Deletion of myosin light chain kinase in endothelial cells has a minor effect on the...

Deletion of myosin light chain kinase in endothelial cellshas a minor effect on the lipopolysaccharide-inducedincrease in microvascular endothelium permeability inmiceYang Yu1,2, Ning Lv1,2, Zheng Lu1, Yan-Yan Zheng1, Wen-Cheng Zhang2, Chen Chen2,Ya-Jing Peng2, Wei-Qi He2, Fan-Qing Meng3, Min-Sheng Zhu2 and Hua-Qun Chen1

1 Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, China

2 Model Animal Research Center and MOE Key Laboratory of Model Animal for Disease Study, Nanjing University, China

3 Department of Pathology, Drum Tower Hospital, Affiliated to the Medical School of Nanjing University, China

Keywords

endothelial cell; knockout; lipopolysaccharide

(LPS); myosin light chain kinase (MLCK);

permeability

Correspondence

H. Q. Chen, Jiangsu Key Laboratory for

Molecular and Medical Biotechnology,

College of Life Sciences, Nanjing Normal

University, Nanjing 210046, China

Tel: +86 25 85891353

E-mail: [email protected]

M. S. Zhu, Model Animal Research Center

and MOE Key Laboratory of Model Animal

for Disease Study, Nanjing University,

Nanjing 210061, China

Tel: +86 25 58641529

E-mail: [email protected]

(Received 2 August 2011, revised 20

January 2012, accepted 16 February 2012)

doi:10.1111/j.1742-4658.2012.08541.x

There is a current view that myosin light chain kinase (MLCK) plays a

critical role in endothelial permeability. To investigate the functions of

MLCK in endothelial cells in vivo, we generated a mouse model in which

MLCK was selectively deleted by crossing Mylk1 floxed mice with Tie2 ⁄ cretransgenic mice. Knocking out Mylk1 from endothelial cells had no effect

on the global phenotype of the mice, including body weight and blood

pressure. Lipopolysaccharide (LPS)-mediated septic death was also not

altered in the knockout (KO) mice. Consistently, LPS-induced inflamma-

tory injury and the increase in microvascular permeability in the main

organs, including the lung and the kidney, was not significantly attenuated

in KO mice as compared with wild-type mice. However, the LPS-induced

microvascular hyperpermeability of the esophagus and the eyeballs was

attenuated in KO mice. We also found that the LPS-mediated increase in

the number of caveolae in the endothelial cells of the esophagus was signifi-

cantly reduced in KO mice. Our results do not support a role for endothe-

lial cell MLCK in the pathogenesis of inflammatory diseases.

Introduction

The vascular endothelium forms a semipermeable bar-

rier between the bloodstream and interstitial space

[1,2]. The intact endothelial barrier is restrictive to cells,

proteins, fluid, and solutes, thus maintaining vascular

homeostasis and the physiological functions of different

organs [3]. In the development of a variety of inflam-

matory diseases, such as sepsis and atherosclerosis, the

endothelium is disturbed, resulting in microvascular

hyperpermeability [4]. Fluid, molecules and cells move

across the endothelium via a transcellular or a para-

cellular pathway. Small molecules are transported

through a structural junction between adjacent endo-

thelial cells (ECs) by a paracellular pathway. Large

molecules, e.g. albumin, are transported across the

Abbreviations

EBD, Evans blue dye; EC, endothelial cell; H&E, hematoxylin and eosin; IF, immunofluorescence; KO, knockout; L-MLCK, long form of

myosin light chain kinase; LPS, lipopolysaccharide; MLC, myosin light chain; MLCK, myosin light chain kinase; S-MLCK, short form of

myosin light chain kinase; WT, wild-type.

FEBS Journal 279 (2012) 1485–1494 ª 2012 The Authors Journal compilation ª 2012 FEBS 1485

endothelium through the caveolae by a transcytosis

pathway [5].

Inflammatory mediators, such as lipopolysaccharide

(LPS), cytokines, and thrombin, are capable of binding

to their receptors on ECs, leading to disruption of

the junctions, the formation of gaps between adjacent

cells, and, finally, an increase in endothelial perme-

ability and endothelial dysfunction [6]. During this

process, a change in cell shape caused by actomyosin

contraction is speculated to be the primary determi-

nant of increased endothelial permeability [7]. Acto-

myosin contraction is primarily mediated by myosin

light chain (MLC) phosphorylation, which activates

myosin, leading to cross-bridge movement [8,9]. MLC

kinase (MLCK) is a dedicated kinase for MLC phos-

phorylation, and is expressed in ECs [10,11]. MLCK

is considered to be important for the regulation of

endothelial permeability in response to LPS [12,13].

Therefore, it is expected to be a target for pharma-

ceutical intervention against endotoxic sepsis and

other diseases associated with the increase in endothe-

lial permeability.

MLCK activates myosin Mg-ATPase and initiates

actomyosin contraction, thereby functioning in many

cell biological processes [14]. There are at least three

MLCK genes. Mylk1 encodes two isoforms of the

220-kDa MLCK [long MLCK (L-MLCK) and

MLCK210], a 130-kDa MLCK (short MLCK

(S-MLCK)], and telokin. Mylk2 encodes a skeletal

muscle-specific isoform [15,16], and Mylk3 encodes a

cardiac isoform [17]. In adult cells, MLCK210 is

prominently expressed in nonmuscular lineages, includ-

ing ECs, epithelial cells, and neutrophils [11,18–20].

Previous studies have shown that inflammatory medi-

ators activate MLCK in ECs and cause EC contrac-

tion, resulting in barrier dysfunction and endothelial

hyperpermeability [21–24]. This conclusion was sup-

ported by observations made in global L-MLCK

knockout (KO) mice (MLCK210) ⁄ )); the mutation

was shown to have a protective effect against the

inflammatory injury and the increase in lung micro-

vascular permeability induced by LPS [12]. To specifi-

cally elucidate the in vivo functions of MLCK in the

endothelium, we generated a line with a conditional

deletion of Mylk1 in ECs by crossing Mylk1 floxed

mice with Tie-2 ⁄Cre transgenic mice [25]. We found

that deletion of Mylk1 from ECs had no obvious

effect on mouse development, as indicated by the nor-

mal phenotype of KO mice in comparison with that

of wild-type (WT) mice. To our surprise, no apparent

protective effects against LPS-induced septic death

and inflammatory injury in the main organs, including

the lung, liver, and kidney, were observed in KO mice

as compared with WT mice. No significant differences

were seen in LPS-mediated microvascular hyperperme-

ability in the main organs between KO and WT mice.

We found that only increased microvascular leakage

in the esophagus and the eyeballs was attenuated in

Mylk1 KO mice. Our data suggest that MLCK may

not be critical in the upregulation of endothelial per-

meability in response to LPS administration, such as

the hyperpermeability seen in sepsis and other inflam-

matory diseases.

Results

Generation of endothelial-specific MLCK KO

mice and detection of the ablation of MLCK

expression in the endothelium

It was first reported that L-MLCK was the predomi-

nant form expressed in ECs [10]. However, both

L-MLCK and S-MLCK have been shown to be

expressed in primary isolated bovine pulmonary artery

ECs, whereas only L-MLCK was expressed in cultured

ECs [26]. To our surprise, we only observed expression

of S-MLCK in fresh isolated mouse endothelium of

arteries (Fig. 1D). Our observation that S-MLCK was

predominantly expressed in ECs in mice was supported

by our further detection of S-MLCK in endothelium

from other species (Fig. S1) and the recent results of

another group [27]. To further understand the functions

of MLCK in endothelial cells in vivo, we completely

ablated the expression of MLCK by deleting both

L-MLCK and S-MLCK in mice. Female Mylk1flox ⁄ flox

mice were crossed with male Mylk1flox ⁄ +; Tie2 ⁄Cremice to produce Mylk1flox ⁄ flox; Tie2 ⁄Cre mice (KO

mice). Deletion of the floxed region and the presence of

the Cre gene were confirmed by PCR analysis from

genomic DNA (Fig. 1A,B). To determine whether the

deletion of a targeted fragment altered MLCK expres-

sion in the endothelium, we first demonstrated that we

had isolated a pure endothelial sheet by a morphology

assay with eosin staining and positive expression of the

EC-specific antibody against CD31 with immunofluo-

rescence (IF) staining (Fig. 1C). To assess the gene KO

efficiency, we determined the expression of MLCK in

the fresh isolated endothelial sheets by western blotting

and semiquantitative RT-PCR. Significant reductions

in both MLCK protein and mRNA in the artery

endothelium were observed (Fig. 1D), and this was

confirmed by IF staining (Fig. 1E).

We next evaluated the specificity of the Tie-2 ⁄Cresystem used in this study by examining the expression

of LacZ in different organs of mice, which were estab-

lished by crossing ROSA26tm1 mice with Tie-2 ⁄Cre

MLCK has a minor effect on microvascular permeability Y. Yu et al.

1486 FEBS Journal 279 (2012) 1485–1494 ª 2012 The Authors Journal compilation ª 2012 FEBS

mice according to a previous report [28,29]. The result-

ing double-positive mice showed LacZ expression

when the ‘stop’ signal in b-galactosidase was deleted,

which was revealed through blue staining in the ECs

of the artery, lung tissue, esophagus, and kidney,

indicating positive expression of Cre in the floxed mice

(Fig. 1F). The dark blue signal on the inner surface of

the esophagus in Fig. 1F may be nonspecific staining

of the mucosa substance, which is observed frequently

in control mice.

In this study, the birth of pups, including Mylk1

floxed mice with (Mylk1flox ⁄ flox; Tie2 ⁄Cre and

Mylk1+ ⁄ flox; Tie2 ⁄Cre) or without (Mylk1flox ⁄ flox and

Mylk1+ ⁄ flox) Cre recombinase, occurred in the

expected Mendelian ratio. All of the mice with differ-

ent genotypes reached adulthood without any obvious

abnormalities in body size or behavior. In our sub-

sequent experiments, we used Mylk1+ ⁄ flox; Tie2 ⁄Crelittermates as the WT mice and Mylkflo ⁄ flox; Tie2 ⁄Cremice as the KO mice.

Global phenotypic analysis of the KO mice showed

normal morphology (data not shown) and histology of

the lung, the liver, the kidney, the spleen, the esopha-

gus, and the small intestine (Fig. S1A). The KO mice

showed normal body weights (P > 0.05) (Fig. S1B),

normal blood pressures (KO, 118 ± 20 mmHg; WT,

124 ± 15 mmHg; P > 0.05), normal electrocardio-

grams and normal heart rates as compared with

the WT mice (KO, 560 ± 45 beatsÆmin)1; WT,

556 ± 56 beatsÆmin)1; P > 0.05) (Fig. S1C).

Fig. 1. Targeted disruption of Mylk1 in the endothelium. (A) PCR genotyping for mice. Cre is shown in the left panel (+, Tie-2 ⁄ Cre+, ), WT);

loxP is shown in the right panel (+ ⁄ +, Mylk1+ ⁄ +; + ⁄ ), Mylk1flox ⁄ +; ) ⁄ ), Mylk1flox ⁄ flox). (B) Deletion of Mylk1 in the endothelium as deter-

mined by PCR. The 0.4-kb bands correspond to the deleted allele, and the 1.7-kb bands correspond to the floxed allele. (C) The isolated

sheets were determined to be endothelium by IF staining with antibody against CD31 (green, right) and eosin staining (red, left). Scale bars:

5 lm. (D) Deletion of Mylk1 in the endothelium in KO mice as determined by western blotting (left panel) (n = 5) and semiquantitative

RT-PCR (right panel) (n = 3). MLCK protein expression in mouse lung tissue was used as a positive control. Expression of both MLCK pro-

tein and mRNA in the endothelium in KO mice was aberrant. (E) Deficiency of MLCK protein in the artery endothelium as determined by IF

staining with a K36 antibody (red). The endothelial layer was confirmed by IF staining with an antibody against CD31 (green). The nucleus

was stained with 4¢,6-diamidino-2-phenylindole (blue). Arrows indicate the endothelium or smooth muscle. EC, endothelium; SM, smooth

muscle. (F) b-Galactosidase activity was shown by blue X-gal staining in the ROSA26R reporter allele activated by the Tie2 ⁄ Cre transgene in

the artery, esophagus, lung, and kidney (CL, capillary loop; G, mature glomeruli, S, S-shaped glomeruli; V, vessel). Arrows or thin lines indi-

cate endothelium. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Y. Yu et al. MLCK has a minor effect on microvascular permeability


Deletion of MLCK in endothelium does not

decrease LPS-induced mortality

To determine the effects of MLCK in the endothelium

on LPS-induced septic death, we treated the mice with

a lethal dose of LPS (25 mgÆkg)1 body weight) and

assessed their survival fraction. As shown in Fig. 2,

there was no significant difference in the survival rate

between the KO (n = 22) and the WT (n = 12)

groups after LPS treatment (P > 0.05, log-rank test),

indicating that the specific deletion of MLCK in the

endothelium does not protect mice from LPS-induced

septic death. One hundred percent of both the WT

mice and KO mice survived for more than 17 h. The

survival time of the KO mice ranged from 18 to 140 h

or longer, and the survival time of the WT mice

ranged from 19 to 140 h or longer.

KO mice are not apparently protected from

LPS-mediated inflammatory injury

The pathological results showed that 16 h of treatment

of LPS at a lethal dose induced serious inflammatory

injury in lung tissue (Fig. 3, upper panel), which was

indicated by interstitial hemorrhage, interstitial infiltra-

tion of inflammatory cells, and a thickened alveolar

wall. In the kidney, there was very slight hemorrhage

in the renal cortex and the medulla in both groups of

mice (Fig. 3, lower panel). We did not observe kidney

tubular dilatation in the mice in response to LPS treat-

ment, in agreement with other studies [30]. Our data

suggest that MLCK in ECs does not play an impor-

tant role in LPS-induced inflammatory injury in lung

and kidney tissues in mice.

MLCK has a minor effect on the LPS-induced

increase in microvascular permeability

LPS-mediated sepsis is characterized by serious micro-

vascular hyperpermeability and edema in multiple

organs. Evans blue dye (EBD) is able to bind to albu-

min, and thereby to cross the endothelium under certain

condition [31,32]. The leakage of EBD is extensively

used as an important index of permeability. We assessed

the microvascular permeability of KO mice by measur-

ing the amount of leakage of EBD and comparing the

wet ⁄dry tissue ratio of mice. We first measured the

amount of EBD leakage from the microvessels in

the main organs of mice with LPS administration. We

found that LPS (10 mgÆkg)1 body weight) mediated a

significant increase in EBD leakage in the liver, small

intestine, eyeballs and esophagus (Fig. 4A–C)

(P < 0.05 or P < 0.01) in WT mice, but not in the lung

and the kidney tissue (Fig. 4A) (P > 0.05). However,

only EBD leakage in the microvessels of the eyeballs

and esophagus was significantly attenuated by MLCK

deletion in the ECs in KO mice as compared with WT

mice (P < 0.01) (Fig. 4B,C). In this study, we did not

find an apparent increase in EBD leakage in the lung tis-

Fig. 2. Survival rate of mice in response to LPS. KO mice (n = 22)

and WT mice (n = 12) (20 ± 1 g, 9 weeks old) were treated with

an intraperitoneal injection of LPS (25 mgÆkg)1 body weight), and

the survival time for each mouse was recorded. The survival rate

was expressed as a percentage of surviving mice over total mice.

A B C

D E F

A B C

D E F

Fig. 3. LPS-induced inflammatory injury as shown by H&E staining.

The lungs and the kidneys of KO (n = 3) or WT (n = 3) mice were

isolated, fixed in formalin, and embedded in paraffin. The histologi-

cal assay was performed by H&E staining. Inflammatory injury in

lung tissues resulting from LPS exposure, as indicated by interstitial

hemorrhage, interstitial infiltration of inflammatory cells, and a thick-

ened alveolar wall, were shown in both KO and WT mice (upper

panel) [(A, D) WT mice treated with saline; (B, E) WT mice treated

with LPS; (C, F) KO mice treated with LPS]. Subtle hemorrhaging

in the cortical part (B, C) and the modular part (E, F) of the LPS-

treated kidneys in both KO and WT mice is shown (lower panel).



sue with LPS treatment by intraperitoneal injection. The

reason was not known. To eliminate possible errors in

the experiment, we performed the same assay in the lung

tissues of the mice with intratracheal administration of

LPS (2.5 mgÆkg)1 body weight). After LPS treatment

and EBD injection, the color of lung tissue of WT mice

became obviously blue, and a dramatic increase in EBD

leakage in the lung tissues was detected as compared

with the mice treated with saline (P < 0.01), indicating

LPS-induced hyperpermeability in the endothelium in

these mice. No obvious difference in the EBD leakage in

microvessels was seen in the lungs between WT mice

and KO mice (Fig. 4D).

We next evaluated the severity of edema in the main

organs in response to LPS treatment. The wet ⁄dryratio was apparently elevated in the lung, esophagus,

small intestine and liver tissues in mice after 6 h of

LPS treatment as compared with the control saline

treatment groups (P < 0.05) (Fig. 4E). There was no

apparent increase in the wet ⁄dry ratio in the kidneys

(P > 0.05). Consistent with the EBD leakage results,

there was no significant difference in edema mediated

by LPS in the main organs between WT mice and KO

mice (P > 0.05), except for the esophagus (P < 0.05).

However, we did not examine edema in the eyeballs,

because this was difficult to perform.

Taken together, our findings show that deletion of

MLCK in endothelial cells was not able to attenuate

the LPS-induced increase in endothelial permeability in

the intestine, lung, liver and kidney in mice, but was

able to in the eyeballs and esophagus, showing a minor

effect of endothelial MLCK on LPS-associated patho-

physiological changes in mice.

MLC phosphorylation level in KO endothelium

was not altered

As MLC is the only substrate of MLCK, we next

measured the phosphorylated and total MLC in the

mutant endothelium. The relative amount of MLC

Fig. 4. LPS-mediated endothelium hyperpermeability. (A, B) After treatment with LPS, the amount of EBD extravasation across the endothe-

lium was significantly increased in the esophagus (P < 0.01), small intestine, liver (P < 0.05), and eyeballs (P < 0.01). There was no apparent

difference between KO mice and WT mice, except in the esophagus and eyeballs. (C) Obvious microvascular EBD leakage in the esophagus

in mice, as indicated by the blue color. (D) Tracheal injection of LPS induced severe acute lung injury, as indicated by EDB leakage in lung

tissues. (E) LPS induced a significant increase in endothelial permeability, as assessed by the wet ⁄ dry ratio in WT mice (P < 0.05). There

was no apparent difference in the main organs between KO mice (n = 6) and WT mice (n = 6), except for the esophagus. *P < 0.05;

**P < 0.01.



phosphorylation in the endothelium isolated from the

mutant mice without LPS treatment was about 86% of

that in the control (85.7 ± 6.7% versus 100 ± 8.7%,

n = 3, P > 0.05); the amount of MLC phosphoryla-

tion in the endothelium isolated from the KO mice with

pretreatment with LPS was comparable to that in the

control (100 ± 34% versus 100 ± 5.5%, n = 3,

P > 0.05). Unexpectedly, under our experimental con-

ditions, LPS pretreatment did not increase the MLC

phosphorylation level of either mutant or control endo-

thelium. This may be an effect of mechanical manipula-

tion of the endothelium. Figure 5 shows typical results

for MLC phosphorylation.

Deletion of MLCK attenuates the increase in

caveolae number in the esophageal endothelium

in response to LPS challenge

Genetic KO of L-MLCK has been reported to prevent

epithelial tight junction disruption and protein leakage

[18]. We next examined the ultrastructure of the endo-

thelium of different tissues in both WT and KO mice.

With or without LPS treatment, no apparent alteration

in endothelial tight junction structure in lung tissue or

esophagus tissue was observed in KO mice as com-

pared with WT mice (Fig. 6). Caveolae-mediated trans-

cytosis of acute-phase plasma proteins, such as

albumin, across the endothelium has been reported to

contribute to the regulation of LPS-induced microvas-

cular permeability [33,34]. We found that the number

of caveolae in esophageal ECs in WT mice with LPS

challenge was obviously greater than that in KO mice

(KO, 16.47 ± 1.32 per lm2; WT, 22.48 ± 1.25 per

lm2; P < 0.001). However, we did not find apparent

caveolae in the ECs in the lung tissues.

Discussion

Several studies using in vitro models and pharma-

ceutical inhibitors have suggested that endothelial

permeability may be regulated by MLCK [21–23]. As

ECs have been previously reported to express exclu-

sively L-MLCK in vitro, and specific deletion of

L-MLCK in mice prevented LPS-mediated endothelial

permeability and inflammatory injury, there is a cur-

rent view that MLCK in endothelium is a critical regu-

lator of endothelial barrier function [10,12,21–24,26].

However, in this study, we found that mice with spe-

cific deletion of total MLCK (both long and short) in

endothelium appeared to have normal physiological

phenotypes. No protective effect of MLCK against

LPS-induced lethality and inflammatory injury was

found in endothelium-specific gene KO mice. We also

found that the LPS-induced increase in microvessel

endothelial permeability was significantly attenuated

only in the esophagus and eyeballs in KO mice, and

not in other main organs, such as the liver, lung, and

kidney. Therefore, we argue that MLCK is required

for the maintenance of normal physiological functions

of endothelium, and our data suggest that it has only

a minor effect on endothelial permeability regulation

in response to LPS stimulation. The difference between

previous conclusions and ours may be attributable to

the following factors: first, the MLCK inhibitors used

previously are not specific enough [21–24]; second, the

in vitro endothelial barrier model is different from the

physiological endothelial barrier in vivo; and third,

the endothelium in vivo does not express L-MLCK, as

described in this article, and hence the protection

against LPS in L-MLCK KO mice may be attributable

to nonendothelial cell types (e.g. epithelial cells and

smooth muscle cells around blood vessels).

Despite the minor effect mentioned above, MLCK

deletion was able to greatly affect the microvascular per-

meability of the esophagus and eyeballs. This suggests

that MLCK may regulate endothelial permeability

in vivo in ways that vary with the source of endothelium.

Such differential regulation may be attributable to the

heterogeneity of ECs in different tissues [35,36].

MLCK is a dedicated kinase for MLC phosphoryla-

tion [14]. We found no apparent alteration of MLC

phosphorylation in endothelium with MLCK deletion,

which was consistent with the minor effects on endo-

thelial permeability. This finding also suggests a

compensatory effect of other signaling modules for

MLC phosphorylation. The possible candidates are

RhoA ⁄ROCK modules, which are capable of inhibit-

ing MLC phosphatase activity and hence maintaining

MLC phosphorylation [37–40]. According to our

observations in this study, the overformation of

caveolae in ECs might underlie the inhibition of endo-

thelial permeability in the MLCK-deficient esophagus

and eyeballs [33,34], but more evidence is required.

Fig. 5. MLC phosphorylation in the endothelium. The mice were

intraperitoneally injected with 10 mgÆkg)1 LPS 3 h prior to endothe-

lium isolation. The fresh endothelial layers from WT (n = 3) and KO

(n = 3) mice were sampled [46] for western blot analysis of MLC

phosphorylation. Specific antibodies against MLC and phosphory-

lated MLC (p-MLC) were used as primary antibodies, and total

MLC protein was used as an internal control.



Previous reports have suggested that both conven-

tional deletion of L-MLCK and MLCK inhibitor

could protect mice from LPS-induced and ventilation-

induced acute lung injury, and inhibit the increase in

endothelial permeability [12,41]. Given the critical roles

of MLCK in the regulation of endothelial permeabil-

ity, it was thought to be an attractive drug target for

the development of treatment strategies for related dis-

eases. However, our results suggest that MLCK has

only minor effects on endothelial permeability, and it

may not be a critical drug target in endothelial perme-

ability.

Experimental procedures

Generation of conditional Mylk1-deletion mice

with the Cre ⁄ loxP system

We have established a mouse strain with loxP sites flanking

exons 23–25 of the coding region for the ATP-binding site

of MLCK [42]. To ablate Mylk1 gene expression specifi-

cally in ECs, we crossed female Mylk1flox ⁄ flox mice (back-

crossed to C57BL ⁄ 6 for six generations) with male

Mylk1flox ⁄ +; Tie2 ⁄ Cre mice (Jackson Laboratories, C57BL ⁄ 6background) to produce Mylk1flox ⁄ flox; Tie2 ⁄ Cre (KO) mice.

All of the mice were maintained in the National Resource

Center for Mutant Mice (China). Animal experiments were

conducted in accordance with the Animal Care and Use

Committee of the Model Animal Research Center of

Nanjing University.

Genotyping of the mice

The existence of the Cre gene in Tie2 ⁄Cre crossed

Mylk1flox ⁄ flox mice was examined by PCR amplification

with genomic DNA extracted from mouse tails as a

template. The primer pair was 5¢-TGCCACGACCAAGT

GACAGCAATG-3¢ and 5¢-AGAGACGGAAATCCAT

CGCTCG-3¢. The deletion of the Mylk1 alleles was

confirmed by PCR for the loxP sites. The primer pair was

5¢-TAGTGCGAGTGTCACTGTTG-3¢ and 5¢-TGACTG

GAAAAGGAGCCA-3¢. Deletion of Mylk1 in the ECs

of Mylk1flox ⁄ flox; Tie2 ⁄ Cre mice was determined by PCR

detection of genomic DNA. The primer pair was 5¢-TAG

TGCGAGTGTCACTGTTG-3¢ and 5¢-CCCCATGATTT

GCCTCTAGT-3¢.

Determination of the expression of Cre in

endothelial cells by use of ROSA26R

b-galactosidase reporter staining

ROSA26tm1 reporter mice [29] were purchased from the

Jackson Laboratory and maintained in the National

Resource Center for Mutant Mice. After crossing of these

mice with Tie2 ⁄Cre transgenic mice, the progeny were

genotyped by PCR for Cre and ROSA26tm1 PCR, with

primers described previously [28,29]. The mice were anes-

thetized, and the organs were harvested, frozen directly in a

Tissue-Tek OCT compound, and stored at )80 �C until

cutting. The tissues were cut into 10-lm sections and

stained for b-galactosidase activity with X-gal (MD Bio,

Shanghai), according to a previous report [29].

Preparation of fresh endothelial layer

Approximately 0.5–1 cm of blood vessel was obtained by

cutting the aorta along the longitudinal axis. The endothe-

lial sheet of the aorta was isolated with fine forceps,

and spread out carefully on an APES-pretreated

(Boster, Wuhan, China) glass coverslip under a dissecting

microscope (Leica MZ16F, Wetzlar, Germany).

Characterization of the endothelium by eosin

staining and IF

The endothelial sheet was fixed with 4% paraformaldehyde

and subjected to eosin staining or IF staining. For the IF

staining, the slides were incubated with rat anti-CD31 IgG

Fig. 6. LPS-induced increase in caveolae number in ECs of the esophagus. (A) Electronmicrographs show the ultrastructure (tight junction)

of the endothelium in the lung tissue with or without LPS treatment. (B) Electronmicrographs show the caveolae in the endothelium of the

esophagus in WT mice and KO mice. Arrows and stars indicate the tight junctions and the caveolae (left), respectively. The number of

caveolae was significantly decreased in KO mice (n = 3) as compared with WT mice (n = 3). ***P < 0.001.



(550274; BD Biosciences, Franklin Lakes, NJ, USA) over-

night at 4 �C, and then with secondary antibody [fluores-

cein isothiocyanate-labeled donkey anti-rat IgG (A11006;

Invitrogen, Carlsbad, CA, USA)]. The slide was examined

under a confocal microscope (Leica DMIRE2), and images

were obtained.

Detection of MLCK expression in the artery

endothelium by IF

A fragment of artery from the mice was harvested, frozen

directly in the Tissue-Tek OCT compound, and stored at

)80 �C. The artery was cut into 10-lm sections and stained

with a monoclonal antibody against MLCK (K36; Sigma)

or an antibody against CD31. The slide was examined, and

images were obtained.

Western blot assay

The freshly isolated endothelial sheet was immediately

placed in 2 · SDS sample buffer for preparation of the

whole cell lysate sample. The sample was separated by 6%

SDS ⁄PAGE, and the proteins were transferred to a

poly(vinylidene difluoride) membrane (Millipore). The

membrane was probed with antibody against MLCK, MLC

(kindly supplied by J. T. Stull, UT South Western Medical

Center) or phosphorylated MLC (Sigma), and this was

followed by incubation with a horseradish peroxidase-

conjugated secondary antibody. Signal development was

performed with an ECL chemiluminescence kit (Amersham

Com., Amersham, UK), according to the manual. Optical

densities were analyzed with imagequant software (GE

Healthcare, Chalfont St. Giles, UK).

Semiquantitative RT-PCR

Total RNA of endothelium was extracted with TRIzol

reagent (Invitrogen) and reverse transcribed with Prime-

Script RT reagent kit (TaKaRa, Dalian, China). The

cDNA samples were subjected to semiquantitative PCR for

amplification of the fragment of exon 23–25 of Mylk1

(319 bp). The primer pair was 5¢-GATGAAGTGGAAG-

TGTCCGA-3¢ and 5¢-CCAGAACCATGACAATGTTG-3¢.

LPS administration and analysis of inflammatory

injury

To study the effects of MLCK on inflammatory injury

and on the mortality of severe sepsis in mice, LPS

(0111:B4; Sigma) was administered at a dose of 25 mgÆkg)1

by intraperitoneal injection. The mice were regularly moni-

tored, and the survival time of the mice after LPS injection

was recorded. The organs were harvested, the paraffin-

embedded tissues were prepared, 4-lm sections were cut,

and staining with hematoxylin and eosin (H&E) was

performed. A minimum of five fields at · 40 magnification

were examined for each section. An assessment of hemor-

rhage and inflammation was performed, which included

the presence of red blood cells and inflammatory cells in

the tissues.

Evaluation of microvascular permeability

Microvascular permeability was evaluated by measuring

the leakage of EBD (E2129; Sigma) extravasated from the

microvessel into the peripheral tissues according to previ-

ously described methods [43]. After 6 h of LPS treatment

(10 mgÆkg)1 body weight), EBD was administered

(40 mgÆkg)1 body weight) by tail vein injection. The ani-

mals were anesthetized with 200 mgÆkg)1 pentobarbital by

intraperitoneal injection. The lungs perfused with NaCl ⁄Pi,

the livers, the kidneys and other organs were collected

and homogenized in NaCl ⁄Pi (1 mL per 100 mg of tissue),

incubated with two volumes of formamide for 18 h at

60 �C, and then centrifuged at 5000 g for 30 min. The

A620 nm of the supernatant was determined with a spectro-

photometer. The concentration of leaked EBD in tissues

was calculated against a standard curve (micrograms EBD

per gram tissue).

Determination of wet ⁄ dry ratio

For microvascular hyperpermeability measurement, the

mice were injected in the tail vein with LPS (0.5 mgÆkg)1),

and the control mice were treated with an equivalent vol-

ume of saline. Six hours later, the animals were anesthe-

tized with 200 mgÆkg)1 pentobarbital by intraperitoneal

injection. The wet weight of the organ was determined

immediately after isolation from the mouse body. The dry

weight was measured after heating of the organ at 80 �Cfor 72 h. The wet ⁄ dry ratio was determined by dividing the

wet weight by the dry weight.

Transmission electron microscopy

Tissue samples were fixed in 2.5% glutaraldehyde, postfixed

in 1% osmium tetroxide, dehydrated through graded

acetone solutions, and embedded in Epon 812 (Sigma,

St. Louis, MO, USA). Ultrathin sections were observed

with a transmission electron microscope (Hitachi H-7650,

Hitachi High-technologies, Tokyo, Japan) after staining

with uranyl acetate and lead citrate.

Statistical analysis

Data are presented as the mean ± standard error of the

mean. The significance of the differences between groups

was determined with Student’s t-test.



Acknowledgements

We are grateful to Chang Liu and Shan Lu for critical

reading of this manuscript and helpful comments on it.

This work was supported by the Natural Science

Foundation of China (30971540 and 30570911), the

Natural Science Foundation of Jiangsu Province

(BK2009404), and a project funded by the Priority

Academic Program Development of Jiangsu Higher

Education Institutions (PAPD).

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Supporting information

The following supplementary material is available:

Fig. S1. The global phenotype of KO mice is normal.

Fig. S2. The expression of MLKC in endothelium

from different species.

This supplementary material can be found in the

online version of this article.

Please note: As a service to our authors and readers,

this journal provides supporting information supplied

by the authors. Such materials are peer-reviewed and

may be reorganized for online delivery, but are not

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should be addressed to the authors.



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