Gaudio et al. 1

For consideration of publication in Am J Physiol February 7, 2006

ADMINISTRATION OF r-VEGF-A PREVENTS HEPATIC ARTERY LIGATIONINDUCED BILE DUCT DAMAGE IN BILE DUCT LIGATED RATS

Eugenio Gaudio, M. D.7#

Barbara Barbaro, Ph. D.2, 6, 7#

Domenico Alvaro, M. D.6

Shannon Glaser, M. S.3

Heather Francis, B. S.3

Antonio Franchitto, B. S.7

Paolo Onori, M. D.8

Yoshiyuki Ueno, M. D., Ph. D.5

Marco Marzioni, M. D.4

Giammarco Fava, M. D.4

Julie Venter, B. S.2

Ramona Reichenbach, B. S.2

Ryun Summers, B. S.2

Gianfranco Alpini, Ph. D.1, 2, 4

From 1Central Texas Veterans Health Care System, 2Department of Medicine and4Systems Biology & Translation Medicine, 3Division of Research and Education, Scott& White Hospital and The Texas A&M University System Health Science Center,

College of Medicine, Temple, TX 76504, 5Div. Gastroenterol, Tohoku University School

of Med, Aobaku, Sendai, Japan, and 6Div. Gastroenterol and 7Anatomy, University "La

Sapienza", Rome, Italy and 8Dept. Experimental Medicine, University of L’Aquila, Italy.This work was supported by a grant award to Dr. Alpini from Scott & White Hospital andThe Texas A&M University System, by a Grant Award from Scott & White Hospital toShannon Glaser, and by a VA Merit Award, a VA Research Scholar Award and the NIHgrants DK58411 and DK062975 to Dr. Alpini and by a Grant from MIUR (COFIN 2003,

# 2003060498_002 and COFIN 2005 # 2005067975_002) Dr. Domenico Alvaro and by agrant from MIUR (PRIN 2003 and ex 60%) to Prof Eugenio Gaudio and from MIURBiomedicina, Cluster04, progetto n.5 to Prof. E. Gaudio / P. Onori and by a grant fromHealth and Labor Sciences Research Grants for the Research on Measures for Intractable

Diseases (from the Ministry of Health, Labor and Welfare of Japan) and from Grant-in

Aid for Scientific Research C (16590573) from JSPS to Dr. Ueno. #Dr. Gaudio and Dr.Barbaro equally contributed to this study.

Short title: VEGF regulation of cholangiocyte functions

Key words: cAMP; ductal secretion; intrahepatic biliary epithelium; mitosis;microcirculation; secretin.

Page 1 of 45Articles in PresS. Am J Physiol Gastrointest Liver Physiol (March 30, 2006). doi:10.1152/ajpgi.00507.2005

Copyright © 2006 by the American Physiological Society.

Gaudio et al. 2

Abbreviations used: BDI = bile duct incannulation; BDL = bile duct ligation; cAMP =adenosine 3', 5’-monophosphate; PCNA = proliferating cellular nuclear antigen; r-VEGF-A = recombinant-VEGF-A; VEGF = vascular endothelial growth factor.Address correspondence to: Gianfranco Alpini, Ph. D.

VA Research Scholar Award RecipientProfessor, Medicine and Systems Biology &Translation MedicineDr. Nicholas C. Hightower Centennial Chair ofGastroenterologyCentral Texas Veterans Health Care SystemThe Texas A & M University System HealthScience Center College of MedicineMedical Research Building702 SW H.K. Dodgen Loop, Temple, TX, 76504Phone: 254-742-7044 or 254-742-7058Fax: 254-724-5944 or 254-742-7130E - m a i l : g a l p i n i @ t a m u . e d u o rgalpini@medicine.tamhsc.edu

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Gaudio et al. 3

ABSTRACT

The hepatic artery, through the peribiliary vascular plexus, nourishes the intrahepatic

biliary tree. During obstructive cholestasis, the nutritional demands of intrahepatic bile

ducts are increased as a consequence of enhanced proliferation; in fact, the peribiliary

plexus displays adaptive expansion. The effects of hepatic artery ligation (HAL) on

cholangiocyte functions during cholestasis are unknown although ischemic lesions of the

biliary tree complicate the course of transplanted livers and are encountered in

cholangiopathies. We evaluated the effects of HAL on cholangiocyte functions in

experimental cholestasis induced by bile duct ligation (BDL). By using BDL and BDL +

HAL rats or BDL + HAL rats treated with r-VEGF-A for 1 week, we evaluated liver

morphology, the degree of portal inflammation and peri-ductular fibrosis,

microcirculation, cholangiocyte apoptosis, proliferation, and secretion. Microcirculation

was evaluated by scanning electron microscopy vascular corrosion cast technique. HAL

induced in BDL rats: (i) the disappearance of the peribiliary plexus; (ii) increased

apoptosis and impaired cholangiocyte proliferation and secretin-stimulated ductal

secretion; and (iii) decreased cholangiocyte VEGF secretion. The effects of HAL on

peribiliary plexus and cholangiocyte functions were prevented by r-VEGF-A that, by

maintaining the integrity of the peribiliary plexus and cholangiocyte proliferation,

prevents damage of bile ducts following ischemic injury.

INTRODUCTION

Cholangiocytes, the epithelial cells lining the intrahepatic biliary epithelium (6), modify

bile, originally secreted at the bile canaliculus (43), by a series of absorptive and

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Gaudio et al. 4

secretory events regulated by a number of factors including gastrointestinal

hormones/peptides, bile salts and nerve receptor agonists (4-7, 11, 31, 38). The

gastrointestinal hormone secretin increases ductal secretion by interaction with specific

receptors (expressed only by cholangiocytes) (10), an interaction that induces an increase

in intracellular adenosine 3',5'-monophosphate (cAMP) levels (4, 7, 25, 26, 31, 38).

Increased intracellular cAMP levels induces activation of the CFTR Cl- channels (9, 31)

and Cl-/HCO3- exchanger activity (7, 11, 31), a series of events that leads to secretin-

stimulated bile and bicarbonate secretion (5, 26).

In normal liver, cholangiocytes have low basal DNA synthesis (4, 51). However,

cholangiocytes proliferate in a number of experimental models of cholestasis including

bile duct ligation (BDL) (4, 5, 12, 25, 37, 38). Cholangiocyte proliferation in the course

of cholangiopathies compensates for the loss of injured ducts and, in fact, proliferating

cholangiocytes display enhanced basal and secretin-stimulated ductal secretory activities

(4-6, 26, 31, 38). A number of studies in rats (3-6, 8-10, 25, 36-40) and humans (45)

have shown that changes in cholangiocyte proliferation are associated with parallel

modifications in secretin receptor gene expression, secretin-stimulated cAMP levels and

secretin-induced bile and bicarbonate secretion. For example, we have shown in rats that

in pathological conditions associated with increased cholangiocyte proliferation (e.g.,

following BDL or partial hepatectomy) (4, 5, 25, 38), there is enhanced secretin receptor

gene expression and augmented basal and secretin-stimulated cAMP levels and bile and

bicarbonate secretion. On the other hand, reduced cholangiocyte proliferation or

enhanced cholangiocyte loss (e.g., following vagotomy, acute administration of CCl4 or

depletion of endogenous bile acid pool) (3, 36, 39) is coupled with decreased basal and

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Gaudio et al. 5

secretin-stimulated cAMP levels and bile and bicarbonate secretion. In humans, an

impaired response to secretin was observed in cholestatic conditions (45).

Cholangiocyte proliferation is regulated by neuropeptides, hormones and growth factors

including vascular endothelial growth factor (VEGF) (6, 21, 37). Rat cholangiocytes

express the protein for VEGF-A and secrete VEGF, and express the VEGF receptor

subtypes, VEGFR-2 and VEGFR-3 but not VEGFR-1 (21). VEGF secretion is enhanced

in proliferating cholangiocytes from BDL rats, where it stimulates, by autocrine

mechanisms, cholangiocyte proliferation (21).

The peribiliary plexus (PBP) stems from the hepatic artery, nourishes the biliary tree and

sustains a contercurrent of substances reabsorbed from bile toward hepatocytes (23). A

true microvascular plexus vascularizes larger ducts, whereas around the smaller ducts the

plexus gets progressively simpler (up to a single capillary) and thinner (23). In normal

rats, where cholangiocytes are in a quiescent status (39), ligation of the main hepatic

artery by its own is not sufficient to induce bile duct damage, suggesting that accessory

arteries, collateral vessels or anastomosis between PBP and portal system may overcome

the interruption of arterial flow in the main hepatic artery (16, 17). Consistently,

interruption of blood flow to intrahepatic bile ducts (by short-term ligation of the hepatic

artery of normal guinea pigs) does not alter cholangiocyte secretion (50). The

extrahepatic and intrahepatic PBP in the normal liver has been the subject of a number of

studies using three-dimensional observations (22-24). Changes in intrahepatic bile duct

mass are always associated with changes of the PBP architecture (22, 23). After BDL,

the increase in intrahepatic bile duct mass is followed by a parallel growth of the PBP

(23), which is fundamental in sustaining the enhanced nutritional and functional demands

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Gaudio et al. 6

of proliferating cholangiocytes (4-6). Nevertheless, the proliferation of the PBP occurs

only after the hyperplasia of the intrahepatic biliary epithelium (23). This finding

suggests a cross-talk mechanism between cholangiocytes and endothelial cells, an

interaction that mediates the adaptive changes of these cells during liver damage.

However, limited information exists on the role of blood supply through the hepatic

artery in pathological conditions characterized by cholangiocyte proliferation/loss (14,

48). This concept has clinical implications since ischemic bile duct lesions are

considered possible causes of cholestatic disorders, in particular after liver

transplantation, hepatic surgery and intra-arterial chemotherapy (13, 15).

MATERIAL AND METHODS

Materials

Reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise

indicated. The substrate for -glutamyltranspeptidase ( -GT), N ( -L-glutamyl)-4-

methoxy-2-naphthylamide was purchased from Polysciences (Warrington, PA). Porcine

secretin was purchased from Peninsula (Belmont, CA). The antibodies against

proliferating cellular nuclear antigen (PCNA), VEGF-A and the VEGF receptor subtypes

VEGFR-1, VEGFR-2 and VEGFR-3 were purchased from Santa Cruz Biotechnology

Inc. (Santa Cruz, CA). The recombinant mouse VEGF (r-VEGF-A) was purchased from

Leinco Technologies Inc. (St. Louis, MO).

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Gaudio et al. 7

Animal Models

Male Fischer 344 rats (150 to 175 gm) were purchased from Charles River (Wilmington,

MA). The animals were kept in a temperature-controlled environment (22˚C) with a 12-

hour light-dark cycle and fed ad libitum rat chow. The studies were performed in: (i)

normal rats and normal rats + HAL (for evaluation of cholangiocyte apoptosis and

proliferation in liver sections); (ii) 1 week BDL (for isolation of cells) (4, 5) or bile duct

incannulated (BDI, for bile collection) (5) rats; and (iii) rats that (immediately after BDL

or BDI + HAL) were treated by IP implanted Alzet osmotic minipumps with 0.2% bovine

serum albumin (BSA) or r-VEGF-A (2.5 nmol/kg/hour with 0.2% BSA) for 1 week. The

dose (nM range) of r-VEGF-A administered to BDL + HAL rats was chosen according to

the concentration (nM range) of VEGF found in the serum of rats and human in other

studies (12, 52). The group of BDL + HAL rats was studied since we observed impaired

secretion of VEGF in BDL cholangiocytes after HAL. Since we observed impaired

secretion of VEGF in BDL cholangiocytes after HAL, the group of BDL + HAL + VEGF

rats was chosen to evaluate whether the effects of HAL observed in BDL rats are

prevented by chronic administration of r-VEGF-A. BDL and BDI were performed as

described (5). HAL was performed as described (29). Before each procedure, animals

were anesthetized with sodium pentobarbital (50 mg/kg body weight, IP). Study

protocols were performed in compliance with the institution guidelines.

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Gaudio et al. 8

Isolation of Hepatocytes and Cholangiocytes

Hepatocytes were isolated as described (2). Cholangiocytes (97-100% pure by -GT

histochemistry) (46) were purified by immunoaffinity separation (4, 28). Cell number

and viability (greater than 97%) was assessed by trypan blue exclusion.

Body Weight, Liver Morphology, Necrosis, Inflammation and Peri-ductular

Fibrosis

We evaluated the effect of BDL, HAL and HAL + r-VEGF-A administration to BDL +

HAL rats on body weight, liver morphology, necrosis, inflammation and peri-ductular

fibrosis. We evaluated, by H&E staining of paraffin-embedded liver sections (4 µm

thick, 6 slides evaluated for each group), the degree of portal inflammation (18), necrosis

and lobular morphology (disarrangement of hepatocytes). At least 10 different portal

areas were evaluated. Following H&E staining, liver sections were examined in a coded

fashion with an Olympus BX-40 (Tokyo, Japan) microscope equipped with a camera.

We evaluated, by Masson’s trichrome staining of paraffin-embedded liver sections (4 µm

thick, 6 slides evaluated for each group), the degree of fibrosis around proliferating ducts

from BDL, BDL+HAL and BDL+HAL+r-VEGF-A treated rats. At least 6 different

microscopic fields (10X and 20X) for each slide were analysed in a coded fashion with

an Olympus BX-51 microscope (Tokyo, Japan) equipped with a Videocam (Spot Insight,

Diagnostic Instrument, Inc. Sterling Heights, MI) and processed with an Image Analysis

System (IAS - Delta Sistemi, Roma- Italy). Peri-ductular fibrosis was measured as the

volume fraction of the entire liver tissue specimen (% volume fraction of the green

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Gaudio et al. 9

stained collagen fibres less volume fraction occupied respectively by portal triads and by

the parenchyma).

Evaluation of Liver Microcirculation

Following anesthesia, the abdomen of the selected animal was opened and a cannula

(Inpharven diameter 1.4 mm, Inphardial, Italy) was inserted into the aorta and fixed with

2 silk ties. Before flushing the vascular bed with heparinized saline solution (23), the

thorax was opened and the right atrium incised to allow the efflux of the perfusate. When

the outflow fluid appeared clear of blood, Mercox CL2R resin, diluted with methyl

methacrylate monomer (4:1) (34) and mixed with a standard amount of its catalyzer (up

to 2 ml catalyzer per 20 ml of base compound), was injected at room temperature. A

constant pressure control was maintained (by a CONEL electronic manometer, Rome,

Italy) through the lateral port of the cannula’s injection valve until resin polymerization

was visible. The animals were left at room temperature for 24 hours and after the

polymerization of the resin, the livers were removed and macerated in 20% NaOH

solution at room temperature. After rinsing in distilled water, the liver casts were placed

in 5% trichloroacetic acid solution to free the cast from tissue remnants. The casts were

isolated, frozen in distilled water and frozen-dried. They were then glued onto stubs by

means of Silver Dag and gold coated in an Sl50 sputterer (Edwards, London, UK). The

prepared casts were examined with a Hitachi S4000 Field Emission scanning electron

microscope (Hitachi Ltd., Tokyo, Japan) operating at 5-8 kV (23).

Evaluation of Cholangiocyte VEGF Protein Expression and Secretion

Page 9 of 45

Gaudio et al. 10

Protein expression for VEGF-A, VEGFR-1, VEGFR-2 and VEGFR-3 was evaluated by

immunohistochemistry in liver sections (5 µm thick, 6 slides evaluated per group)

mounted on glass slides coated with 0.1% poly-L-lysine. Following staining, sections

were analyzed in a coded fashion with an Olympus BX-51 microscope (Tokyo, Japan)

equipped with a Videocam (Spot Insight, Diagnostic Instrument, Inc. Sterling Heights,

MI) and processed with an Image Analysis System (IAS - Delta Sistemi, Roma- Italy).

The intensity and distribution of immunostaining were assessed in a coded fashion.

Following isolation, hepatocytes or cholangiocytes (1x106) were incubated at 37oC for

zero or six hours, centrifuged at 1,500 rpm for 10 minutes, the supernatant (100 µl)

transferred to a tube and analyzed for VEGF concentration by ELISA (Peninsula

Laboratories, Inc, San Carlos, CA). VEGF secretion (ng/1x106 cells) was calculated as

the difference between the amount of VEGF detected at six hours and the amount

detected at time zero.

Cholangiocyte Apoptosis and Proliferation

We evaluated cholangiocyte and hepatocyte apoptosis by TUNEL analysis (39) in liver

sections (3 slides evaluated for each group, 5 µm thick) from the selected group of

animals. Following counterstaining with Hematoxylin solution, liver sections were

examined by light microscopy with an Olympus BX-40 microscope (Tokyo, Japan)

equipped with a camera. Approximately 100 cells per slide were counted in a coded

fashion in ten non-overlapping fields.

Cholangiocyte and hepatocyte proliferation was evaluated by measuring the number of

PCNA- and hepatocyte-positive cholangiocytes. Cholangiocyte growth was also

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Gaudio et al. 11

evaluated by measuring the % of CK-19- and -GT-positive ducts in liver sections (3

slides evaluated for each group of animals, 5 µm thick) (39). Sections were

counterstained with hematoxylin and examined in a random, blinded fashion with an

Olympus BX 51 light microscope (Tokyo, Japan). Data were expressed as number of: (i)

PCNA-positive cholangiocytes or hepatocytes per each 100 cells measured; and (ii) % of

C 19-positive ducts area evaluated in ten different fields (10X or 20X) of slide taken

from each 6 blocks randomly taken from medial lobe. Histochemistry for -GT-positive

ducts (46) was performed in frozen sections (5 µm thick, 3 slides evaluated for each

group).

Measurement of Basal and Secretin-Stimulated Ductal Secretion

At the functional level, cholangiocyte proliferation was evaluated by measurement of

basal and secretin-stimulated cAMP levels (32, 39) in purified cholangiocytes and bile

and bicarbonate secretion (5) in bile fistula rats, two functional indices of cholangiocyte

proliferation) (4, 5, 20, 25, 26, 36-39).

For the measurement of cAMP levels, purified cholangiocytes were incubated for 1 hour

at 37°C (32) and incubated for 5 minutes at room temperature (32) with 0.2% BSA

(basal) or 100 nM secretin with 0.2% BSA. Intracellular cAMP levels were measured by

commercially available RIA kits (Amersham Life Science) (32).

Following anesthesia, rats were surgically prepared for bile collection as described by us

(5). One jugular vein was incannulated with a plastic cannula to infuse either Krebs

Ringer Henseleit (KRH) or secretin (100 nM) dissolved in KRH. Biliary bicarbonate

concentration (measured as total CO2) was determined by an ABL 520 Blood Gas

System (Radiometer Medical A/S, Copenhagen, Denmark).

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Gaudio et al. 12

Statistical Analysis

All data are expressed as mean ± SEM. Differences between groups were analyzed by

the Student unpaired t test when two groups were analyzed and analysis of variance

(ANOVA) when more than two groups were analyzed, followed by an appropriate post

hoc test.

RESULTS

Effects of HAL and r-VEGF-A on Body Weight, Liver Morphology, Necrosis and

Inflammation and Peri-ductular Fibrosis

No changes in body weight were observed among BDL (161.7 ± 4.0 gm), BDL + HAL

(172.3 ± 7.0 gm) and BDL + HAL + r-VEGF-A (165.6 ± 5.6 gm) rats. There were no

differences in the amount of lobular damage among BDL rats and rats that (immediately

after BDL + HAL) were treated with 0.2% BSA or r-VEGF-A for 1 week (see

representative Figure 1 a and Table 1). The degree of necrosis, portal inflammation and

the % volume fraction of peri-ductular fibrosis observed in BDL rats were reduced in

BDL+HAL compared to the value of BDL rats (Figure 1 a-b and Table 1). Following the

administration of r-VEGF-A to BDL + HAL rats, the degree of necrosis, portal

inflammation and the % volume fraction were similar than that of the BDL rat (see

representative Figure 1 a-b and Table 1).

Effect of HAL and r-VEGF-A on the Peribiliary Plexus and VEGF Expression

The peribiliary plexus, observed in BDL rats (23), was not demonstrated in BDL + HAL

rats by scanning electron microscopy corrosion cast technique. We did not observe PBP

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Gaudio et al. 13

in BDL+HAL rats because the PBP is nourished by the hepatic artery. In normal rats,

PBP was observed more easily in large portal tracts (not shown) (23). In the small portal

tracts, the PBP was characterized by single layer of capillaries or even by a single

capillary; in BDL+ HAL we do not observed PBP. Administration of r-VEGF-A to

BDL+HAL rats prevented the HAL-induced microvascular modification (absence of

PBP) (Figure 2). The microvascular pattern observed after administration of r-VEGF-A

to BDL + HAL rats demonstrated the presence of a PBP (Figure 2) with similar

characteristics previously described in BDL rats (23). In vascular corrosion casts, we

observe only vascular tree because the tissue is completely digested. Therefore in Figure

2, we did not observe bile duct (completely digested) but only the absence of peribiliary

plexus.

Immunohistochemistry in BDL liver sections shows that bile ducts express VEGF-A,

VEGFR-2 and VEGFR-3 (Figure 3 a-b and Table 2). HAL induced a decrease in the

number of cholangiocytes positive for VEGF-A and VEGFR-2 and VEGFR-3 receptors

compared to liver sections from 1 week BDL rats (Figure 3 a-b and Table 2). Following

the administration of r-VEGF-A to BDL + HAL rats, the expression of VEGF-A and

VEGFR-2 and VEGFR-3 was similar or higher than that of the BDL rat (Figure 3 a-b and

Table 2). VEGFR-1 was not expressed by cholangiocytes (Table 2). VEGF-A was

predominantly expressed by hepatocytes of centrolobular zone (Table 3). Hepatocyte

VEGF-A protein expression did not increase significantly in BDL liver sections

compared to normal sections, decreased after HAL and returned to values similar to those

of BDL rats (Table 3).

Normal rat hepatocytes and cholangiocytes secrete VEGF (Figure 4). Following BDL,

there was an increase in VEGF secretion in cholangiocytes (Figure 4, top). VEGF

Page 13 of 45

Gaudio et al. 14

secretion significantly decreased in BDL hepatocytes compared to normal hepatocytes

(Figure 4, bottom). In cholangiocytes and hepatocytes from BDL+HAL rats, there was

decreased VEGF secretion (Figure 4) compared with cholangiocytes and hepatocytes

from BDL rats (Figure 4 b). Administration of r-VEGF-A prevented the decrease of

cholangiocyte VEGF secretion induced by HAL in cholangiocytes and hepatocytes

(Figure 4).

Cholangiocyte Apoptosis and Proliferation

Parallel to previous studies (41), TUNEL analysis showed a few apoptotic bodies in the

liver sections of normal (results not shown) and BDL rats (Figure 5). The number of

cholangiocytes undergoing apoptosis increased in liver sections from BDL + HAL rats

compared to BDL rats (Figure 5). HAL had no effect on cholangiocyte apoptosis of

normal rats (not shown). Administration of r-VEGF-A prevented the increase in

cholangiocyte apoptosis induced by HAL in BDL rats (Figure 5). The number of

apoptotic hepatocytes was not changed in the different groups of animals (BDL = 2.87 ±

0.13; BDL + HAL = 3.47 ± 0.16; BDL + HAL + r-VEGF-A = 3.39 ± 0.16).

Following HAL, the number of PCNA-positive cholangiocytes and the % of CK-19 and

-GT-positive ducts decreased compared to liver sections from BDL rats (Table 4 and

representative Figure 6 showing the % of CK-19-positive ducts in liver sections).

Administration of r-VEGF-A prevented the inhibitory effect of HAL on the number of

PCNA-positive cholangiocytes and the % of CK-19 and -GT-positive ducts, values that

were similar to that of BDL rats (Table 4 and Figure 6). HAL did not alter the number of

PCNA-positive cholangiocytes and the % of CK-19 and -GT-positive ducts in normal

liver sections (not shown). The proliferation of hepatocytes (PCNA positive) was similar

Page 14 of 45

Gaudio et al. 15

in the different groups of animals (BDL = 3.44 ± 0.19; BDL + HAL = 3.04 ± 0.16; BDL

+HAL + r-VEGF-A = 3.68 ± 0.14.

Basal and Secretin-stimulated cAMP Levels and Ductal Secretion

In agreement with previous studies (25), secretin increased intracellular cAMP levels of

cholangiocytes from BDL rats (Figure 7). HAL significantly reduced basal

cholangiocyte cAMP levels and inhibited secretin-stimulated cAMP levels of

cholangiocytes compared to cholangiocytes from BDL rats (Figure 7). Administration of

r-VEGF-A prevented the inhibition of basal and secretin-stimulated cAMP levels induced

by HAL (Figure 7).

Secretin stimulated bile flow, bicarbonate concentration and secretion of BDI rats (Table

5). In HAL + BDL rats, secretin did not increase bile flow and bicarbonate concentration

and secretion of BDI rats (Table 5). Administration of r-VEGF-A prevented the

inhibition of secretin-stimulated bile and bicarbonate concentration and secretion, which

were similar to that of BDL rats (Table 5).

DISCUSSION

The study demonstrates that in BDL rats, HAL: (i) induced the absence of the peribiliary

plexus; (ii) decreased the immunolocalization of VEGFR-2 and VEGFR-3 receptors and

VEGF-A in liver sections, and VEGF secretion in purified cholangiocytes and

hepatocytes; (iii) induced loss of intrahepatic bile ducts in BDL but not normal rats,

caused by both increased apoptosis and decreased cholangiocyte proliferation; no

changes in hepatocyte apoptosis and proliferation were observed in BDL rats and rats that

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Gaudio et al. 16

(immediately after BDL + HAL) were treated by IP implanted Alzet osmotic minipumps

with 0.2% BSA or r-VEGF-A for 1 week; and (iv) decreased basal and secretin-induced

intracellular cAMP synthesis and impaired basal and secretin-stimulated bile flow and

bicarbonate secretion. The adverse effects of HAL on peribiliary plexus, VEGF

expression and secretion (in cholangiocytes and hepatocytes), and on cholangiocyte

proliferative and secretory activities in BDL rats were all prevented by chronic

administration of r-VEGF-A to BDL + HAL rats.

After BDL, the intrahepatic biliary epithelium undergoes cholangiocyte proliferation (4-

6, 25), which leads to bile duct mass expansion, which is followed by an adaptive

proliferation of the PBP (23). Proliferating bile ducts are characterized by enhanced

cholangiocyte secretory and proliferative activities (5). Therefore, the adaptive

proliferation of the PBP (and its circulating factors including VEGF) is fundamental to

sustain the enhanced functional and nutritional demands of the proliferating biliary tree.

Since proliferation of the PBP follows in order of time the proliferation of bile ducts (23),

it is reasonable to suppose that proliferating cholangiocytes modulate the adaptive

response of the vascular bed. Consistently, proliferating cholangiocytes express VEGF-A

and secrete VEGF (21), which modulates cholangiocyte proliferation by autocrine

mechanisms. The fact that: (i) hepatocytes express and secrete VEGF (35, 49): and (ii)

hepatocyte VEGF secretion is decreased by HAL and maintained by administration of r-

VEGF-A raises the possibility that cholangiocyte growth may be also regulated by a

paracrine mechanism. However, we have demonstrated that: (i) hepatocyte VEGF

secretion decreases following BDL and it is much lower than cholangiocyte VEGF

secretion; and (ii) the proliferation/apoptosis of hepatocytes is similar in BDL rats and in

Page 16 of 45

Gaudio et al. 17

rats that (immediately after BDL + HAL) were treated by IP implanted Alzet osmotic

minipumps with BSA or r-VEGF-A for 1 week. Taken together, we propose that

regulation of BDL cholangiocyte apoptosis/proliferation occurs mainly by an autocrine

mechanism (by changes in cholangiocyte VEGF secretion), although a paracrine

mechanism (by hepatocyte or vascular VEGF secretion) cannot be ruled out completely

by this study.

We submitted BDL rats to hepatic artery ligation and evaluated the pathophysiology of

the intrahepatic bile duct system at the morphological and functional levels in comparison

with BDL and BDL + HAL rats. Interestingly, VEGF-A expression and secretion in

cholangiocytes was decreased by HAL. On the basis of this finding, we evaluated

whether the effects of HAL on BDL rats are prevented by chronic in vivo administration

of r-VEGF-A. The dose of VEGF used in our studies is similar to the concentration of

VEGF found in the serum of rats and human (12, 52).

The decreased VEGF expression and secretion by BDL cholangiocytes was an

unexpected finding since, in different tissues, VEGF is usually induced by ischemia (42).

However, VEGF is expressed at low levels by quiescent non-proliferating cholangiocytes

of normal rat liver (21) but markedly expressed and secreted by cholangiocytes

proliferating following BDL (21). This suggests that interruption of hepatic artery blood

supply to proliferating bile ducts, characterized by the vanishing of the peribiliary plexus,

as demonstrated by vascular corrosion cast, compromises VEGF protein synthesis in

cholangiocytes, which is associated with impaired proliferation and with activation of

apoptosis. However, the fact that the reduced VEGF expression and secretion by BDL-

cholangiocytes plays a role in HAL-induced impairment of proliferative and secretory

Page 17 of 45

Gaudio et al. 18

activities of BDL rats was suggested by the findings that all the effects of HAL on

cholangiocyte function of BDL rats were prevented by chronic in vivo r-VEGF-A

administration. Furthermore, the administered dose of r-VEGF-A induced serum levels

of VEGF, which was similar to control BDL rats, both being higher with respects to BDL

rats submitted to HAL.

Interestingly, we found that in BDL rats HAL induced a decrease in the degree of

necrosis, portal inflammation (presumably due to decreased cholangiocyte cytokine

secretion) (19) and peri-ductular fibrosis (may be due to decreased cholangiocyte

secretion of specific growth factors including platelet-derived growth factor) (27). This

finding should be interpreted as a consequence of the reduced cholangiocyte proliferation

following HAL, since portal inflammation and peri-ductular fibrosis in the BDL model

are triggered by cholangiocyte proliferation via secretion of a number of

cytokines/chemokines able to activate stellate cells (27, 44).

HAL induced no effect in the liver of normal rats confirming findings from several

previous studies (16, 17, 30). This has been attributed to accessory arteries or to

anastomosis between the PBP and the portal system, which, if necessary, may overcome

interruption of the blood supply through the main hepatic artery (16, 17, 30). Evidently,

when the intrahepatic bile duct mass is expanded as occurs after BDL (5), blood supply

through the hepatic artery becomes fundamental in sustaining the enhanced nutritional

and functional demands of proliferating intrahepatic biliary epithelium (23). Regarding

the deleterious effects of HAL on BDL cholangiocyte function, VEGF seems to play a

role although we cannot exclude that decreased VEGF expression and secretion is a

consequence of impaired proliferation caused by decreased blood supply. However,

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Gaudio et al. 19

since VEGF is a player in the complex loop of agents sustaining cholangiocyte

proliferation after BDL (6, 37), the impaired synthesis and release of VEGF by

cholangiocytes after HAL certainly has a role in compromising cell proliferation. In

support of this, we have shown (21) that VEGF stimulates cholangiocyte proliferation.

Furthermore, we found that HAL-induced impairment of proliferation is associated with

decreased basal and secretin induced bile flow and bicarbonate secretion, all these effects

being prevented by r-VEGF-A administration. This further supports the role of VEGF in

mediating the effects of HAL on the functions of the intrahepatic biliary epithelium. In

cholangiocytes from HAL + BDL rats, we also observed a decreased level of basal and

secretin-induced cAMP levels that were normalized by r-VEGF-A in vivo administration.

The second messenger, cAMP, plays an important role in modulating cholangiocyte

growth (20, 25, 36). Furthermore, cAMP-related intracellular pathways are

activated/deactivated by different agents involved in the regulation of cholangiocyte

proliferation including the cholinergic system, gastrin, somatostatin and bile salts

including taurocholate and ursodeoxycholate (1, 4, 25, 36). For example, intracellular

cAMP levels are elevated in BDL cholangiocytes compared to normal cholangiocytes (4,

7). Chronic in vivo administration of forskolin, a cAMP activator (33), increases

cholangiocyte proliferation and secretin-stimulated ductal secretion in normal rats (20).

Thus, a complex loop of neuropeptides, hormones and growth factors, which potentiate

each other, sustains cholangiocyte proliferation after BDL (6) and in this loop VEGF is

an important player. Evidently, fall of VEGF after HAL compromises the global

function of the proliferative machinery, including the function of agents acting by cAMP-

related pathways. Thus, the decrease of cAMP levels found in HAL+BDL rat

Page 19 of 45

Gaudio et al. 20

cholangiocytes is consistent with impaired proliferation and increased apoptosis. While

increased basal and secretin-stimulated cholangiocyte secretory activity is coupled with

enhanced ductal hyperplasia (4, 5, 8, 10, 38), impaired ductal secretion is associated with

conditions causing reduction of cholangiocyte proliferation (1, 25, 36, 39). In agreement

with this study, HAL-induced impairment of proliferation is associated with decreased

basal and secretin-induced ductal secretion, all these effects being prevented by VEGF.

After liver transplantation, ischemic lesions of bile ducts may occur presumably by

surgical lesions of hepatic artery (13, 15, 47). In cholangiopathies, mainly primary

sclerosing cholangitis, lesion of hepatic artery and its branches play a causal role in bile

duct damage and in the related ductal cholestasis (13). Our study gives the

pathophysiological basis for these cholestatic conditions since reduction of the blood

supply through the hepatic artery impairs the proliferative and the repairing capacities of

damaged ducts. The beneficial effects of VEGF may provide the experimental

background for using this growth factor in the management of cholangiopathies.

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Gaudio et al. 28

LEGENDS

Figure 1 [a] Light microscopy of liver sections (stained with H&E) from 1 week

BDL rats, and rats that (immediately after BDL + HAL) were treated by IP implanted

Alzet osmotic minipumps with 0.2% BSA or r-VEGF-A with 0.2% BSA for 1 week.

There were no significant differences in the amount of lobular damage in liver sections

from 1 week BDL rats, and rats that (immediately after BDL + HAL) were treated by IP

implanted Alzet osmotic minipumps with 0.2% BSA or r-VEGF-A for 1 week (for

quantitative data see Table 1). In BDL + HAL rats, the degree of necrosis and portal

inflammation decreases compared to BDL rats (for quantitative data see Table 1).

Following the administration of r-VEGF-A to BDL + HAL rats, the degree of necrosis

and portal inflammation was similar than that of the BDL rat (for quantitative data see

Table 1). Orig. magn., x80. [b] Light microscopy of liver sections (Masson’s stain) from

1 week BDL rats, and rats that (immediately after BDL + HAL) were treated by IP

implanted Alzet osmotic minipumps with 0.2% BSA or r-VEGF-A with 0.2% BSA for 1

week. The % volume fraction of peri-ductular fibrosis observed in BDL rats was reduced

in BDL+HAL rats compared to the value of BDL rats. Following the administration of r-

VEGF-A to BDL + HAL rats, the % volume fraction was similar than that of the BDL rat

(for quantitative data see Table 1). Orig. magn., x20.

Figure 2 Scanning electron microscopy vascular corrosion cast from rats (that

immediately after BDL + HAL) were treated by IP implanted Alzet osmotic minipumps

with 0.2% BSA or r-VEGF-A with 0.2% BSA for 1 week. Observe in BDL+HAL rats

Page 28 of 45

Gaudio et al. 29

the presence of sinusoidal network (S) and portal vein (P) and the absence of peribiliary

plexus (orig. magn. 50X); differently, in BDL + HAL+ r-VEGF-A rats the peribiliary

plexus (PBP) that run together with portal tract was observed (orig. magn. 100X).

Figure 3 Immunohistochemistry for [a] VEGF-A and [b] VEGFR-2 and VEGFR-3

in liver sections from 1 week BDL rats and rats that (immediately after BDL + HAL)

were treated by IP implanted Alzet osmotic minipumps with 0.2% BSA or r-VEGF-A

with 0.2% BSA for 1 week. [a] Bile ducts from BDL rats express VEGF-A. The

administration of r-VEGF-A to BDL + HAL rats prevented the decrease in cholangiocyte

VEGF-A expression observed in BDL + HAL rats. Orig. magn. 40X. [b] Following the

administration of r-VEGF-A to BDL+HAL rats, the expression of VEGFR-2 and

VEGFR-3 was similar to that of the BDL rat alone. VEGFR-1 was not expressed by

cholangiocytes. Orig. magn. 40X. For statistical evaluation of the number of VEGF-A-,

VEGFR-2 and VEGFR-3-positive cholangiocytes see Table 2.

Figure 4 VEGF secretion in primary cultures (6 hours) of [top] cholangiocytes or

[bottom] hepatocytes from normal and 1 week BDL rats, and rats that (immediately after

BDL + HAL) were treated by IP implanted Alzet osmotic minipumps with 0.2% BSA or

r-VEGF-A (2.5 nmol/kg/hour with 0.2% BSA) for 1 week. Primary cultures (i.e. 6

hours) of normal rat hepatocytes and cholangiocytes secrete VEGF. Following BDL,

there was an increase in VEGF secretion in primary cultures of cholangiocytes. VEGF

secretion significantly decreased in BDL hepatocytes compared to normal hepatocytes.

In both cholangiocytes and hepatocytes from BDL+HAL rats, there was a marked

Page 29 of 45

Gaudio et al. 30

decrease of VEGF secretion compared with cholangiocytes and hepatocytes from control

1-week BDL rats. Administration of r-VEGF-A prevented the decrease of cholangiocyte

VEGF secretion induced by HAL in cholangiocytes and hepatocytes. Data are mean ±

SE of 7 (cholangiocytes) and 4 (hepatocytes) experiments. [top] *p < 0.05 vs. VEGF

secretion of cholangiocytes from BDL rats. #p < 0.05 vs. VEGF secretion of normal

cholangiocytes. [bottom] *p < 0.05 vs. VEGF secretion of hepatocytes from BDL rats.

#p < 0.05 vs. VEGF secretion of normal hepatocytes.

Figure 5 Measurement of cholangiocyte apoptosis in liver sections by TUNEL

analysis from 1 week BDL, and rats that [immediately after BDL or BDI + HAL) were

treated by IP implanted Alzet osmotic minipumps with 0.2% BSA, or r-VEGF-A (2.5

nmol/kg/hour with 0.2% BSA) for 1 week. TUNEL analysis showed only a few

apoptotic bodies in the liver sections of 1 week BDL rats. The number of cholangiocytes

undergoing apoptosis increased in liver sections from BDL + HAL compared to 1 week

BDL rats. Chronic administration of r-VEGF-A prevented the increase in cholangiocyte

apoptosis (by TUNEL analysis) induced by HAL. Data are mean ± SE of 8 values. *p <

0.05 vs. cholangiocyte apoptosis of all the other groups.

Figure 6 Measurement of cholangiocyte proliferation by quantitative measurement

of the % of CK-19-positive ducts in liver sections from 1 week BDL rats and rats that

[immediately after BDL + HAL) were treated by IP implanted Alzet osmotic minipumps

with 0.2% BSA or r-VEGF-A (2.5 nmol/kg/hour with 0.2% BSA) for 1 week. Following

HAL, the % of CK-19- positive ducts decreased in liver sections compared to liver

Page 30 of 45

Gaudio et al. 31

sections from 1 week BDL rats. Chronic administration of r-VEGF-A prevented the

inhibitory effect of HAL on the % of CK-19-positive ducts, a value that was not

statistically different from that of 1 week BDL rats. For statistical evaluation of the % of

CK-19-positive ducts see Table 4.

Figure 7 Measurement of basal and secretin-stimulated cAMP levels in purified

cholangiocytes from 1 week BDL, and rats that [immediately after BDL + HAL) were

treated by IP implanted Alzet osmotic minipumps with 0.2% BSA, or r-VEGF-A (2.5

nmol/kg/hour with 0.2% BSA) for 1 week. Secretin increased intracellular cAMP levels

of cholangiocytes from 1-week BDL rats. HAL significantly reduced basal

cholangiocyte cAMP levels and inhibited secretin-stimulated cAMP levels of

cholangiocytes compared to cholangiocytes from 1 week BDL rats. Chronic

administration of r-VEGF-A prevented the inhibition of basal and secretin-stimulated

cAMP levels induced by HAL. Data are mean ± SE of 7 experiments. *p < 0.05 vs. its

corresponding basal value. #p < 0.05 vs. basal cAMP levels of cholangiocytes from BDL

rats and BDL + HAL rats treated with r-VEGF-A for 1 week.

Page 31 of 45

Table 1 H&E staining of liver sections from 1 week BDL rats and rats that

(immediately after BDL + HAL) were treated by IP implanted Alzet osmotic minipumps

with 0.2% BSA or r-VEGF-A with 0.2% BSA for 1 week.

Treatment PortalInflammation

Necrosis LobularDamage

Peri-ductularFibrosis

(% volumefraction)

BDL 1 week 1.7 ± 0.1 1.3 ± 0.1 1.5 ± 0.1 12.8 ± 0.8

BDL + HAL +0.2% BSA

1 week

1.2 ± 0.1* 0.7 ± 0.1* 1.2 ± 0.1 7.9 ± 0.7*

BDL + HAL +r-VEGF-A

1 week

1.4 ± 0.1 1.0 ± 0.1 1.3 ± 0.1 14.1 ± 1.0

BDL = bile duct ligated; HAL = hepatic artery ligation. There were no significant

differences in the extent of lobular damage in liver sections (4 µm thick) from the

different groups of animals. In BDL + HAL rats, the degree of necrosis, portal

inflammation and peri-ductular fibrosis decreased compared to BDL rats. Following the

administration of r-VEGF-A to BDL + HAL rats, the degree of necrosis, portal

inflammation and peri-ductular fibrosis were similar than that of the BDL rat. Data are

mean ± SEM of 17 values from the evaluation of 3 slides for each group. *p<0.05 vs. all

the other groups.

Page 32 of 45

Table 2 Immunohistochemical evaluation of cholangiocyte VEGF-A, VEGFR-1,

VEGFR-2 and VEGFR-3 expression in liver sections from 1 week BDL rats, and rats that

(immediately after BDL + HAL) were treated by IP implanted Alzet osmotic minipumps with

0.2% BSA or r-VEGF-A with 0.2% BSA for 1 week.

Treatment VEGF-Aexpression(% positive

cholangiocytes)

VEGFR-1expression(% positive

cholangiocytes)

VEGFR-2expression(% positive

cholangiocytes)

VEGFR-3expression(% positive

cholangiocytes)

BDL 1 week(n = 6)

78.3 ± 3.9 Negative 46.2 ± 3.2 49.4 ± 2.9

BDL + HAL+ 0.2% BSA

1 week(n = 6)

23.1 ± 2.7* Negative 25.6 ± 1.8* 10.5 ± 2.1*

BDL + HAL+ r-VEGF-A

1 week(n = 6)

74.9 ± 2.9# Negative 48.1 ± 3.7 81.6 ± 3.2#

BDL = bile duct ligation; HAL = hepatic artery ligation. The immunohistochemical

cholangiocyte expression of VEGF-A, VEGFR-2 and VEGFR-3 decreased in liver sections

from BDL + HAL rats compared to BDL rats and BDL + HAL rats treated with r-VEGF-A.

Chronic administration of r-VEGF-A prevented HAL-induced loss of cholangiocyte

expression of VEGF-A, VEGFR-2 and VEGFR-3, values that were similar or higher

(#p<0.05) to that of liver sections from 1 week BDL rats. Cholangiocytes did not express

VEGFR-1. *p<0.05 vs. all the other groups. #p<0.05 vs. corresponding value of 1 week

BDL rat liver sections.

Page 33 of 45

Table 3 Measurement of hepatocyte VEGF-A

protein expression in liver sections from normal and 1

week BDL rats and rats that (immediately after BDL +

HAL) were treated by IP implanted Alzet osmotic

minipumps with 0.2% BSA or r-VEGF-A with 0.2% for 1

week.

Treatments VEGF-A(% positive hepatocytes)

Normal(n = 6)

20.3 ± 1.7

BDL 1 week(n = 6)

24.5 ± 2.1

BDL + HAL +0.2% BSA 1 week

(n = 6)

13.2 ± 1.6*

BDL + HAL +r-VEGF-A 1 week

(n = 6)

28.5 ± 2.5

BSA = bovine serum albumin; HAL = hepatic artery

ligation. Hepatocyte VEGF-A protein expression did not

increase significantly in BDL liver sections compared to

normal sections, decreased after HAL and returned to

values similar to those of BDL rats in BDL + HAL rats

treated with r-VEGF-A. *p<0.05 vs. corresponding value

of liver sections from 1 week BDL rats and BDL + HAL

rats.

Page 34 of 45

Table 4 Measurement of cholangiocyte proliferation by quantitative

measurement of the number of PCNA-positive cholangiocytes and the % of

CK-19- and -GT-positive ducts in liver sections from 1 week BDL, and rats

that (immediately after BDL + HAL) were treated with 0.2% BSA or r-

VEGF-A with 0.2% BSA for 1 week.

TreatmentPCNA-positivecholangiocytes

(per portal tract)(n = 12)

% CK-19-positiveducts

(per portal tract)(n = 10)

% -GT-positiveducts

(per portal tract)(n = 14)

BDL 1 week 16.4 ± 1.2 5.6 ± 0.6 7.2 ± 0.5

BDL + HAL+ 0.2% BSA

1 week

4.6 ± 05* 3.7 ± 0.4* 1.1 ± 0.1*

BDL + HAL+ r-VEGF-A

1 week

11.7 ± 0.6 6.4 ± 0.6 5.6 ± 0.5

Following HAL, the number of PCNA-positive cholangiocytes and the % of

CK-19 and -GT-positive ducts decreased in liver sections compared to liver

sections from BDL rats and BDL + HAL rats treated with r-VEGF-A.

Chronic administration of r-VEGF-A prevented the inhibitory effect of HAL

on the number of the number of PCNA-positive cholangiocytes and the % of

CK-19 and -GT-positive ducts, values that were not statistically different

from those of 1 week BDL rats. Data are mean ± SE of 8 values. *p < 0.05

vs. all other groups.

Page 35 of 45

Table 5 Measurement of basal and secretin-stimulated bile flow, bicarbonate concentration and secretion in

1-week BDI rats and rats that (immediately after BDI + hepatic artery ligation) were treated by IP implanted Alzet

osmotic minipumps with 0.2% BSA or r-VEGF-A for 1 week.

Bile Flow Bicarbonate Concentration Bicarbonate Secretion

Treatment Basal(µl / min /Kg BW)

Secretin(µl / min / Kg

BW)

Basal(mEq / Liter)

Secretin(mEq / Liter)

Basal(µEq / min /

Kg BW)

Secretin(µEq / min /

Kg BW)

BDI(n = 8)

98.6 ± 6.5 138.7 ± 6.5# 38.1 ± 1.8 55.0 ± 3.4# 3.7 ± 0.3 7.6 ± 0.7#

BDI + HAL +0.2% BSA

(n = 8)

96.6 ± 12.7 111.8 ± 6.7ns 39.5 ± 1.4 42.4 ± 1.5ns 3.8 ± 0.5 4.7 ± 0.4ns

BDI + HAL +r-VEGF-A

(n = 6)

82.8 ± 8.7 122.1 ± 7.5# 44.88 ± 2.1 53.4 ± 4.1# 3.6 ± 0.3 6.4 ± 0.3#

BDI – bile duct incannulation; HAL = hepatic artery ligation. When steady spontaneous bile flow was reached (60-

70 minutes from the infusion of Krebs Ringer Henseleit (KRH), rats were infused for 30 minutes with secretin

followed by a final infusion of KRH for 30 minutes. After the rats were surgically prepared for bile flow

experiments, bile was collected every 10 minutes in pre-weighed tubes and used for determining bicarbonate

concentration. Data are mean ± SEM. #p < 0.05 vs. their corresponding basal values of bile flow, bicarbonate

concentration or bicarbonate secretion. nsvs. corresponding basal value of bile flow, bicarbonate concentration or

bicarbonate secretion of BDI rats. Differences between groups were analyzed by the Student unpaired t test when

two groups were analyzed and analysis of variance (ANOVA) when more than two groups were analyzed.

Page 36 of 45

Figure 1 a

BDL BDL + HAL BDL + HAL + r-VEGF-A

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BDL BDL+HAL BDL+HAL+r-VEGF-A

Figure 1 b

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BDL + HAL BDL + HAL + r-VEGF-A

Figure 2

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Figure 3 a

BDL BDL+HAL BDL+HAL+r-VEGF-A

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