (10, 162). By converting

international unit to mass unit with the factor of 0.548 µg retinyl palmitate (RP)/IU, we

calculated the VA doses to be 10.96 µg RP (or 6 µg retinol) /g BW, scaled to body weight

of neonatal rats. The amount of RA was based on previous usage shown to induce lung

septation in neonatal rats (97): 500 µg RA ip/kg body weight, and we adjusted the

amount due to the estimation that about 80% of dose will be absorbed by oral delivery.

VA and RA were mixed at molar ratio of 10:1 and the concentration for each one was

0.05 M and 0.005 M, respectively. Am580 was prepared at the same molar concentration

2.4.2 Dose preparation

38

as RA, and mixed with VA at the same molar ratio of VARA. Canola oil was used as

placebo (control).

The volume of each dose provided to pup was 0.4 µl/g body weight and the exact volume

of dose was determined based on the pup’s body weight (20 nmol retinol and 2 nmol of

acidic retinoid, depending on treatment group, per gram of body weight). In studies I and

II, a single dose was given to pups on day 4 or day 14, respectively. In the study III,

repeated doses were given on day 4, 7, 11 and 14. Pups were killed with carbon dioxide

(CO2) 6 hours after treatment in the study I and II, and 24 hours after the last treatment in

the study III. The lung tissues were removed, trimmed and weighed. All samples were

frozen in liquid nitrogen immediately and then stored -80oC for later analysis.

2.4.3 Experimental methods and tissue collection

39

Figure 2.1 Animal experimental design. Three studies were conducted which differed

in how many doses were given and how old the neonatal rats were. In study I and II, a

single oral dose was given to pups on postnatal day 4 (Study I), and on postnatal day 14

(Study II). In study III, multiple dosing were given on day 4, 7, 11, 14. Tissues were

collected 6h after the single treatment in study I and II, and 24h after the last treatment in

study III. In each study, animals were randomly assigned to 6 groups, and received oil

(vehicle), VA, RA, VARA, Am580 and VAAm580, respectively.

40

Portions of the lung tissue were cut, weighed and extracted in chloroform:methanol, 2:1

v:v, overnight. Then, samples were processed by the Folch washing procedure (163).

After the final wash, the extract was dried down under argon; the samples were

redissolved in 2 ml hexanes. A portion of the hexanes volume was dried again and then

underwent a hydrolysis reaction by a saponification procedure. A known amount of an

internal standard, trimethylmethoxyphenyl-retinol (TMMP) was added to each sample

and the samples were dried under argon and reconstituted in 100 µl of methanol for

HPLC analysis. Portions of each sample (usually 18-22 µl) were injected onto a C-18

HPLC column and eluted with a gradient of 92.5:7.5 methanol:water at a flow rate of 1.5

ml/min for 5 min. The eluate was monitored by a Waters 960 photodiode array detector

and the areas of the peaks for TMMP and retinol were analyzed by Millenium-32

(Waters) software.

Total RNA from lung tissues from individual pups was extracted using a guanidine

extraction method and reverse transcribed into its complementary DNA (cDNA). The

diluted reaction product was used for real-time PCR (rt-PCR) analysis. Primers designed

2.4.4 Retinoid analysis

2.4.5 Gene mRNA level determination

41

to detect mRNA expression were: 5´-ATA GGA TCC TGA CCA ACA CTA CAT CCT

CTC-3´ (forward) and 5´-ATT CTC GAG TCT AAG TTT ATT GAA ACC CCA GA-3´

(reverse) for rat LRAT (NM_022280.2); 5'-TTG AGG GCT TGG AGT TGG T-3'

(forward) and 5'-AAC GTT GCC ATA CTT CTC GC-3' (reverse) for rat CYP26B1

(NM_181087); 5'-GTG CCA GTG ATT GCT GAA GA-3' (forward) and 5'-GGA GGT

GTC CTC TGG ATG AA-3' (reverse) for rat CYP26A1; 5'-CCG ATC CTG GAC AGT

TCC TA -3' (forward) and 5'-CCA CCT GGT AAG TGG CTG TT -3' (reverse) for rat

STRA6 (NM_0010029924.1); 5’-TTG TCG CTG GTA TCA AGT GC-3’(forward) and

5’-CAG CCC CTA TCA TTC CAT GT-3’ (reverse) for rat SPA1 (sftpa1)

(NM_017329.1); 5’-GAT GAT GGG GAA GCT GGT AA-3’ (forward) and 5’-ACC

ATT GGC ACC TTT AGC AC-3’(reverse) for rat collagen (Col1a1) (NM_053304.1);

5’-GAC TTG GGG CGT GTA CAG AT-3’(forward) and 5’-GCA TGA CCA TAG CAG

AAG CA-3’(reverse) for rat laminin (Lamb1) (NM_001106721.1); 5’-GAG GAA AGC

CTG GGA AAG TT-3’ (forward) and 5’-TCC ACC TCT GGC TCC ATA CT-3’

(reverse) for rat elastin (Eln) (NM_012722.1); 5’-ACC ACC CAG AAC TAC GAT GC-

3’ (forward) and 5’-TCT CCC AGG AGT CAC CAA TC-3’ (reverse) for rat fibronectin

(fn1) (NM_019143.2); 5’-GCC CAT GAA GTG AAG TT-3’ (forward) and 5’-TTT CTT

GCG CTT TCG TTT TT-3’ (reverse) for rat Vegfa (NM_031836.2). The mRNA

expression level of each sample was corrected by calculating mRNA-to-ribosomal 18S

RNA ratio. Data were normalized to the average value for the control group, set at 1.00,

prior to statistical analysis.

42

Data are presented as group means ± SEM (standard error of the mean). Group

differences were tested by one-factor ANOVA followed by Fisher’s protected least

significant difference test. The software used for statistic analysis was GraphPad Prism

(San Diego, CA). For comparison, we converted the mean mRNA value of the lung

control group to a value of 1, and the mean values of the other groups were converted

accordingly. To reduce variance of each group mean, values were transformed to log10

form before statistical analysis. Differences with p≤0.05 were considered significant.

Multiple dosing resulted in a higher, cumulative increase in lung RE contents for all

treatments. Previously we have shown that VA combined with acidic retinoids

synergistically increases retinol uptake and RE formation in neonatal rat lung. In the

present project, we designed three studies according to the timing of the dosing

throughout the period of lung septation: an early treatment study (a single dosing on day

4), a late treatment study (a single dosing on day 14) and a multiple treatment study

(multiple dosing on day 4, 7, 11, 14), to compare the level of lung RE formation

influenced by timing and numbers of times of dosing (Fig. 2.2). In each of the study,

2.4.6 Statistical analysis

2.5 RESULTS

43

neonatal rats were assigned to 6 groups and animals in each group received oral dose of

oil (control), VA alone, RA alone, VARA, Am580 alone, or VAAm respectively. In both

of the single dosing studies, VARA and VAAm promoted higher lung RE formation

compared with treatment of VA alone. The results of the early treatment studies agree

with the result of our previous study, in which VARA and VAAm580 could

synergistically increased lung RE content as early as 6 h after dosing. The treatment on

day 14 showed a similar result as the early time (day 4) administration, indicating a

consistent mechanism of VA metabolism throughout the lung septation period. Relative

to the results of the single dosing studies, multiple dosing resulted in a higher,

accumulative increase in lung RE contents for all treatments. The synergy with VARA or

VAAm was still significant in multiple dose studies. Notably, the repeated treatment of

VAAm580 exaggerated the effect of VARA in promoting RE formation, which is a 2

fold increase in lung RE. This is because of the Am580 is resistant to metabolism and its

sustaining activity on gene regulation alter neonatal lung retinoid metabolism. In

conclusion, multiple treatments of VA and acidic retinoids in combination during the

septation period greatly increased neonatal lung RE content in a synergistic and

accumulative manner.

44

Figure 2.2 Lung RE concentration after a single treatment or multiple treatments

with VA alone, all-trans-retinoic acid (RA) alone, combination of VA and RA

(VARA), Am580 alone, and combination of VA and Am580 (VAAm). Neonatal rats

were treated with an early single dose on day 4 (A), late single dose on day 14 (B), and

multiple doses on day 4, day 7, 11, 14 (C). Lung total retinol contents were determined

by HPLC after treatments. Data are presented as group means ± SEM; Groups were

compared by one-factor ANOVA. Data were transformed by log10 prior to ANOVA.

Tukey’s Least square mean test was used after ANOVA. Groups with different letters

differed significantly, p<0.05.

45

Multiple treatments of VA and acidic retinoids in combination during the septation

period had no cumulative effects on the expression of lung homeostatic genes. But

VAAm can prolong the effect.

Our previous studies had shown that acidic retinoids combined with retinol could

increase retinol uptake and esterification in neonatal lung by upregulating the expression

of lung LRAT and STRA6 genes. In the present study, we examined how the long-term

administration of acidic retinoids affects the expression of retinoid homeostatic genes.

In both early and late single treatment studies, LRAT mRNA was significantly induced

by acidic retinoids at 6 h. The stable retinoid, Am580, compared with RA, had much

stronger effect of inducing LRAT mRNA compared with RA (Fig. 2.3). These results are

consistent with the results of our previous 6 h treatment study (67). However, in the

multiple treatment study, LRAT mRNA was reduced to the basal level 24 h after the last

dosing in the lungs of RA and VARA-treated neonates. By comparison with RA, Am580

dramatically increased LRAT mRNA at 6 h and maintained this level at 24 h. This result

suggests that even multiple treatments with RA still increase mRNA level transiently,

probably because RA was quickly metabolized by CYP26B1 (RA hydroxylase). In

contrast, due to its resistance to oxidative metabolism, Am580 shows its sustained

regulatory activity on LRAT gene expression even 24 h after the treatment. The extended

increase in LRAT mRNA coincided with the dramatic increase in RE content in the lung

of VAAm580 treated pups. Am580 continues to exert its regulatory activity due to its

46

resistance to oxidative metabolism, and CYP26B1 is dramatically upregulated, to a

higher extent than LRAT.

The rt-PCR results for CYP26B1 showed similarity with LRAT (Fig. 2.4). In both single

dose studies, the level of CYP26B1 mRNA was elevated 3–25-fold by all of the

treatments at 6 h, compared with control. In the multiple dose study, the mRNA level in

RA or VARA treated pups come down to basal level at 24 h, where as Am580 treatment

maintains its level at 24 h. These results indicated that CYP26B1 gene also could be

transiently induced by RA but persistently by Am580.

Then we determined STRA6 gene expression levels (Fig. 2.5). By contrast to our

pervious results, the regulation of STRA6 in the lung of 4-day old neonates by RA or

Am580 was not as strong as in the previous analysis from 7~8-day old rats, but it still

showed a similar trend of increasing in the pups treated with acidic retinoids. A

comparison of 4-day old and 2 week old rats showed that STRA6 is developmentally

regulated since the older pups, even in the vehicle group, had lower STRA6 expression

levels than younger pups.

47

Figure 2.3 Expression level of Lung LRAT gene after treatments. Neonatal rats

received an early single dose on day 4 (A), a late single dose on day 14 (B) and multiple

doses on day 4, 7, 11, 14 (C). Lung tissue from neonatal rats treated with oil, VA, RA,

VARA, Am and VAAm was processed for total RNA isolation and subjected to rt-PCR

analysis. Data was normalized to 18S rRNA and the average value for the control group

was set to 1 for each experiment. Data are presented as group means ± SEM; Groups

were compared by one-factor ANOVA. Data were transformed by log10 prior to

ANOVA. Tukey’s least square mean test was used after ANOVA. Groups with different

letters differed significantly, p<0.05.

48

Figure 2.4 Expression level of lung CYP26B1 gene after treatments. Neonatal rats









49

Figure 2.5 Expression level of lung STRA6 genes after treatments. Neonatal rats









50

Retinoid treatments did not perturb the expression of lung structural and functional

genes.

Lung surfactant protein A1 (SPA1), collagen, laminin, elastin, fibronectin and vascular

endothelial growth factor A (VEGF-a) are important to lung structure and function during

lung development and maturation. We next examined whether the expression of these

genes was affected by the retinoid treatments used. Primers for SPA1, collagen, laminin,

elastin, fibronectin and VEGF-a were designed and used to analyze each of these genes

necessary for normal lung functioning, determined by rt-PCR. We tested SPA1 and

collagen genes with sample from individual pup, and laminin, elastin, fibronectin,

VEGF-a genes with pooled cDNA samples for each group. The results showed that all

these genes remained nearly constant with treatment. Laminin and fibronectin genes were

developmentally down-regulated since the older pups, even in the vehicle group, had a

lower expression level compared with younger pups. These results suggest that our

treatments did not perturb the expression of these important lung structural and functional

genes.

51

Figure 2.6 Expression level of lung functional and structural genes after treatments.

Neonatal rats received an early single dose on day 4 (A), a late single dose on day 14 (B)

and multiple doses on day 4, 7, 11, 14 (C). Lung tissue from neonatal rats treated with oil,

VA, RA, VARA, Am and VAAm was processed for total RNA isolation and subjected to

rt-PCR analysis. The analyses of SPA1 and collagen genes were based on sample from

individual pups, while laminin, elastin, fibronectin and VEGF-a were based on pooled

sample of each group. Data was normalized to 18S rRNA and the average value for the

control group was set to 1 for each experiment. Data are presented as group means ±

SEM; Groups were compared by one-factor ANOVA. Data were transformed by log10

prior to ANOVA. Tukey’s Least square mean test was used after ANOVA. Groups with

different letters differed significantly, p<0.05.

52

2.6 DISCUSSION

It is known that the lung is immature at birth. It still needs a process of septation which is

dependent on the signals provided by RA. Severe VA deficiency leads to abnormal

cellular differentiation and proliferation in the epithelium of the respiratory tract (164).

Infants with extremely low birth weight usually have low VA concentrations in plasma

and tissues, and this population is more susceptible to various respiratory diseases and

infections. Therefore, strategies to improve the VA status of newborns in the postnatal

period are of great clinical interest. In our project, we used neonatal rat lung as our model

because lung development of rodents in the first two postnatal weeks parallels lung

development in the human from week 24 of gestation through the first 2 years of age

(165), therefore, it is a good model of preterm infant. Our previous studies have reported

that VA given orally to neonatal rat in combination with RA increase lung RE contents

more effectively than the same amount of VA given alone (160, 161). The administration

of RA promotes retinol uptake and esterification by upregulating several retinoid

homeostatic genes, LRAT, CYP26B1 and STRA6, and thus, redirects part of the flow of

supplemental VA in the neonate lungs, as supported by increased [3H]retinol uptake in

VARA treated neonates (67). The use of Am580, a metabolism-resistant analog of RA,

shows an extended activity in regulating retinoid homeostatic genes and further supports

the effects of RA on lung VA metabolism (67).

In the present study, we determined the effects of multiple treatments with VARA on

promoting lung RE formation during postnatal period. The dose of VA was based on a

53

previous VA supplementation study in newborn infants (10, 162). The amount of 50,000

IU VA was scaled to the body weight of neonatal rats, which is ~21 µmol of retinol per 1

kg body weight. The dose of RA was based on the amount previously used to induce lung

septation in neonatal rats, which is 1.67 µmol/kg (97). We adjusted the dose of RA to 2.1

µmol/kg to compensate for a ~20% loss during intestinal absorption. Indeed, the dose of

VARA was the exact mixture of the VA dose and the RA dose discussed above and thus

had a molar ratio of 10:1 retinol/RA. To evaluate the effect of multiple dosing, we also

conducted two single dose studies at the same time. In both of these studies, a single dose

was administrated to neonatal rats on postnatal day 4 (early treatment study) or postnatal

day 14 (late treatment study), which are the beginning and the end of the septation period,

respectively.

The early and late single treatment did not differ from each other with respect to lung RE

formation. In contrast, multiple treatments showed a higher, cumulative increase in lung

RE content for all treatments. Compared with RA, Am580 exerts a persistent and

enhanced effect on lung VA accumulation, thus, repeated treatments with VAAm580 led

to a much higher increase in RE contents than treatment with VARA (Fig.2.2). Multiple

dose of RA or Am580 given alone also increased lung RE content to a extent even

slightly higher than multiple dose of VA (Fig.2.2). Since there was no retinol mass in the

dose, the possible origin of retinol for newly formed lung RE is the liver. Thus, we

speculate that repeated administrations of RA or Am580 during postnatal period mobilize

the liver VA reserve and deliver retinol to the lung to meet the demands for lung

development, thus changing the distribution of retinol in different tissues.

54

In our single treatment studies, we determined expression of retinoid homeostatic genes

at 6h after dosing, because VARA or VAAm had a rapid effect on retinol uptake and RE

accumulation in the lungs. The gene results agree with the results of our previous 6-h

retinoid treatment study (67), in which LRAT, CYP26B1 and STRA6 were significantly

induced by RA of Am580. Although responses of these genes to the treatment with

retinoids are rapid, a 12 h study has suggested activity of RA is just transient, because

RA is quickly oxidized by induced CYP26B1 (67). To evaluate how long-term retinoids

administration affects expression of retinoid homeostatic genes, we determined gene

levels 24h after the last treatment. The data still showed the similar results to that of 12 h

study (Fig. 2.3, 2.4, 2.5). This suggests the significant increase in lung RE is the outcome

of several temporary promotions of RE formation by RA, after each dose. In contrast,

extended activity from Am580 not only maintains the expression of genes at a high level

but also exaggerates the effects observed for RA (Fig. 2.2, 2.3, 2.4, 2.5).

RA has been implicated in normal lung development and repair mechanism by promoting

expressions of several important structural and functional genes, such as surfactant

proteins (SP), collagens, elastins, etc. (157, 166-171). However, these effects usually

were observed in VA deficient animal models or cultured lung explants. In our study,

neonatal rats were delivered by mothers fed normal diet. Therefore, the VA status in

these rats before the treatments is in a normal range that is adequate to maintain a normal

lung development and functioning. We examined expression of several lung structural

and functional genes to test our hypothesis that multiple administrations of acidic

retinoids might affect their normal expression. However, our results showed that all these

55

genes remained nearly constant with all treatments and suggest that RA or Am580 did

not perturb the expression of these genes. This finding increases the feasibility of using

VARA in a clinical setting for the goal of promoting lung VA status, as no perturbing

effects were observed on important structural and functional genes.

In summary, these studies have shown that multiple treatments of VARA and VAAm

during the period of lung septation increased lung RE contents in neonatal rat lung in a

synergistic and cumulative manner. The effect is accompanied by repeated induction of

retinoid homeostatic genes, LRAT, CYP26B1 and STRA6, by RA or Am580. However,

multiple treatments had no cumulative effects on the expression of these genes. In

contrast, Am580 produced a stronger and sustained induction of LRAT and CYP26B1

mRNAs, even 24 h after the last dose, which may contribute to a higher RE formation in

the neonatal lung. The repeated administrations of acidic retinoid show no influence on

several important lung structural and functional genes. Our findings provide some clues

for a more efficient and better therapeutic strategy in clinic use that may improve lung

RE formation in neonatal lungs.

56

Chapter 3

LPS-INDUCED INFLAMMATION STUDY

Vitamin A plays an important role in postnatal lung development and maturation. Our

previous study has suggested that the principal metabolite, retinoic acid (RA),

supplemented simultaneously with VA (VARA) to neonatal rats synergistically increases

lung retinol uptake and storage by upregulating several important retinoid homeostatic

genes, LRAT (retinol esterification), CYP26B1 (RA hydroxylase) and STRA6 (retinol-

binding protein receptor). However, whether inflammation impacts expression of these

genes and thus affects retinol homeostasis in neonatal lungs is not clear. In this study, we

investigated whether LPS-induced inflammation could affect RE formation in neonatal

rat lung, promoted by VARA. 7~8 day-old neonatal rats were treated concurrently with

(6-h study) or 12 h after (18-h study) LPS administration. Lung tissues were collected 6 h

after VARA treatment. Results suggested that in both studies, LPS slightly reduced the

VARA-mediated increase in lung RE formation. Rt-PCR data indicated that in the 6-h

study, LPS attenuated the induction in LRAT by VARA, but not the CYP26B1 and

STRA6. In the 18-h study, LPS had little effects on the expression of all these genes.

Overall, these results suggest that acute inflammation modestly reduces the RA-mediated

induction in LRAT mRNA in the neonatal lung, in turn, it slightly attenuates the

3.1 ABSTRACT

57

synergistic increase in RE formation promoted by VARA. Nevertheless, VARA

increased lung RE under conditions of mild inflammation.

Vitamin A storage is low in the human lung at birth. Usually, premature delivery is one

of the major factors that contribute to severe VA deficiency and put preterm infants at

high risk for developing pulmonary diseases, such as RDS and a chronic lung disease,

BPD. Supplementing VA to the newborns directly not only improve VA status but also

produces promising results for reducing lung injury and dysfunction (109, 152, 153).

Administration of RA to neonatal rats has been shown to induce alveolar formation,

repair epithelial lesions and increase surfactant synthesis in lung (172-174).

It is believed that RA exerts its influence in development and cell differentiation through

the binding of nuclear retinoid receptors (RAR), which forms heterodimers with RXR

and subsequently interacts with one or several retinoic acid-responsive elements (RARE)

that are located in the regulatory regions of direct targets, thereby activating gene

transcription (175-177). Several important genes that are involved in retinoid homeostasis

are also regulated by RA activity. Lecithin:retinol acyltransferase, CYP26B1 (RA

hydroxylase) which catalyzes the esterification of retinol and oxidation of RA,

respectively, and STRA6 (retinol-binding receptor) which mediates retinol uptake into

tissue, are known to be tightly regulated by RA in the lung (67). Interruption of the

3.2 INTRODUCTION

58

expression of these genes might alter the balance of the retinol metabolism in the lung

tissue.

Our previous study has shown a more efficacious way in increasing VA storage in the

lung by administrating VA orally in combination with all-trans-RA (VARA) to neonatal

rats (160, 161). VA and RA were mixed in the ratio of 10:1 and the dose produces a

synergistic effect in promoting RE formation, about 4 fold as compared with same

amount of VA given alone. A metabolic study showed that the administration of RA

redirects part of the flow of supplemental [3H]retinol in the lungs in VARA-treated

neonates (67). The molecular mechanism of VARA synergy was investigated and

revealed that including RA in the dose acts as a regulator of VA homeostasis by rapidly

upregulating retinoid homeostatic genes, LRAT, CYP26B1 and STRA6 in the lung (67),

thus promoting RE formation more efficiently.

Inflammation is very often observed in preterm infants or infants with a weakened

immune system. In the process of inflammatory responses, proinflammatory cytokines

such as tumor necrosis factor-α (TNF-α), IL-1β and IL-8 are secreted by alveolar

macrophages, fibroblasts, type II pneumocytes and endothelial cells in the early

inflammatory response upon the stimulation of inflammatory agents (178). These

cytokines recruit more circulating neutrophils and macrophages to the local sites of injury

or infection and subsequently release higher amount of cytokines and chemokines to

initiate a sequence of injurious events (179). As an organ with large area exposed to the

outside, the lung is more vulnerable to be attacked by exogenous pathogens, allergens or

chemicals. The increased mRNA level and secretion of pro-inflammatory cytokines and

59

chemokines have been detected in airway of preterm infants at various stages of

developing BPD (180, 181). Risk factors contributing to the inflammatory responses in

the lung include inappropriate resuscitation, oxygen toxicity, mechanical ventilation,

pulmonary and/or systemic infection, etc. (182).

Inflammation is also known to alter the expression of many genes including genes in the

CYP family (183). Our previous in vitro study reported that suppression of RA-induced

CYP26A1 mRNA expression was observed in LPS treated THP-1 cells (184). An in vivo

study in rat liver suggested that, LPS or poly-IC induced inflammation significantly

opposes the induction of CYP26A1 and CYP26B1 expression by RA (185). It is

unknown whether inflammation (which is often associated with newborn infants)

inhibits, attenuates, or has no effect on VARA synergy in the lungs.

In the present study, we investigated effects of LPS-induced inflammation on VARA

mediated RE formation and RA induced expression of LRAT, CYP26B1 and STRA6 in

neonatal rat lungs. At the same time, we compared the lung RE and gene results of

LPS/VARA in a co-administration study and in a model of LPS pre-administration study

to test whether the time of inflammation has an effect on the VARA synergy. The results

of these studies show that LPS slightly attenuates the induction of RE formation in

neonatal lungs when VARA and LPS are administered simultaneously. Gene data

indicate that the inflammation only decreased the RA-induced increase in LRAT mRNA.

However, when LPS was administered in advance, it has little effect on RE formation and

retinoid homeostatic genes induced by RA.

60

Hypothesis 1: Inflammation markers will be dramatically increased in the lung of LPS

treated pups and inflammation will prevent the increase in RE formation in neonatal rat

lung promoted by VARA.

Hypothesis 2. The expression of retinoid homeostatic genes, LRAT, CYP26B1 and

STRA6, which respond to VARA treatment in neonatal rat lungs, will be interrupted by

LPS-induced inflammation.

Aim 1: To test how the storage of RE in the neonatal rat lung is affected by the LPS-

induced inflammation induced.

The combination of VA and its bioactive metabolite retinoic acid, VARA, has a

synergistic effect in increasing RE contents in postnatal lung tissue (161). We

investigated how LPS-induced inflammation would affect lung RE storage and whether

the inflammation would prevent the synergy of VARA. We conducted two studies, 6-h

study and 18-h study which are based on the timing of the treatments. In the 6-h study,

neonatal pups were treated with LPS and VARA simultaneously. In the 18-h study, pups

received VARA treatment 12 h after LPS administration. For both studies, the lung tissue

was collected 6 h after VARA treatments. Lung total retinol contents (RE plus retinol)

from each lung sample were quantified by HPLC. We also demonstrated the


61

inflammation state in LPS-treated rats by determining the inflammation markers, such as

IL6, CCL2 gene in the lung and C-creative protein (CRP) in plasma.

Aim 2: To determine the response of retinoid homeostatic genes related to VA uptake

and metabolism in the neonatal lungs after treatment with VARA in inflammation states.

Lung retinoid homeostatic genes LRAT, CYP26B1, and STRA6 play important roles in

VA uptake and metabolism in the lung. By analyzing the mRNA levels of these genes,

we may be able to understand how LPS-induced inflammation interfere the expression of

these genes, thus better understand VA metabolism in the neonatal lung in inflammation

state. In this study, specific genes like LRAT, CYP26B1 and STRA6 were analyzed for

their transcript levels using rt-PCR.

Animal procedures were approved by the Institutional Animals Use and Care Committee

of The Pennsylvania State University. We conducted two studies according to the time

LPS is administered relative to the time VARA was supplemented (Fig. 3.1). In a short-

term study, LPS and VARA were co-administered 6 h before collection of lung tissue. In

a long-term study, LPS was administered 18 h before and VARA was supplemented 6 h

before tissue collection. In each study, 7-8 day-old Sprague-Dawley rat pups were


3.4.1 Animals and experimental designs

62

assigned randomly to 4 groups (n=4-5/group) and received canola oil (Control), LPS

alone, VARA, and combination of LPS and VARA. ). Neonatal pups were delivered and

nourished by mother rats fed a VA adequate diet. ). Neonatal pups were delivered and

nourished by mother rats fed a VA adequate diet.

63

Figure 3.1 Animal experimental design. Two studies were conducted according to the

time LPS is administered relative to the time VARA was supplemented. In study I (also

termed 6 hr study), LPS and VARA were administered simultaneously 6 h before tissue

collection. In study II (also termed 18 hr study), pups received VARA treatment 12 h

after LPS administration and tissues were collected 6 h later. In each study, 7-8 day-old

Sprague-Dawley rat pups were assigned randomly to 4 groups (n=4-5/group) and

received canola oil+saline (Control), LPS+oil, VARA+saline, and LPS+VARA

respectively.


64

Vitamin A, in the form of all-trans-retinyl palmitate, and at-RA, purchased from Sigma-

Aldrich (St. Louis, MO) were mixed in a molar ratio of 10:1 and the concentration for

each one are 0.05 M and 0.005 M, respectively. The dose of the VARA combination

given orally to neonates was based on body weight (0.4 ul/g body wt). Please refer to the

chapter 2 for the detailed information. Pseudomonas aeruginosa-LPS, purchased from

List Biological Laboratory (Campbell, CA), was prepared in ta concentration of 10 µg/ml

and the dose injected intraperitoneally to neonates was 0.02 ml/g body wt (20 µg

LPS/100g body wt, less than the 50 µg/100 g body weight dose for adults). Canola oil

and saline were used as placebos for VARA and LPS, respectively.

The dose of retinoid treatment given to each pup was delivered directly to the rat pup’s

mouth by a micropipette. Pseudomonas aeruginosa-LPS from a stock concentration of

2.5mg/ml was diluted to 0.01 mg/ml (10 µg/ml) and a single dose of 0.02 ml/g body wt

LPS was injected intraperitoneally to these neonates. Neonates from both studies were

sacrificed by carbon dioxide (CO2) inhalation 6 hours after VARA treatment for both

studies and then lung tissue was collected and frozen in liquid nitrogen immediately and

then stored -80oC for later analysis.


65

Plasma anti-C Reactive Proteins (CRP) was quantified by a rat CRP ELISA TEST KIT

(Life Diagnostic, Inc., West Chester, PA). The C Reactive Proteins are indicators of

inflammation in the blood. The plasma samples were diluted and incubated in the

microtiter wells for 45 minutes. The microtiter wells were subsequently washed and

incubated with horseradish peroxidase (HRP) conjugate for 30 minutes. Then the wells

were washed to remove unbound HRP-labeled antibodies and TMB reagent was added

and incubated for 20 minutes at room temperature. Color development was stopped when

the color changed to yellow. Optical density was measured spectrophotometrically at 450

nm. A standard of serially diluted control serum was included, and titers of antibody were

calculated based on this standard curve. One titer unit was defined as the dilution fold

that produced 50% of the maximal optical density for the standard sample.

Lung tissue was processed and lung total retinol was quantified in the same way as in

chapter 2. Please refer to the chapter 2 for the detailed information.

3.4.4 Plasma anti-CRP antibody enzyme-linked immunosorbent assay

3.4.5 Retinoid analysis

66

Please refer to the chapter 2 for detailed information of LRAT, CYP26B1 and STRA6

primers. Primer sets designed to detect mRNA expression were: 5’-TGT GCA ATG

GCA ATT CTG AT-3’ (forward) and 5’-TGG TCT TGG TCC TTA GCC AC-3’ for rat

IL6 (NM_012589.1); 5’-AGG TGT CCC AAA GAA GCT GT-3’ (forward) and 5’-TGC

TTG AGG TGG TTG TGG AA-3’ (reverse) for rat CCL2 (NM_031530.1). The mRNA

expression level of each sample was corrected by calculating mRNA-to-ribosomal 18S

RNA ratio. Data were normalized to the average value for the control group, set at 1.00,

prior to statistical analysis.

Please refer to the Chapter 2 for the detailed information of statistical analysis.

LPS-induced inflammation slightly reduced the VARA-mediated increase in lung

total retinol

3.4.6 Gene mRNA level determination

3.4.7 Statistical analysis

3.5 RESULTS

67

In this study, we determined lung total retinol concentration after LPS and VARA

administration. We also examined how the time of the initiation of inflammation affected

retinoid homeostasis by conducting a short-term study (6-h study) and a long-term study

(18-h study) which differed according to the time VARA was administered relative to the

time of LPS administration. In the short-term study, LPS and VARA were provided to

neonatal rats concurrently, while in the long term study, LPS was administered 12 hours

before VARA treatment. In both studies, VARA significantly increased lung RE

formation 6 h after VARA supplementation. LPS alone has no effect in changing lung RE

level. In the short-term study, LPS attenuated the increase in RE by VARA by an ~38%

reduction (Fig. 3.2A). In the long-term study, LPS also slightly but not significantly

reduced the increase due to VARA (Fig. 3.2B). In the 18 hr study, we conducted a

metabolic study by adding a tracer of [3H]retinol to the dose and investigating how much

newly absorbed retinol was taken up by lung tissue. We found that the percentage of

newly absorbed 3H in the lungs treated with VARA did not differ between the normal

state and inflammation state (data not shown). These results suggested that an acute

inflammation induced by LPS slightly affects lung RE formation promoted by VARA.

68

Figure 3.2 Lung RE concentration after a single treatment with oil or VARA in the

presence or absence of LPS induced inflammation state. In each study, 7-8 day-old

neonatal rats were randomly assigned to 4 groups, and received a single dose of

saline+oil (control), Saline+VARA, LPS+oil, LPS+VARA. In study I (6 hr study), LPS

and VARA were administered simultaneously (Fig 2A), as in study II (18 hr study), LPS

was administered 12 h before VARA supplementation (Fig 2B). Lung total retinol

contents were determined by HPLC after treatments. Data are presented as group means

± SEM; Groups were compared by one-factor ANOVA. Data were transformed by log10

prior to ANOVA. Tukey’s Least square mean test was used after ANOVA. Groups with

different letters differed significantly, p<0.05.

69

The increase in inflammation markers indicated the state of inflammation

To check whether the neonatal rats are in a state of inflammation, we determined the

expression of certain inflammation markers in the neonatal lung by rt-PCR and the level

of C-reactive protein (CRP) in the serum by ELISA. Interleukin-6 (IL-6) and hemokine

(C-C motif) ligand 2 (CCL2) are inflammatory mediators in the early inflammatory

response and in the evolution of inflammatory events. In the lung of neonatal rats, the

expression of these two genes was increased by LPS at 6 h (Fig. 3.3A, 3.4A). But 18 h

later, the increase returned to the control level (Fig. 3.3B, 3.4B). On the other hand, the

increase in plasma CRP level was only observed at 18 h, not 6 h (Fig. 3.5A, 3.5B). These

results demonstrated that neonatal rats that received LPS administration were

experiencing an inflammation state in both the short-term and the long-term studies.

However, due to the time of gene response and protein response to inflammation, the

expression level of each inflammation indicator could be very different.

70

Figure 3.3 Expression level of lung IL6 gene 6 h or 18 h after LPS administration..

Lung tissue from neonatal rats treated with saline+oil (control), Saline+VARA, LPS+oil,

LPS+VARA were collected 6h after VARA treatment and processed for total RNA

isolation and subjected to rt-PCR analysis. In study I (6 h study), LPS and VARA were

administered simultaneously (Fig 3A), as in study II (18 h study), LPS was administered

12 h before VARA supplementation (Fig 2B). Data was normalized to 18S rRNA and the

average value for the control group was set to 1 for each experiment. Data are presented

as group means ± SEM; Groups were compared by one-factor ANOVA. Data were

transformed by log10 prior to ANOVA. Tukey’s Least square mean test was used after

ANOVA. Groups with different letters differed significantly, p<0.05.

71

Figure 3.4 Expression level of lung CCL2 gene 6 h or 18 h after LPS administration.

Lung tissue from neonatal rats treated with saline+oil (control), Saline+VARA, LPS+oil,


isolation and subjected to rt-PCR analysis. In study I (6 hr study), LPS and VARA were

administered simultaneously (Fig 3A), as in study II (18 hr study), LPS was administered






72

Figure 3.5 Plasma C Reactive Proteins (CRP) level 6 h or 18 h after LPS

administration. The plasma samples from neonatal rats treated with saline+oil (control),

Saline+VARA, LPS+oil, LPS+VARA were collected 6h after VARA treatment and

subjected to ELISA to test plasma CRP level. Groups were compared by one-factor

ANOVA. Data were transformed by log10 prior to ANOVA. Tukey’s Least square mean

test was used after ANOVA. Groups with different letters differed significantly, p<0.05.

73

LPS-induced inflammation did not interrupt the induction of STRA6, LRAT and

CYP26B1 mRNA by VARA treatment

LRAT, CYP26B1 and STRA6 are the most important genes that regulate retinoid

homeostasis in the neonatal lung. RA is able to up-regulate these genes to enhance lung

uptake and storage in the lung. We next determined the expression level of these retinoid

homeostatic genes in the lung of LPS treated rats. Gene expression was determined by

quantitative PCR.

Treatment with LPS alone has no effects on STRA6 expression, but VARA was able to

significantly increase its expression regardless of addition of LPS and what time LPS is

administered (Fig. 3.8). Similarly, VARA significantly induced LRAT and CYP26B1

expression respectively in both the 6-hour and 18-hour studies (Fig 3.6, 3.7). Only in the

6-h study, LPS slightly reduced the induction of LRAT by VARA which is

corresponding to a slight reduction of RE formation in LPS/VARA treated group. In both

studies, expression of all retinoid homeostatic genes was significantly and rapidly

increased above the control level 6 h after VARA supplementation. These results

suggested that LPS initiated inflammation has little effect in interrupting the VARA

induced gene induction in neonatal rat lungs.

74

Figure 3.6 Expression level of lung LRAT gene 6 h after VARA treatment. Lung

tissue from neonatal rats treated with saline+oil (control), saline+VARA, LPS+oil,

LPS+VARA were collected 6 h after VARA treatment and processed for total RNA

isolation and subjected to rt-PCR analysis. In study I (6 h study), LPS and VARA were

administered simultaneously (Fig 3A), as in study II (18 h study), LPS was administered






75

Figure 3.7 Expression level of lung CYP26B1 gene 6 h after VARA treatment. Lung

tissue from neonatal rats treated with saline+oil (control), Saline+VARA, LPS+oil,









76

Figure 3.8 Expression level of lung STRA6 gene 6 h after VARA treatment. Lung

tissue from neonatal rats treated with saline+oil (control), Saline+VARA, LPS+oil,









77

BPD is one of the lung disorders that most commonly occur in premature infants who

received prolonged mechanical ventilation and oxygen therapy to treat RDS. It is

characterized by chronic inflammation and impaired abnormal structural changes (alveoli

and vascular) in the lung. In the state of inflammation, inflammatory cells are recruited to

the airways and lung tissues to produce pro-inflammatory mediators such as TNF-α, IL-1,

IL-8, which leads to a series of injurious immune responses. As a major organ for

inflammatory responses and VA homeostasis, liver has been investigated for the

relationship between inflammation and retinoid homeostasis. It was reported that

expression of cytochrome P450 (RA hydroxylase) mRNA can be suppressed by

inflammation or infection in rat hepatocytes (183). Another study has indicated that

inflammation induced by LPS opposes the induction of CYP26A1 and CYP26B1 to RA

in the rat liver when RA and LPS were administered at the same time or LPS was

administered 4 h after RA treatment (185). These studies suggest inflammation might

affect normal VA homeostasis by interfering with expression of retinoid homeostatic

genes.

As a major extrahepatic organ that require VA’s activity for normal development and

function, the lung has potential for storing VA after VA administration (27). When RA is

supplemented simultaneously with VA, the dose produced a synergy in increasing lung

3.6 DISCUSSION

78

RE level, which is not observed in the liver (160, 161). Therefore, VA level can be

elevated in a tissue-specific manner. However, little is known about retinoid homeostasis

in the lung under inflammation condition. In our present studies, we used a model of

LPS-induced systemic inflammation to study whether inflammation has any influence on

retinoid homeostasis in the lungs. The results indicated that the influence from LPS-

induced inflammation on VA homeostasis is mild and only slightly attenuates the

increase in lung RE by VARA. LPS-induced inflammation reduced ~38% RE increase

when LPS and VARA were administered concomitantly, and reduced only ~24% when

LPS was administered 12 h before retinoids treatment. The induction in IL-6 and CCL2

mRNA (indicators of early inflammatory responses) in the lung and the increase in CRP

protein in the plasma (marker of acute inflammation) confirmed the inflammation state in

the animals. The gene data also indicated that inflammation state had little influence on

the expression of lung retinoid homeostatic genes.

In our study, we used LPS, an outer membrane component of Gram negative bacteria, to

induce inflammation in neonatal rats. Although LPS-induced inflammation only

attenuates the VARA mediated increase in RE formation modestly, it is still interesting

for us to understand the possible mechanism that causes the attenuation. LPS is able to

initiate immune responses via binding to toll-like receptor 4 (TLR4) and subsequently to

stimulate several signaling pathways that in turn activate a variety of transcription factors

including nuclear factor kappa-B (NF-κB) and AP-1, etc (186, 187). The activated

transcription factors in the cytoplasm translocate into the nucleus where they bind to

specific response elements on target genes and induce many genes encoding

79

inflammatory mediators (187). Although our experiments showed little influence from

LPS on the expression of retinoid homeostatic genes, co-administration of LPS and

VARA still reduced expression of LRAT mRNA 6 h after treatment, which perhaps

explains the reduction in VARA promoted RE formation. On the other hand, RA was

reported to be able to attenuate immune responses by opposing LPS and/or TNF-α

induced STAT expression and lowered the binding activity of STAT-1 and NF-κB to

response elements (184). Other in vitro and in vivo studies suggested that RA is able to

reduce NF-κB activity and DNA binding (86, 188). Therefore, RA’s activity in inhibiting

TLR4 downstream signaling pathways might also contribute to attenuate inflammatory

responses in neonates.

Animal and adult human studies have shown that long term respiratory inflammation

results in increased VA consumption and depletion of hepatic stores (189, 190). But in

our studies, there is no significant reduction in the lung RE and liver RE (data not shown)

observed in LPS treated group. We speculated that the short term inflammation shows

little impact on retinoid consumption. Meanwhile, the animals we used were naturally

delivered, healthy neonatal rats. They probably have a better anti-inflammatory

mechanism or immune defense than animals and humans whose health is compromised

by chronic inflammatory lung diseases. However, it would be interesting to understand

whether long-term inflammation will prevent the synergy of VARA.

However, there are limitations to this study. First, while LPS-treated newborn rat is a

common reproducible inflammation model, the systemic inflammation produced in this

model may not simulate the local inflammation like human BPD. As our study was

80

designed to evaluate the effects of postnatal lung inflammation on lung RE formation and

gene regulation, we did not observe marked decrease in lung RE content after VARA

treatment and the changes in expression of retinoid homeostatic genes. We also failed to

see significant differences in lung RE and gene levels between the 6h and 18h studies. To

better evaluate the effects of local inflammation on lung VA homeostasis, in future, we

need to establish a pneumonia model in neonatal rats which has more strong

inflammatory reaction in the lung and is more similar to the condition of BPD in preterm

infants. Second, the does of LPS administered to pups may be too low to induce the

inflammatory conditions that can compromise VARA promoted RE formation in

neonatal rat lung. To avoid this situation, an appropriate dose of LPS or other pathogen

should be determined in our future studies.

In summary, these studies provide insights into how acute inflammation during the early

postnatal period affects retinoids homeostasis in the neonatal rat lung. We demonstrated

that the LPS-induced inflammation has little influence on the induction of retinoid

homeostatic genes by RA and only slightly attenuates the synergistic increase in RE by

VARA. These results suggest that, even in a state of inflammation, VARA could still be

an effective therapeutic strategy for improving RE status in neonatal lungs.

81

Chapter 4

ACIDIC RETINOIDS DILUTION STUDY

In contrast to VA alone, the combination of VA and 10% RA (10:1 molar ratio)

administered orally is able to increase lung RE contents synergistically in neonatal rats.

However, the lowest RA concentration to cause the same promotion is not known. In this

part of this thesis, we designed two studies to test the lowest concentration of RA in

VARA dose that still is able to promote high RE formation in neonatal rat lung using a

single dosing (day 5) vs. multiple dosing schedule throughout the period of lung septation

(day 4, 7, 11, 14). In the first study, a single oral dose of oil, VA, VARA (10% RA) and

VA with a series of diluted RA (5%, 2%, 1%, 0.5% RA) was administered to 5 day-old

pups. In the second study, doses of oil, VA, VARA (10%, 2%, 1% RA) were given to

neonatal pups on each of days 4, 7, 11, and 14. We also examined the effect of

substituting Am580 (a stable analog of RA) for RA in the multiple dosing protocol by

giving oil, VA, VAAm580 (10%, 2%, 1% Am580), with the same feeding schedule as

above. Lung tissues were collected 6 hours after the last treatment and analyzed by HPLC

for lung total retinol level. The results showed that although the concentration of RA was

reduced to 1%, it still increased lung RE to the same level 10% RA does. However, this

effect is only observed in the single dosing study. The multiple doses of diluted RA (2%)

4.1 ABSTRACT

82

did not show the same effect. However, in the Am580 dilution study, which was parallel

to the multiple dosing RA study, even 1% Am580 showed the same effect of 10%

Am580 in promoting lung RE formation. We conclude that RA even in more diluted

concentration combined with the same dose of VA still promote high retinol uptake and

RE formation in neonatal lungs at a short time (6 h) compared with VA given alone. But,

this effect is not seen after multiple treatments which might be due to a quick metabolism

of a reduced amount of RA.

VA storage in the lung shows a specific pattern during the perinatal period. It rises

significantly in late gestation, starts to decline before birth and continues into the early

postnatal period (92). The utilization of VA stores strongly implies the dependence on

VA for postnatal lung development. Improving lung VA status in the early postnatal life

has been shown to be beneficial for lung development and reducing lung injury and

dysfunction (95, 173, 174, 191, 192).

VA is stored in tissues primarily in the form of retinyl esters (RE). Although a high dose

of VA administration is able to improve VA status, it has limited capability of increasing

RE concentration in the lung, because most of the supplemented VA is distributed to the

liver, the major organ for VA storage. Our previous study has suggested that a nutrient-

metabolite combination of VA and RA, VARA, mixed in a 10:1 molar ratio, shows great

potential for elevating RE level in neonatal lung (160, 161). When VARA was repeatedly

4.2 INTRODUCTION

83

administered to neonatal rats throughout the lung septation period, the increase was

augmented not only in a synergistic but also in a cumulative manner (Fig. 2.2). In a dose

dilution study, the VARA dose diluted to 25% of its original concentration still showed

same ability of increasing lung RE as that of the standard VA dose (160). The standard

dose of VA and RA used in our previous study was based on prior usage of VA in human

infants and RA in neonatal rats (160, 161). These findings reveal great potential of RA to

promote lung RE formation, and provide a more efficient method for improving lung VA

status.

In this study, we continued to test RA’s ability to induce lung RE formation during the

postnatal period. In contrast to the past dose preparation in VARA dilution study, we

only reduced the concentration of RA but used the same amount of VA in each of the

standard and diluted doses. By using the same dose, we examined the effects of a single

treatment and multiple treatments on VA stores in the lungs of neonatal rats and found

that the synergy of increasing lung RE only could be observed after a single dose, not the

multiple doses. We also compared effects of RA and Am580, a stable analog of RA, to

test our hypothesis that the absence of VARA’s synergy in multiple treatments study is

due to the quick metabolism of RA. The results of the study revealed the great potential

of RA in promoting RE formation and provided a better understanding of retinoid

metabolism in neonatal lung during the postnatal period.


84

Hypothesis 1: A single dose of RA in more diluted concentration combined with the

same (standard) amount of VA still can produce a synergistic effect in increasing retinol

uptake and RE formation in neonatal lungs, compared with the standard VA dose.

Hypothesis 2: In contrast to a VA dose alone, diluted RA combined with VA still can

promote RE formation significantly in the lung after multiple treatments throughout the

lung septation period.

Hypothesis 3: Am580 substituted for RA in the combination dose will show a stronger

capability of inducing lung RE formation compared to the same amount of RA after

multiple treatments.

Aim 1: To test whether a single dose of RA in low concentration is still able to

promote high retinol uptake and RE formation in neonatal lungs.

In this study, the amount of RA in the VARA combination was reduced to different

concentrations as the dose of VA was kept the same. We tested how 5 d-old pups respond

to diluted doses at 6 h. The lung tissue were collected and subjected to HPLC for lung

total retinol quantification. The effects of VARA dose containing different levels of RA

were evaluated.

Aim 2: To test whether multiple doses of RA in low concentrations are still able to

promote high retinol uptake and RE formation in neonatal lungs.

In this study, the doses were prepared in the same way as in aim 1, and administered to

neonatal pups repeatedly throughout postnatal lung septation period on d4, d7, d11 and

85

14, respectively. Six hours after the last dose, lung tissue was collected and lung total

retinol from each sample was quantified by HPLC. The effect of multiple treatments

with diluted RA were analyzed and compared with the single dose.

Aim 3: To test effects of multiple treatments of VA and diluted Am580 in lung RE

formation.

In this study, we substituted Am580 for RA in multiple dose study. Diluted Am580

combined with the same amount of VA were administered to neonatal rats in the same

way as in aim 2. Lung total retinol from each sample was quantified by HPLC following

the treatments. We compared the effects of diluted Am580 and diluted RA doses in

promoting lung RE formation.

Protocols for all studies were approved by the Animal Care and Use Committee of

Pennsylvania State University. Pregnant female rats (Sprague-Dawley strain) were

purchased or obtained from natural mating. Dams were fed with semipurified AIN-93G

diet containing 4 mg retinol/kg (193), which is a VA adequate diet. After birth, pups

within litters were randomly distributed to each treatment group and sex was evenly

distributed as well. We conducted two RA dilution studies: single dosing (d 5) and


4.4.1 Animals and Experimental Design

86

multiple dosing throughout the period of lung septation (d 4, 7, 11, 14) (Fig. 4.1). In the

single dosing study, 5 day-old neonatal rats received a single oral dose of oil (control),

VA alone, VARA (10% RA) and VA combined with a series of diluted RA (5%, 2%, 1%,

0.5%). In the multiple dosing study, neonatal rats received multiple doses of oil, VA,

VARA (10%, 2%, 1% RA). We also conducted a multiple Am580 dilution study which

was parallel to the multiple RA dilution study (Fig. 4.1). Before each treatment, the pups

were weighed and the volume of each dose provided to pup was adjusted to 0.4 µl/g

bodyweight.

87

Figure 4.1 Animal experimental design. Two studies were conducted to evaluate the

effect of a single treatment and multiple treatments of diluted RA in VARA dose. In the

single dose study, the doses of VA combined with a series of diluted RA were orally

supplemented to 5 d-old pups. In the multiple dosing study, multiple doses were given

consecutively on d4, d7, d11 and d14. Pups were randomly assigned to several groups to

receive oil (vehicle), VA and VARA doses (n=5-6/group). In the multiple dose study, an

Am580 dilution study was included to be compared with the effect of VARA. The dose

of VAAm580 was prepared and administered in the same way as in multiple VARA dose

study. Tissues were collected 6h after the last treatment.

88

The standard dose was prepared in the same way as in chapter 2. Please refer to the

chapter 2 for detailed information of how the standard doses were prepared. For the RA

or Am580 diluted doses, VA and acidic retinoids (at-RA and Am580) were first prepared

as a 2x stock solution in canola oil at 0.1 mmol/g and 0.01 mmol/g, and diluted with the

same volume of oil to form 1x oral dose of VA and 1x RA/1x Am580 respectively. The

2x stock solution were then mixed 1:1 to form the dose referred to as VARA10% and

VAAm10%. Similarly, 1x RA was further diluted with oil to form 0.4x, 0.2x and 0.1x

solutions to mix with the same volume of 2x VA stock solution to form the dose referred

to as VARA5%,VARA2%, VARA1% and VARA0.5%. The combination dose of VA

and Am580 was prepared in the same way to form VAAm10%, VAAm2% and

VAAm1% solutions. Canola oil was used as placebo (control).

The volume of each dose provided to pup was 0.4 µl/g body weight and the exact volume

of dose was determined based on the pup’s body weight. In the single dose study, a single

dose was given to pups on day 5. In the multiple dose study, multiple doses were given

on day 4, 7, 11 and 14. Pups were killed with carbon dioxide (CO2) 6 hours after the last



89

in both studies. The lung tissues were removed, trimmed and weighed. All samples were

frozen in liquid nitrogen immediately and then stored -80oC for later analysis.

Lung tissue was processed and lung total retinol was quantified in the same way as in

chapter 2. Please refer to the chapter 2 for the detailed information.

Chapter 2 provides the detailed information for statistical analysis.

A single dose of RA in more diluted concentration still promotes high lung RE

formation in younger pups

Our previous study suggested that VA and RA mixed with a molar ratio of 10:1 (RA is

10% in concentration relative to VA) for a single oral treatment is able to produce a

synergistic increase in RE contents in neonatal rat lung compared with VA given alone.

However, it was not clear whether the dose of RA is appropriate or excessive for the

synergy. In the present study, we tested whether RA in more diluted concentration still

4.4.4 Retinoid Analysis

4.4.5 Statistical Analysis

4.5 RESULTS

90

promotes high lung RE formation. In the dose of VARA, the concentration of RA was

diluted to different levels while the concentration of VA was kept the same as in our

previous study. Five-day old neonatal rats received a single oral dose of oil, VA and

VARA. For the treatment of VARA, RA was added in a series of diluted concentration

10%, 5%, 2%, 1% and 0.5%. Six hours after treatment, pup lung was collected and lung

RE was analyzed by HPLC. The result showed that RA concentration as low as 1% still

increased lung RE content to the same extent as 10% RA (Fig. 4.2). The increase did not

differ between VA and VARA0.5% treatments (Fig. 4.2). These results showed the

strong potential of RA in promoting lung RE formation.

Multiple doses of diluted RA does not promote high lung RE formation in older

pups while Am580 still does

Given the potential beneficial effects and safe use of long-term treatment with low

concentration of RA to neonates, in the following study we investigated the effect of

multiple doses of VA combined with diluted RA (RA was added in concentration of

10%, 2% and 1% relative to VA) administered to neonatal pups on d4, 7, 11 and 14. The

lung tissue was collected 6 hours after the last treatment and subjected to HPLC for total

lung retinol analysis. The result showed that, the dose of VA alone increase lung RE level

~3 folds higher than the control level. When VA combined with 10% RA, the dose

increase lung RE content significantly; this is consistent to the result of our multiple-

dosing study (Fig. 4.3). However, VARA2% and VARA1% produced no further increase

in lung RE compared with VA dose alone group (Fig. 4.3). These results indicated that

91

multiple doses with diluted RA have a much reduced ability to promote lung RE

formation. This might be explained by the transient effect of RA and quick depletion of

reduced RA mass in the lung by CYP26B1.

To interpret the lost synergy with multiple treatments, we substituted Am580 for RA in

the in the combination dose. After the multiple dose treatment, lung RE was increased

significantly by all of the diluted Am580 treatments, even with 1% Am580, which still

increased the RE level higher than VA given alone (Fig. 4.3). By contrast to VARA,

VAAm580 produced a stronger and longer induction of RE formation. This could be due

to the stability of Am580 which is resistant to CYP26B1 metabolism, thereby leading to a

sustained induction on retinol uptake and RE formation in the lung.

92

Figure 4.2 Lung RE concentration after a single treatment with VA alone, VARA

10%, VARA 5%, VARA 2%, VARA1% and VARA0.5%. Neonatal rats were

treated with an early single dose on d5. Lung total retinol contents were determined by

HPLC 6h after treatment. Data are presented as group means ± SEM; Groups were

compared by one-factor ANOVA. Data were transformed by log10 prior to ANOVA.

Tukey’s least square mean test was used after ANOVA. Groups with different letters

differed significantly, P<0.05.

93


VARA10%, VARA2% and VARA1%. Neonatal rats were treated with multiple doses

on d4, d7, d11 and d14. Lung total retinol contents were determined by HPLC 6h after

the last treatment. Data are presented as group means ± SEM; Groups were compared by

one-factor ANOVA. Data were transformed by log10 prior to ANOVA. Tukey’s least

square mean test was used after ANOVA. Groups with different letters differed

significantly, P<0.05.

94


VAAm10%, VAAm2% and VAAm1%. Neonatal rats were treated with multiple doses

on d4, d7, d11 and d14 (designated older pups based on day of final dosing). Lung total

retinol contents were determined by HPLC 6h after the last treatment. Data are presented


transformed by log10 prior to ANOVA. Tukey’s Least square mean test after ANOVA.

Groups with different letters differed significantly, p<0.05.

95

In previous work, we showed that VARA, a combination of VA and 10% RA (we

referred to as VARA10% in the current study), increases RE content synergistically in the

lungs of neonatal rats (67, 160, 161). In this study, we examined the effects of dose with

same amount of VA but a reduced amount of RA. The amount of RA in our standard

VARA10% dose is based on the dose previously used to induce alveolar formation in

dexamethasone-treated neonatal rats during the lung septation period (99). The

mechanism by which VARA10% promotes higher RE content in the lung was

investigated and the findings suggested that 10% RA in the dose plays a role in

upregulating LRAT (retinol esterification) and STRA6 genes, which direct more of the

flow of supplemental VA into the lung tissue (67).

We started our study from a single dose study, in which a single dose of standard

VARA10% dose or VA combined with diluted RA (referred as VARA5%, 2%, 1%,

0.5%) was administered to rat pups on postnatal day 5. The increase in lung RE induced

by a single dose of VARA10% is consistent with our previous results (67). Diluted RA as

low as 1% in VARA dose still showed the effect equal to that of the VARA10% (Fig.2).

This result indicated that with the certain amount of VA, the amount of RA we used

before to produce the synergy in a short period (6 h) is saturating with respect to lung RE

formation, because a lower concentration of RA in the dose is as effective as standard

VARA dose for promoting lung RE formation.

4.6 DISCUSSION

96

Given the great potential of a single dose of VARA for elevating lung RE level at 6 h, we

expected to see the similar effect after multiple treatments with VA and diluted RA,

because in our multiple-dosing study, the lung showed great capability for RE storage

after multiple treatments with VARA10%. In the second study, we determined the effects

of the repeated doses of VARA10%, VARA2% or VARA1% throughout the lung

septation period. The standard VARA10% dose administered repeatedly to neonatal rat

has been demonstrated to be effective in increasing lung RE significantly in the multiple-

dosing study and in this study (Fig. 4.3). But VARA2% and VARA1% failed to show the

synergy that we observed in the single dose study. Both of these RA diluted doses did not

differ from an equal dose of VA only (Fig.4.3).

To interpret the discrepancy between the single- and multiple- dosing studies, we

substituted Am580 for RA in the VARA combination dose. Am580, a synthetic analog of

RA, which is known for its resistance to metabolism (73), has shown its ability to

regulate lung retinoid homeostasis and it replicates the effects of RA on lung RE

accumulation (67). In the present study, VA and Am580 were mixed in the same way

VARA was prepared (VAAm10%, VAAm2% and VAAm1%). Our results showed that

the lung RE level was elevated 3~5-fold by all VAAm doses compared with VA dose.

Based on the comparisons between single dose vs multiple dose, VARA treatments vs

VAAm treatments, we suggest several possible factors that might result in the absence of

VARA synergy in the diluted VARA formula. 1) A low amount of RA is depleted faster

than a higher amount of RA, thus losing its activity for LRAT and STRA6 induction; 2)

The lung during septation period is in high need of VA for development, therefore, the

97

temporarily improved RE content by diluted VARA is consumed rapidly; 3) The lung

responds to RA treatment more sensitively in younger rats than in older rats. To test these

hypotheses, a metabolic study to examine the distribution of supplemented retinol

combined with diluted RA, and a dose response study to test the effect of a single diluted

VARA dose at different time points during postnatal period would need to be done in the

future.

In summary, these studies were designed to test RA’s potential to induce lung RE

formation in the early time of life. The results showed that a single dose of RA in much

reduced concentration, combined with VA, still produced the same effect on RE

formation in young pups as the standard dose of RA. However, multiple treatments with

the same dose failed to increase lung RE formation synergistically, possibly due to the

transient action of RA and high demand for retinol in developing lungs. The higher

increase in RE storage caused by diluted Am580 provided evidence for our interpretation

of RA’s transient activity. Overall, we conclude that the combination of VA and diluted

RA still has great potential to induce RE formation at an early time after the treatment.

However, this effect is just transient. RA in reduced concentration is not adequate to

accumulate RE contents over time.

98

Chapter 5

LOCALIZATION OF LUNG RETINOID HOMEOSTATIC PROTEIN

STRA6, LRAT and CYP26B1 are the most important VA metabolism genes that regulate

retinoid uptake, storage and oxidation in the lung, but little is known about their protein

distribution pattern in lung tissue. Previous studies reported that the lipofibroblast is a

vitamin A-storing cell and STRA6 has been shown to be expressed in endothelial cells of

blood-organ barriers of other tissues. Due to the lack of specific antibody to CYP26B1

before, extremely limited information about this protein was available. Based on this

information, we hypothesized that LRAT is most likely expressed in lung lipofibroblast

where it can catalyze retinol esterification, while STRA6 and CYP26B1 are possibly

localized in the endothelial cells of the vascular network to mediate retinol uptake and

RA clearance. To test these hypotheses, we determined the localization of retinoid

homeostasis proteins by conducting immunofluorescence staining on lung sections from

neonatal rats. The results showed that LRAT is co-localized with adipocyte

differentiation-related protein (ADRP), the marker protein of lipofibroblast cells in lung

interstitium, STRA6 is co-localized with platelet endothelial cell adhesion molecule-1

(PECAM-1), the marker of endothelial cells, in the blood vessels and CYP26B1 is

exclusively expressed in the epithelium of bronchioles and the smooth muscle of small

5.1 ABSTRACT

99

airway and blood vessels. Overall, our results demonstrate the localization of LRAT in

lipofibroblasts and STRA6 in endothelial cells, while in addition, for the first time, we

report the localization of CYP26B1 in bronchiole epithelium. This information might

prove helpful for better understanding retinoid metabolism mechanisms in neonatal rat

lungs.

The lung is a complex tissue composed of over 40 different cell types which serve

various roles in lung development and functions. The most common lung cells include

alveolar epithelial type I, type II cells, endothelial cells, macrophage, and fibroblast, etc

(Fig. 5.1). It is known that VA plays an important role in normal lung development and

function. Vitamin A homeostasis in regulated by several important genes, LRAT,

CYP26B1 and STRA6 in the lung (67). But very limited information about the

localization and expression patterns of these proteins in the lung is available. It is

important to study this because a good understanding of the spatial and temporal

expression pattern of these proteins will provide insight on how VA is taken up, stored

and oxidized within the lung tissue.

The lung fibroblast is one type of the pulmonary mesenchymal cells that synthesizes

extracellular matrix and collagen. It plays an important role in maintaining the structural

framework for lung tissue. At the same time, there is coordinate signaling between the

epithelial cells and fibroblasts that determines normal alveolar development and mediates

5.2 INTRODUCTION

100

tissue injury repair via epithelial-mesenchymal interactions (133, 143-147). Lung

fibroblasts also provide lipid substrate for the synthesis of surfactant phospholipid in type

II alveolar epithelial cells (148). There are different populations of fibroblasts which are

classified based on the biochemical and functional characteristics of each type. The

lipofibroblast is one population that shows expression of a marker glycoprotein molecule,

Thy-1, on the cell surface (194, 195), and is characterized by abundant lipid droplets and

a high content of glycogen (137). It is located in the alveolar interstitium close to the

central region of the alveolar septum (137). Lipofibroblasts start to be present in rat lungs

around gestational day 16, and after that time the lipid content in the whole lung tissue

starts to rise significantly, especially between gestational days 17 and 19 and at birth, and

peaks during the second postnatal week (196). Compared with Thy-1 negative

fibroblasts, Thy-1 positive lipofibroblasts appear to be more abundant in the lung and to

perform a repair function after injury (194). Lipofibroblasts are also known as vitamin A-

storing cells containing retinyl esters (RE). In an animal study, feeding a VA sufficient

diet resulted in a great increase in lipid droplets size and number in lung fibroblasts (141).

Okabe et al identified that VA is contained in the isolated lung lipofibroblast and still is

present in the progeny cells after cell divisions (134). In rats, significant increase in the

lung RE storage starts from the late gestation, which is paralleled to lipid storage in the

lungs, peaks right before birth, and the depletion of this store begins and continues into

the early postnatal life (92), indicating a broad and rapid utilization of RE store for early

postnatal development of the lung. McGowan et al. have shown that the contents of

retinol and RA, the biologically active forms of vitamin A, in the lungs of rats increases

in early postnatal life (94). Concurrently, the level of cellular retinoic acid-binding

101

protein (CRABP) increases in whole lung tissue and lung lipofibroblasts (47). Also, the

increases of RARβ and RARγ were observed in lipofibroblasts from late gestational

period to early postnatal period (197, 198). It was also demonstrated that, later on,

lipofibroblasts are able to produce and secrete at-RA at the sites of secondary septum

projections, thereby providing endogenous at-RA for other lung cells (135). Overall,

these studies strongly suggest that the lipofibroblast is the cell type where expression of

the LRAT gene is localized and where it is able to generate the biological activities of

acidic retinoids in lung alveoli.

STRA6, a cell membrane protein, was recently identified as a cell-surface receptor for

RBP that mediates uptake of retinol into cells (33). Both in situ hybridization (ISH) and

immunohistochemistry (IHC) studies suggested strong expression of STRA6 could be

detected in blood-organ barriers which are made of epithelial or endothelial cells, for

example, the choroid plexus and the brain microvasculature, RPE cells in the eye, Sertoli

cells in the testis and kidney, spleen, etc. (63). These barriers play a role in facilitating

uptake of many nutrients, including retinol. Although the expression of STRA6 in the

lung is quite low, our previous studies showed that oral supplementation with RA could

increase the level of STRA6 mRNA in neonatal rat lungs (67). Based on its function and

localization in other organs, STRA6 is most probably expressed in the lung vascular

system, especially the small arterioles and the capillary network which forms the air-

blood barrier with the alveolar epithelium.

Due to the lack of proven specific antibodies against CYP26 proteins before, limited

information about CYP26 protein distribution in any organs has been demonstrated. As

102

the major function of CYP26 is to control cellular exposure to RA by inactivating RA in

cells, it is possible that CYP26B1 protein is expressed in endothelial cells of capillary in

the lung, where it might mediate the excretion of polar metabolites of RA into blood

stream. It is also possible that CYP26B1 is expressed in the lung epithelial cells where

RA activity is highly required.

We hypothesized that LRAT is expressed in the lipofibroblastic cells–a lung retinol

storing cell (135), CYP26B1 and STRA6 are expressed in the lung vascular endothelial

cells, where they might play a role in controlling the uptake of retinol and excretion of

RA. In our previous studies, these genes could be highly induced in the neonatal rat lung

by a short-term treatment (6h) with RA. However, this action is transient because RA is

quickly metabolized by the product of the induced CYP26 gene. Based on previous

findings, we speculate that multiple treatments with RA over a period of time (throughout

the postnatal period) might maintain a higher expression level of retinoid homeostatic

proteins compared with a 6-h single dose treatment.

Aim: To determine the spatiotemporal expression pattern of the retinoid

homeostatic proteins LRAT, CYP26B1 and STRA6 in neonatal rat lung.

We tested this aim by giving multiple treatments of VARA to neonatal rats throughout

the stage of lung alveolarization, and conducting immunofluorescence staining with lung


103

sections from treated rats. Aim 1a): We localized spatial expression of the LRAT,

STRA6 and CYP26B1 proteins in the lungs. Aim 2b): We compared the change in

protein expression in the absence or presence of VARA treatment throughout the

postnatal period.

The neonatal rats used in this study were under a protocol approved by the Institutional

Animal Use and Care Committee of the Pennsylvania State University. Neonatal pups

were delivered and nourished by mother rats fed a VA adequate diet. Throughout the

period of lung septation (days 4-14), the pups received 4 oral doses of VARA, one each

on day 4, 7, 11 and 14. Before each treatment, the pups were weighed and the dose was

adjusted to 0.4 µl/g bodyweight.

To inflate the lungs, the trachea was cannulated with a plastic needle. Optical cutting

temperature (OCT) was diluted 3:1 and used for inflation of the lung (1-2 ml) through the


5.4.1 Animals

5.4.2 Preparation of lung tissue

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needle until the lungs filled the pleural cavity. The trachea was then tied and the lungs

were dissected out and quickly minced into small pieces, subsequently. The freshly

dissected lung tissue was placed into pre-labeled plastic freezing molds and covered with

OCT. Filled molds were snap-frozen on dry ice and frozen blocks were stored at -80oC

until ready for sectioning.

Frozen tissue blocks were transferred to a cryotome cryostat (Shandon SME Cryotome

Cryostat) and the temperature of the frozen tissue block was equilibrated to the

temperature of the cryotome cryostat. Then the frozen lung tissue was cut into 6-µm

sections and mounted onto superfrost plus (Fisher) slides. Sections were dried overnight

at room temperature and then stored at -80oC for later use. Frozen tissue sections were

warmed and air dried at room temperature, fixed in pre-cooled acetone (-20oC) for 10

min and then placed at room temperature for > 20 min to allow the acetone to evaporate.

They were then washed in 10 mM phosphate buffered saline (PBS) at a neutral pH for 2

changes, 5 min each, and incubated in 0.3% H2O2 solution in PBS at room temperature

for 10 min to block endogenous peroxidase activity. Afterwards, sections were washed in

PBS again 2x5 min, and blocked with blocking buffer (10% fetal bovine serum in PBS)

in a humidified chamber at room temperature for 1 h. After blocking, sections were

incubated with the mixture of two primary antibodies in a humidified chamber overnight

at 4°C. On the second day, the sections were washed and incubated in the mixture of two

5.4.3 Preparation of tissue sections

105

fluorescent-conjugated secondary antibodies in 1% BSA in PBS in the dark for 30 min at

room temperature. After washing in PBS, a drop of anti-fading fluorescent mounting

medium (VECTASHIELD HardSet Mounting Medium with DAPI) was dispensed onto

the section to mount the tissue, and they were then cover slipped. Sections were viewed

using an Olympus Fluoview 1000 Confocal Laser Scanning Microscope (Olympus

America Inc.; Lehigh Valley, PA). Images were analyzed using Fluoview software.

The following antibodies were used as primary reagents for the detection of LRAT,

STRA6, CYP26B1 and PECAM-1 and ADRP as the marker proteins of endothelial cells

and lipofibroblasts, respectively.

Anti-STRA6, polyclonal antibody made in goat; anti rat, mouse, was obtained from

Sigma-Aldrich (St Louis, MO).

Anti-LRAT, monoclonal antibody made in mouse; anti human, was obtained from

Sigma-Aldrich (St Louis, MO).

Anti-CYP26B1, polyclonal antibody made in rabbit; anti human, was obtained from

Thermo Scientific Inc. (Rockford, IL).

Anti-PECAM-1, monoclonal antibody made in mouse; anti rat, was obtained from Santa

Cruz Biotechnology (Santa Cruz, CA).

5.4.4 Antibodies for section immunostaining

106

Anti-ADRP, polyclonal antibody made in goat; anti mouse, provided by the laboratory of

Dr. Constantine Londos, NIH.

Secondary fluorescent conjugated antibodies included:

Alexa Fluro 488-conjugated anti-goat IgG

Alexa Fluro 488-conjugated anti-mouse IgG

Alexa Fluro 568-conjugated anti-mouse IgG

Alexa Fluro 594-conjugated anti-rabbit IgG

These antibodies were obtained from Molecular Probes, Inc. (Eugene, OR).

We investigated the distribution of retinoid homeostatic proteins by performing

fluorescent immunostaining of lung sections from 14-day old neonatal Sprague-Dawley

rats and using confocal laser scanning microscopy to analyze the results. To test the

hypotheses that LRAT is exclusively or nearly exclusively expressed in lung

lipofibroblasts and CYP26B1 and STRA6 are expressed mainly in lung vascular

networks, we stained ADRP, a marker protein of lipofibroblasts, and PECAM1, a marker

protein of endothelial cells, simultaneously with their target proteins respectively.

5.5 RESULTS

107

Staining with respective 2nd antibody did not show any immunoreactivity (Fig 5.2.P, Q,

Fig 5.3M, N, Fig. 5.4Q, R).

Confocal laser microscopy revealed that in the lungs of 14-day old rats moderate

immunoreactivity of LRAT (red) was present in the interstitial region. The

immunostaining of ADRP, a signature lipofibroblast marker protein (green), overlapped

with LRAT, indicating the localization of LRAT in lung lipofibroblast cells (Fig. 5.2.E,

J). The immunoreactivity of LRAT was not detected in alveolar areas, bronchioles or

blood vessels (Fig. 5.2.O).

PECAM-1, a marker protein of endothelial cells, was clearly detected (red) in the

endothelium of small blood vessels, capillaries that surround connective tissues and

alveolar sacs, indeed everywhere that might be in contact with the vascular network (Fig.

5.3.B, F, J). The detected immunoflorescence of STRA6 (green) in the lung suggested

that this protein is expressed in the same area where PECAM-1 is expressed (Fig. 5.3.C,

G, K). The merged confocal images showed the co-localization of the STRA6 and

PECAM-1 expression, indicating the expression of STRA6 in the endothelial cells of

capillaries and blood vessels (Fig. 5.3.D, H, L).

Similar to the result for STRA6 localization, PECAM-1 staining (green) was easily

detected almost everywhere that is in contact with the vascular system. However, the

merged images showed a completely non-overlapping of CYP26B1 (red) and PECAM-1

immunostaining in the lung. In contrast to PECAM-1 staining, CYP26B1 was primarily

detected in the epithelium of bronchioles, terminal bronchioles and respiratory

108

bronchioles (Fig. 5.4.C, G, K). Less intense staining could be detected in the smooth

muscle of bronchioles, terminal bronchioles walls, as well as in the smooth muscle in the

media of small arteries and arterioles (Fig. 5.4.C, G). However, there is no detectable

staining of CYP26B1 in alveolar sacs and lung interstitium (Fig 5.4.O). Merged images

show non-colocalization of the two proteins.

109

Figure 5.1 Localization of LRAT protein in 14d old rat lungs. Confocal laser scanning

microscopy of immunoreactivity for LRAT (red; C, H, M) on 14 day old rat lungs in

combination with ADRP (green; B, G, L) and DIC display (A, F, K). Merged images (E,

J, O) indicate that ADRP and LRAT proteins are co-expressed and slightly detected in

the interstitium of the lung (black arrows in E and J). But the immunoreactivity of both

proteins is absent in most alveolar area, air-ways and blood vessels (O). Figure P and Q

show the negative control of the secondary antibody and DAPI for nuclear staining.

(as=alveolar sac; B=bronchiole; V=vessel)

110

Figure 5.2 Localization of STRA6 protein in 14d old rat lungs. Confocal laser

scanning microscopy of immunoreactivity for STRA6 (green; C, G, K) on 14-day old rat

lungs in combination with PECAM-1 (green; B, F, J) and DIC display (A, E, I). In the

lung, PECAM-1 is present in whole vascular network, especially the endothelium of

small blood vessels (F, J). Merged images indicate that STRA6 co-localize with PECAM-

111

1 in the vascular network. Figure M and N show the negative control of the secondary

antibody and DAPI for nuclear staining. (as=alveolar sac; B=bronchiole; TB=terminal

bronchiole; RB=respiratory bronchiole; V=vessel)

112

113

Figure 5.3 Localization of CYP26B1 protein in 14d old rat lungs. Confocal laser

scanning microscopy of immunoreactivity for CYP26B1 (red; C, G, K, O) on 14-day old

rat lungs in combination with PECAM-1 (green; B, F, J, N) and DIC display (A, E, I, M).

In the lung, PECAM-1 is present in whole vascular network, especially the endothelium

of small blood vessels (white solid arrows in B, F and J). CYP26B1 is primarily

expressed in the epithelium of bronchioles, terminal bronchioles and respiratory

bronchioles (white arrows in C, G, K). Moderate expression of CYP26B1 could be

detected in the smooth muscle of bronchiole and terminal bronchiole walls (white dashed

arrows in C and G), as well as the smooth muscle in the media of small arteries and

arterioles (yellow dashed arrows in C and G). Merged images show that CYP26B1 is not

overlapped with PECAM-1, indicating no expression of CYP26B1 in vascular network

(D, H, L). CYP26B1 immunoreactivity is also absent in alveolar area (O, P). Figure Q

and R show the negative control of the secondary antibody and DAPI for nuclear

staining. (as=alveolar sac; B=bronchiole; TB=terminal bronchiole; RB=respiratory

bronchiole; V=vessel)

114

Over the past years, significant progress has been made in elucidating the mechanism of

retinoid metabolism in various tissues, and improving the understanding of the roles that

retinoid homeostatic enzymes play in the lung during development. However, a clear

profile of the spatial and temporal expression pattern of these lung retinoid homeostatic

proteins has not been obtained yet. In our study, we used 14 d-old neonatal rats, because

the lungs have just experienced a rapid process of alveolization. During this stage, RA

action is required for normal alveolar development. Therefore, VA homeostasis is

important to maintain. Where is VA taken up into the lung tissue? Where is VA locally

stored? And where is RA cleared? Using immunofluorecence staining, our present

experiments report the localization of the retinoid homeostatic proteins, LRAT,

CYP26B1 and STRA6, in neonatal rat lung. The presence of LRAT in interstitial tissue

corresponds to the presence of ADRP, a marker protein of lipofibroblast, suggest the

possible localization of LRAT in lipofibroblasts. STRA6 expressed in the vascular

endothelium could ideally play a role in taking up VA from the blood stream. Our results

also clearly indicate the expression of CYP26B1 in the epithelium of bronchioles and

smooth muscle of small airways and blood vessels.

5.6 DISCUSSION

115

It is well known that CYP26 functions to prevent the action of RA and deplete RA in

tissue. But, significant gaps exist in understanding the spatio-temporal expression of the

CYP26 enzymes in lung tissue. The primary oxidized metabolites of at-RA are polar

metabolites that could be eliminated through the blood stream, thus we hypothesized that

CYP26B1 is localized in the endothelial cells of the vascular network, which is believed

to be an ideal place for a ready clearance of RA. However, in our experiment results, the

immunostaining of CYP26B1 protein suggests its presence in the epithelial cells of

bronchioles, not the endothelial cells. The possibilities to explain this are that, first,

CYP26B1 protein is produced locally in epithelial cells to prevent RA action in this

tissue, and excess RA is trafficked to epithelial cells for metabolism, or second, that

CYP26B1 produced in other cell types is transferred to epithelial cells to clear excess RA

here. But we are not sure why CYP26B1 is only expressed in epithelium of bronchioles.

One possibility is that the basal level of CYP26B1 is too low to be detected in other

areas, like alveolar walls, or even vascular endothelial cells, and a longer time might be

required for detectable expression of CYP26B1 after RA or Am580 administration.

Furture studies with longer time of treatment and gene localization study (e.g. ISH) are

needed to further determine where the CYP26B1 gene is expressed and the enzyme is

localized.

Our previous study reported that the mRNAs of retinoid homeostatic genes in the

neonatal rat lungs could be quickly increased by oral treatment of VARA as early as 6h

(67). However, we did not observe any changes in protein level between oil (control) and

VARA treated rat lung samples (data not shown). This could be due to the late protein

116

expression after gene induction. Although the signals of CYP26B1, LRAT and CYP26B1

proteins could be detected in the lung and marker proteins provide evidence and

information of where they are localized, it is still very important to see the changes in

protein level that are regulated by RA, if they exist. To solidify our findings on the

localization of lung retinoid homeostatic proteins and their responses to RA regulation,

protein levels in lung tissue with a longer time of treatment should be examined.

In conclusion, based on immunofluorescence staining, we have described a possible

distribution of retinoid homeostatic proteins in neonatal rat lungs. The dietary VA

transported to the lungs is taken up by STRA6 which is expressed in endothelial cells,

and then VA is esterified and stored in lipofibroblast cells by LRAT which is produced

locally. The excess RA is metabolized by CYP26B1 in epithelial cells of bronchioles.

This documentation provides some new clues for a better understanding of overall VA

homeostasis in the neonatal lung.

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Chapter 6

DISCUSSION

VA is essential for the growth and development of the lung. During the late gestation

(day 14 – 18), lung RE concentration increases significantly and then starts to deplete

before birth and throughout the postnatal period (92). And this depletion is accompanied

by a morphological maturation of the lungs (93), indicating an increased demand for

retinoids for the process of postnatal lung development.

Given that VA has shown its essential role in lung development, supplementation of VA

during late fetal life and early postnatal life to maintain adequate lung VA storage is of

great biological advantage. Before birth, the fetus is exclusively dependent on the

maternal supply of VA. When a large dose of retinyl palmitate (50,000 I.U.) was given to

pregnant rat on day 16, the levels of RE in the lungs of fetus and neonatal rats were

significantly elevated (159). Although this supplementation also increased the RE

concentration in the liver of the offspring, this increase was smaller than the increase in

the lung, suggesting a more efficient mechanism of VA uptake in the fetal lung during the

late gestation. Prematurity in infants is usually associated with severe VA deficiency in

the lungs, which might be attributed to the shortage of maternal VA supply during late

6.1 VITAMIN A SUPPLEMENTATION DURING LUNG SEPTATION PERIOD

118

pregnancy and the relatively immaturity of the liver for RBP synthesis. Animal and

human studies have shown that VA supplementation of lactating mothers during the

postnatal period can improve VA status in their offspring (199-201). Direct oral VA

supplementation of the offspring also can increase the lung RE level. However, the

uptake of retinol by the lung is not as sufficient as by the liver (160, 161). This is because

the liver, as the major organ for VA storage, stores most of excess dietary VA. Other

studies in adult rats treated with RA, the principal active metabolite of retinol, have

shown that the storage of VA in the lung can be increased by RA treatment as well (202,

203). Since RA can not be reversibly reduced to produce retinol, the increased amount of

RE in the lungs maybe originate from the liver RE store, and RA may play a regulatory

role facilitating retinol uptake and RE formation in the lung.

We previously tested the effects of VA and RA combination (10:1 molar mixture of VA

and RA) in promoting lung RE formation and the results showed that the administration

of this nutrient-metabolite combination for 3 consecutive days (p5-7) can synergistically

increase lung RE, at least 4-fold more than the same amount of VA dose given alone

(160). The VA dose used in our studies is based on the dose (50,000 IU) of retinol used

in human studies that has been shown to reduce mortality of newborns in the first year of

life (10, 162). The dosage we selected for RA is based on previous usage shown to induce

lung septation in neonatal rats (97). In a metabolic study, we found that the

administration of RA direct more of the orally supplemented [3H]retinol into the lung

(161).

119

Following these findings, we investigated the molecular mechanism by which VARA

promotes lung RE formation. We examined the expression of lung retinoid homeostatic

genes, LRAT, CYP26B1 and STRA6, as they can control the balance of the retinoid

metabolism by mediating cellular retinol uptake and converting retinol to its storage for,

or by oxidizing RA to inactive polar metabolites. We found that all of these three gene

were upregulated by RA administration at 6 h (67) and concluded that the redirection of

VA flow into the lung by VARA is a result of the upregulation of LRAT and STRA6

genes. The other evidence that demonstrated the cooperation of LRAT and STRA6 in

mediating retinol uptake and storage is from the study of retina and retinal pgment

epithelial cells, in which STRA6 and LRAT are co-expressed. These cells take up retinol

more efficiently, suggesting a driving force for retinol uptake resulting from the

conversion of retinol into RE by LRAT (33). However, by 12 h after dose administration,

the regulatory activity of RA is no longer evident, and this might be attributed to the

simultaneous induction of CYP26B1 by RA at 6 h which prevent RA’s action by

oxidizing it (67).

The experiments described above were designed to examine the response to VARA at

early postnatal life (p7~p8) and over relative short times (6 h after treatment). A kinetic

study checked the consumption of lung RE store in the pups treated with VARA on days

5, 6, and 7, showed that although RE declined between days 8 and 16, the VARA-derived

RE store was retained during the period of postnatal lung septation (161). In the present

studies, we determined the effect of multiple treatments with VARA throughout the

septation period on promoting RE formation in neonatal rat lung and found that multiple

120

treatments increased lung RE contents in a synergistic and cumulative manner (Fig. 2.2).

In this study, we included the usage of Am580, a stable analog of RA, to compare with

RA. From the results, VARA and VAAm did not differ from each other with respect to

lung RE formation after a single dose, but VAAm produced a stronger effect than VARA

in increasing RE after multiple doses (Fig. 2.2). The data of gene expression provides a

good explanation for the difference between VARA and VAAm treatments that the

constant induction on lung retinoid homeostatic genes by Am580, which can persist even

24 h after the dosing leads to an exaggerated effects of VARA on lung RE promotion

(Fig. 2.3, 2.4).

Retinoids have been implicated in lung development by promoting alveolar septation,

angiogenesis, and surfactant synthesis (172). RA has also shown promising effects in

lung repair by regulating genes involved in lung functioning, cell proliferation,

differentiation, cell-cell communication, and cell-matrix interactions (204). Products of

such genes like surfactant proteins, collagen, fibronectin, tropoelastin, β-laminin, as well

as VEGF are all required for normal lung structure and function (204, 205). Given that

RA has shown its regulatory activity on these important lung functional and structural

genes, we assessed the impact of VARA treatments on these genes. The results of this

experiment revealed that the expression of lung functional and structural genes was not

affected by either a single dose or multiple doses of VARA (Fig. 2.5). It is not surprised

to see this result because we used a model of full-term healthy rat pups which were

delivered and nourished by mother rats fed a VA adequate diet. The lungs of these pups

were undergoing or had completed the process of development by the time the gene

121

expression level was analyzed. This result suggests that under physiological condition,

our VARA treatments did not perturb the expression of the important lung structural and

functional genes.

In summary, VARA is more effective than VA alone or RA alone in increasing RE

content in neonatal lungs, and it has little effect on lung structural and functional genes.

These properties make VARA a promising therapeutic option in clinic medicine for

increasing RE formation.

In the fasting state, >95% of retinoid in the circulation is found as retinol bound to RBP,

while the rest is comprised of a small portion of RA bound to albumin and RE contained

in chylomirons. In the fed state, the proportion of RE in the circulation is significantly

increased (206). The precise mechanism of how retinoids are taken up by lung tissue is

not defined yet. But this process possibly involves the direct uptake of retinoids in all

these three forms.

The uptake of retinol bound to RBP mediated by a cell-surface receptor for RBP has been

hypothesized for many years until recently Kawaguchi et al. provided solid evidence

which demonstrated the existence of a RBP receptor, STRA6. STRA6 is a

transmembrane protein, which shows high affinity to RBP and facilitates uptake of

retinol in most of tissues (33). In vitro and in vivo studies have shown that the STRA6

6.2 MOLECULAR MECHANISM OF RETINOL UPTAKE INTO THE LUNG

122

gene can be upregulated by RA (63-67). However, in the multiple treatment study, the

expression level of lung STRA6 is not evidently increased by either RA or Am580 (Fig.

2.5). This result suggested that although STRA6 is involved in the process of retinol

uptake, it may functions at a basal level to transport retinol into lung cells, no matter

whether the RA action is present. In contrast, LRAT gene expression is rapidly and

significantly induced by RA (Fig. 2.2), suggesting that LRAT is the more important

contributing factor that leads to RE formation in lung cells.

RE or retinol present in the circulating chylomicrons may be another important source for

lung RE store after meal or oral VA supplementation. One of our previous studies

suggested that although supplementation with VA or VARA at early postnatal age can

elevate RE markedly in the lungs of neonatal rats, and the plasma retinol was maintained

at the same level by either VA-deficient or VA-adequate diet after weaning, these rats are

still dependent on the VA-adequate diet to prevent the drop of lung RE store during the

postweaning growth period (45). These results imply that chylomicron-associated RE or

retinol served as a secondary source of VA for lung RE store, in addition to that provided

by retinol bound to RBP. Previous study had shown that LPL is able to hydrolyze

chylomicron-associated RE to retinol and facilitate retinoid uptake by adipocytes (46).

However, the direct evidence for the uptake of chylomicron-associated RE in the lung is

still lacking, and whether this process is regulated by RA is not clear.

6.3 THE POTENTIAL OF RA TO PROMOTE LUNG RE FORMATION

123

The concentration of RA in the VARA dose used for our animal experiments is 5 mM,

and the dose administered to neonatal pups is 2 nmol per gram of body weight. Both the

single dose and the multiple doses of VARA can increase lung RE content significantly

in neonatal lungs (Fig. 2.2). Is the concentration of RA over-saturated (more than

necessary) for producing the same effect? In the RA dilution study, we tested the

potential of RA to induce lung RE formation by diluting its concentration. We found that

the RA reduced down to 0.5 mM can still synergize with the same (standard) amount of

VA to increase lung RE in younger pups (5-day old) after a single dose. Interestingly, this

effect is not observed in older pups (14-day old), which received multiple doses (Fig.4.3).

Our previous 12 h experiment had suggested that the activity of RA is just transient

because it is quickly oxidized. Therefore, we speculated that the loss of synergy in the

multiple dose study is due to the quicker elimination of the small amount of RA by lung

or other tissues. This hypothesis was supported by a parallel multiple dose experiment in

which RA was substituted by Am580. The synergistic effect of VAAm was at least

partially retained with the 1% concentration of Am580 (Fig. 4.4), indicating a prolonged

regulatory activity of Am580.

Nevertheless, some other possible mechanisms that leading to the discrepancy between

the single dose study and the multiple dose study may also exist. For example, the lung

tissue may respond to RA or Am580 regulation with different sensitivity at different

ages, or there may be a high rate of RA clearance in the lungs of older rat pups. Whether

the VARA synergy in neonatal lung is time-dependent should be tested in the future.

124

It is well known that VA deficiency increases the risk of infectious diseases in young

children, which can lead to increased morbidity and mortality (207, 208). VA

supplementation has been shown to effectively reduce the severity of infectious diseases,

morbidity and mortality (209-212). Although it is already well established that retinoids

help maintain integrity of the immune system (213) and have shown anti-inflammatory

activity (214), little is known about how inflammation affects retinoid metabolism.

It has been reported that inflammation is able to alter the expression of several

cytochrome P450 genes in the liver (183). In a previous study in which adult rats were

treated with LPS and RA simultaneously, we found that LPS can abrogate the RA-

induced increase in CYP26A1 and CYP26B1 mRNA in the liver (185). Similarly, we

tested the effects of inflammation on retinoid homeostasis in neonatal lungs. In the LPS

study, we used a model of LPS-induced inflammation in neonatal rats to determine

whether the inflammation can prevent RE promotion by VARA, and opposes the

response of LRAT, CYP26B1 and STRA6 to RA in the lung, in which CYP26B1 mRNA

is more abundant. The results of our experiments suggested that the acute inflammation

just modestly decreased but did not prevent VARA-promoted RE formation in the lung,

and did not significantly alter the retinoid homeostatic genes expression pattern.

6.4 THE RELATIONSHIP BETWEEN INFLAMMATION AND RETINOID

METABOLISM IN NEONATAL LUNGS

125

Inflammation can reduce the production of RBP in the rat liver as well as the RBP-retinol

complex concentration in the plasma (215), which might contribute to the slight reduction

in the VARA-promoted RE formation. Despite this, the synergistic effect of VARA was

still very obvious in the inflammatory state. Our results also demonstrated that LPS

administration alone has little effects on the basal level of RE, and the expression of

LRAT, CYP26B1 and STRA6 genes in the lung, suggesting effective maintenance of

retinoid homeostasis under the inflammatory stimuli.

Alveologenesis is associated with dramatic changes not only in lung morphological

maturation but also in the metabolism of endogenous retinoids from storage forms, such

as RE, to oxidized metabolites (93, 94, 216). Important components involved in the

retinoid metabolism pathway include STRA6, a RBP receptor which mediate cellular

retinol uptake, LRAT, an enzyme which convert retinol to RE, retinoid binding proteins

(CRBP, CRABP), cellular proteins which regulate the biological action of retinol and

RA, retinoid oxidizing enzymes (RDH, RALDH and cytochrome P450), and RA

receptors (RARs, RXRs), nuclear proteins which act as transcription factors to regulate

downstream gene expression, etc. Previous studies have shown the distribution of CRBP-

I and CRABP-I proteins in lipofibroblasts in postnatal lungs and their upregulation

during alveolarization (94, 217), suggesting the appearance or production of retinol and

6.5 SPATIAL DISTRIBUTION OF THE RETINOID HOMEOSTATIC

PROTEINS IN NEONATAL LUNGS

126

RA in lung lipofibroblast cells. RAR expression also has been reported to be localized in

bronchial epithelium, bronchial and vascular smooth muscle, pleura, and scattered cells

within the alveolar regions (217), implying RA’s activity at these sites.

In the localization study, we chose to determine the spatial expression of LRAT,

CYP26B1 and STRA6 in postnatal lung. Our confocal microscopy images revealed that

STRA6 is specifically expressed in the endothelial cells, and LRAT is expressed in the

lipofibroblast (Fig. 5.2, 5.3). Interestingly, the distribution of CYP26B1 in bronchial

epithelium and smooth muscle of bronchioles and vessel is similar to the distribution of

RAR. It is well known that VA deficiency can result in morphological alteration in the

bronchial epithelium, a tissue known to be very retinoid sensitive (11, 77) and this effect

suggests that RA signaling is required for maintaining the integrity of airway epithelium.

Contradictorily, the CYP26B1 was identified strongly at the same site, which may mean

there is a prevention of RA’s action through RA nuclear receptors. It is not clear what

role CYP26B1 plays in this tissue, possibly, a mechanism with subtle controlling on

cellular concentration and biological activity of RA may exist.

The lipofibroblast cells in postnatal rat lung are primarily localized in the alveolar

interstitium. They are found adjacent to septa junctions when septa are being formed

(137). This cell type might play a role as a retinoid signaling center not only due to its

localization, but also because many components of the retinoid signaling pathway

including receptors and binding proteins are contained in these cells (94). The formation

of septa occurs in the rat mainly between postnatal day 4 and 14 (114). In our

experiments, we used the samples from 14-day old pups. The staining of a lipofibroblast

127

marker protein, ADRP, and LRAT protein is not evident in alveolar wall. This is might

be due to low formation of septa at the end of alveolar stage.

Based on our findings and information from previous studies, we propose a possible

model of retinoid metabolism in the lung (Fig. 6.1): plasma retinol bound to RBP is taken

up by lung tissue through a membrane receptor, STRA6, which is expressed in the

endothelial cells of capillaries. Subsequently retinol is transported to lipofibroblasts, at

where excess retinol will be converted to RE by LRAT for storage, or metabolized to

produce endogenous RA. The locally synthesized RA thereafter is secreted to

neighboring cells, including type II alveolar cells, endothelial cells and myofibroblasts, in

a paracrine manner to initiate or coordinate the alveolar septal eruption during

alveologenesis. In bronchial epithelium, where CYP26B1 is predominantly expressed,

the excess RA will be cleared to maintain the balance of retinoid metabolism pathway.

In conclusion, this experiment determined the spatial distribution of the retinoid

homeostatic proteins in the lung. Together with the information from previous studies,

our findings further demonstrate the key role of lipofibroblast as a retinoid signaling

center during alveologenesis, and provide better understanding of retinol uptake and

metabolism in neonatal lung tissue.

128

Figure 6.1 Model of VA metabolism in neonatal rat lung. This diagram describes a

proposed pathway of retinoid uptake, esterification, and oxidative metabolism based on

the knowledge of spatial distribution of STRA6, LRAT and CYP26B1 proteins. Retinol

circulating in the blood stream is taken up through a transmembrane protein STRA6,

which is expressed in the endothelial cells and then transferred to the lipofibroblast, at

where LRAT enzyme is localized, for conversion to RE. Endogenous RA produced in the

lipofibroblasts then acts as a paracrine signal to affect the neighboring cells biological

activity. Excess RA will be transported to bronchial epithelial cells, the major tissue

where CYP26B1 is expressed for clearance.

129

It is known that septation occurs mainly between the postnatal day 4 and day 14th

postnatal days in rats (7). Our present studies have determined the localization of retinoid

homeostatic proteins in the lungs of neonatal rat at the age of postnatal day 14, a time

point by which major septation process is completed. To better understand the regulation

of retinoid homeostasis in neonatal rat lungs, we need to investigate the temporal

expression of the retinoid homeostatic proteins during the postnatal septation period. We

believe that retinoid homeostatic proteins, similar to CRBP, CRABP and RARs, which

are tightly regulated in the postnatal lung (216), will be temporally and spatially

associated with alveolar formation, because lung VA status and metabolism undergoes a

significant change during this period.

In the RA dilution study, we observed that diluted RA combined with the same dose of

VA showed different effects in promoting lung RE formation in a single dose study and

multiple dose study. To interpret this phenomenon, we speculate that a response to RA

administration with a high sensitivity only exists at the very beginning of the postnatal

life, maybe even earlier than birth, because a high demand of retinoids is required for the

process of alveologenesis. To test this speculation, we need to examine the effects of a

single dose of VA combined with diluted RA administered at different postnatal ages. If

it is the case, supplementing with VA at an earlier postnatal time may be more

6.6 FUTURE DIRECTIONS

130

meaningful than at a later time, and we should then adjust the treatment strategy

according to a best timing to approach the more efficient method of increasing lung RE

level.

In the LPS study, we examined how inflammation affects retinoids metabolism in

neonatal lungs. However, there are many other factors that might impact lung retinoid

metabolism during postnatal stage, such as mechanical ventilation, dexamethasone

treatment, hyperoxia, pulmonary infections, etc, which are often associated with

neonates, especially newborns with very low body weight. In the future, it will also be of

interest to test the synergistic effect of VARA in promoting lung RE formation under

these conditions.

Since the present study has reported that RA combined with PIC promoted TT-induced

vaccine response in both adult and neonatal mice, the following question will be how to

apply to human vaccination.

The recent data in our lab reported that oral administration of retinyl palmitate (RP)

combined with a small amount of RA (1:10 molar of RP) synergistically increased lung

VA concentration about 5 to 10-fold in rat pups, and this potency was maintained even

though the dose of RP+RA was reduced to half or one fourth (Ross et al, unpublished

data). These data suggest a novel supplementation that VA combined with a small

amount of RA can not only reduce the dosage of VA but also increase the capacity of

improving VA status.

131

More importantly, the combination of VA and RA may be more effective than VA alone

to stimulate immune system in VA-sufficient population. Therefore, in the future study,

we will test the immunoregulatory effect of VA+RA in adult and neonatal mice.

132

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Vita

Lili Wu

EDUCATION 2008- 2011 Ph.D., Major: Nutritional Sciences The Pennsylvania State University, University Park, PA 2005-2008 M.S., Major: Nutritional Sciences The Pennsylvania State University, University Park, PA 1996-2001 B.S., Major: Clinical Medicine Anhui Medical University, Hefei, China PROFESSIONAL EXPERENCE Graduate Research Assistant Jan 2005 – Dec 2010 Department of Nutritional Sciences, The Pennsylvania State University, University Park Investigated Vitamin A metabolism in neonatal rat lungs PUBLICATIONS Wu L and Ross AC, Multiple treatments of acidic retinoids during postnatal period accumulate retinol storage in the lung of neonatal rats. (In preparation) Wu L and Ross AC, LPS-induced inflammation modestly affects retinoic acid promoted retinol storage in neonatal lungs. (In preparation) Wu L and Ross AC (2010) Acidic retinoids synergize with vitamin A to enhance retinol uptake and STRA6, LRAT, and CYP26B1 expression in neonatal lung. J Lipid Res 51: 378-387. Wu L, (2008) Master thesis: Investigation on the mechanism of vitamin A uptake, accumulation and metabolism in the lungs of the neonatal rat. Ross AC, Li NQ, and Wu L (2006) The components of VARA, a nutrient-metabolite combination of vitamin A and retinoic acid, act efficiently together and separately to increase retinyl esters in the lungs of neonatal rats. J Nutr 136: 2803-2807. TEACHING EXPERENCE Teaching Assistant 2009 NUTR 445 (Nutrient Metabolism I) PROFESSIONAL MEMBERSHIP American Society for Nutritional Sciences (ASNS)

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