ORIGINAL RESEARCH PAPER

The p38 mitogen-activated protein kinases modulate endothelialcell survival and tissue repair

Nobuhiro Kanaji • Amy Nelson • Diane S. Allen-Gipson • Tadashi Sato •

Masanori Nakanishi • Xingqi Wang • YingJi Li • Hesham Basma •

Joel Michalski • Maha Farid • Stephen I. Rennard • Xiangde Liu

Received: 21 April 2011 / Revised: 17 October 2011 / Accepted: 11 November 2011 / Published online: 3 December 2011

� Springer Basel AG 2011

Abstract

Objective and design This study is designed to investi-

gate the role of p38 MAPK in modulating human

pulmonary artery endothelial cells (HPAECs) survival and

tissue repair functions.

Methods HPAECs (passage 8–12) were used for all

experiments. Cells were treated with IL-1b (0.5 or 2 ng/ml)

or p38 inhibitor (SB203580 or SB220025, 5 lM each).

Cells were also transfected with 50 nM siRNAs. Cell

length was measured using ImageJ software. Collagen gel

contraction and wound close assay were performed to

evaluate tissue repair functions.

Results IL-1b activated p38 MAPK and induced

morphologic change of HPAECs. The p38 inhibitors further

augmented IL-1b-induced cell morphologic change, pre-

vented cell death, and augmented collagen gel contraction.

Suppression of p38a, c, or d, but not p38b resulted in cell

morphologic alteration, and suppressing any one of p38

isoforms by siRNAs increased cell survival. Suppression of

p38a or d augmented gel contraction. While p38a sup-

pression stimulated cell migration, suppressing the rest of

three isoforms inhibit cell migration. Nuclear factor p65-

siRNA blocked IL-1b-induced cell morphologic change,

but did not affect p38 inhibitor-induced change.

Conclusion These findings suggest that p38 MAPK may

negatively modulate tissue repair functions of endothelial

cells via p65 independent pathway.

Keywords p38 � Interleukin-1 � Endothelial cells �Repair � Apoptosis

Introduction

Airway inflammation is believed to play an important role

in the development of a variety of lung diseases. Endo-

thelial cells are essential in normal development and

angiogenesis and also play an important role in inflam-

matory responses. In this regard, up-regulation of adhesion

molecules on endothelial cells causes leucocytes to attach

to the endothelium at sites of injuries and infection [1]. The

inflammatory cytokine interleukin (IL)-1b can induce these

endothelial cell responses [1]. IL-1b can also contribute to

development of tissue remodeling and fibrosis by modu-

lating endothelial cell functions [2]. IL-1b induces

endothelial cells to undergo a fibroblast-like morphologic

change and increases resistance to apoptosis [3].

Responsible Editor: Liwu Li.

N. Kanaji

Division of Endocrinology and Metabolism, Hematology,

Rheumatology and Respiratory Medicine, Kagawa University,

Kagawa, Japan

N. Kanaji � A. Nelson � D. S. Allen-Gipson � X. Wang �H. Basma � J. Michalski � M. Farid � S. I. Rennard � X. Liu (&)

Pulmonary, Critical Care, Sleep and Allergy Division,

University of Nebraska Medical Center, 985910 Nebraska

Medical Center, Omaha, NE 68198-5910, USA

e-mail: xdliu@unmc.edu

T. Sato

Department of Respiratory Medicine, Juntendo University

School of Medicine, Tokyo, Japan

M. Nakanishi

Third Department of Internal Medicine, Wakayama Medical

University School of Medicine, Wakayama, Japan

Y. Li

Department of Hygiene and Public Health, Nippon Medical

School, Tokyo, Japan

Inflamm. Res. (2012) 61:233–244

DOI 10.1007/s00011-011-0405-7 Inflammation Research

123

Mitogen-activated protein kinases (MAPKs) regulate

diverse cellular programs including embryogenesis, pro-

liferation, differentiation, migration and apoptosis [4].

Among more than a dozen MAPKs in mammals, p38 is

known to respond to a wide range of extracellular cues,

particularly cellular stressors, including growth factors and

inflammatory cytokines such as IL-1b [4, 5]. Recent studies

indicate that p38 MAPK plays a role in the development,

maintenance and/or exacerbation of a number of pulmon-

ary diseases, such as asthma, cystic fibrosis, idiopathic

pulmonary fibrosis, and chronic obstructive pulmonary

disease [6–10]. In addition, p38 MAPK has been reported

to be involved in regulating several critical functions of

endothelial cells including cell migration, vasodilatation,

angiogenesis and chemoattraction [11, 12].

The p38 MAPK family consists of four distinct isoforms,

i.e., p38a (MAPK14), p38b (MAPK11), p38c (MAPK12),

and p38d (MAPK13) [4]. The p38a and b isoforms are

expressed in most tissues, whereas p38c and d appear to

be more tissue restricted [4, 13]. Although several studies

have reported that several isoforms of p38 are expressed

in vascular endothelial cells [14, 15], expression of p38

isoforms in pulmonary artery endothelial cells and their

role in regulating cellular functions have not been

reported.

The current study was, therefore, designed to investigate

the role of p38 MAPK isoforms in mediating inflammatory

cytokine-induced HPAEC morphologic change and effects

on cell survival and tissue repair functions. We here show

that inhibition of p38 MAPK per se causes endothelial cells

to undergo morphologic change from a polygonal-shape

into spindle-shaped fibroblast-like cells, and also modu-

lates viability and repair functions. These effects are

differentially mediated by different p38 isoforms and are

independent of the nuclear factor (NF)-jB pathway.

Materials and methods

Reagents

Recombinant human IL-1b was purchased from R&D

systems (Minneapolis, MN). Anti-p38a, anti-p38b, anti-

p38c, anti-p38d and anti-phosphorylated p38 antibodies

were purchased from Cell Signaling Technology, Inc

(Danvers, MA). Anti-b-actin antibody and FITC–phalloi-

din were purchased from Sigma-Aldrich (St. Louis, MO).

HRP-conjugated anti-mouse and rabbit IgG antibodies

were purchased from Rockland (Gilbertsville, PA). Selec-

tive p38 MAPK inhibitors SB203580 and SB220025

were purchased from Calbiochem (San Diego, CA) and

dissolved in dimethyl sulfoxide (DMSO). The small inter-

ference RNAs (siRNAs) targeting p38 MAPK isoforms

(a, b, c, and d), NF-jB p65 and non-targeting control siRNA

were purchased from Dharmacon (Lafayette, CO).

Cell culture and treatment

Human pulmonary artery endothelial cells (HPAEC) were

purchased from Clonetics, Lonza (Walkersville, MD).

Cells were maintained in endothelial basal medium (EBM-2,

Lonza) supplemented with growth factors and 2% fetal

bovine serum according to the manufacturer’s recommen-

dations (complete EBM). Cells passaged between 8 and 12

were used for all experiments. Cells were treated with

IL-1b (0.5 or 2 ng/ml) or p38 inhibitor (SB203580 or

SB220025 (5 lM)) alone or in combination in a 1:1 mix-

ture complete EBM to EBM-2 resulting in a final

concentration of 1% serum (1:1 EBM) for 2 days. Lower

concentration of IL-1b, that is, 0.5 ng/ml, was used when it

was combined with p38 inhibitors in order to maximize the

p38 inhibitors’ effect. DMSO was used as a solvent control

for the p38 inhibitors. To induce apoptosis, cells were

incubated in EBM-2, which is without any growth factors

or serum, for 4 days in the presence or absence of p38

inhibitors (5 lM) and/or IL-1b (0.5 ng/ml). Cells were

then fixed and stained with PROTOCOL HEMA3 solutions

(Fisher Scientific Company L.L.C., Kalamazoo, MI).

A Nikon Eclipse TE300 microscope (Nikon, Tokyo, Japan)

equipped with a DP71 digital camera and DP Controller

software (Olympus, Tokyo, Japan) was used to take

photomicrographs.

Cell length measurement

To quantify cell morphologic change, cell length was

measured using ImageJ 1.33u software, NIH. A total of 50

cells for each condition was measured. Data are presented

as percent of control.

Immunoblot

Following treatment, cells were washed with ice-cold PBS

and total protein was extracted with lysis buffer (35 mM

Tris–HCl, pH 7.4, 0.1% Triton X-100, 0.4 mM EGTA,

10 mM MgCl2) containing a protease inhibitor cocktail

(Sigma-Aldrich). Lysates were centrifuged at 13,000g for

10 min and the protein concentration was measured using

the BIO-RAD Protein Assay Kit (Bio-Rad, Hercules, CA).

Lysates were diluted with 29 sample buffer (125 mM

Tris–HCl, pH 6.8, 4% SDS, 20% glycerol, 0.01% bromo-

phenol blue, 10% b-mercaptoethanol) and heated at 95�C

for 5 min. Five micrograms of proteins were loaded, sep-

arated by electrophoresis in 7.5% SDS–polyacrylamide

gels, and transferred to a PVDF membrane (Bio-Rad). The

234 N. Kanaji et al.

123

membrane was blocked in 5% skim milk for 1 h at room

temperature and incubated with proper concentrations of

first antibody at 4�C overnight. Targeting proteins were

subsequently detected using horseradish peroxidase con-

jugated IgG with an enhanced chemiluminescence plus

detection system (ECL plus) and Typhoon Scanner

(Amersham Pharmacia Biotech, Little Chalfont, Bucking-

hamshire, UK).

Visualization of F-actin

Cells were plated in a 48-well plate and treated with or

without p38 inhibitors (5 lM) and/or IL-1b (0.5 ng/ml) in

1:1 EBM for 2 days. Cells were fixed with 3.7% formal-

dehyde for 15 min at room temperature and permeabilized

with 0.5% Triton X-100 in PBS for 15 min. After washing

with PBS, cells were blocked with 5% BSA, and then

incubated with FITC–phalloidin (1 lg/ml) at room tem-

perature for 1 h.

RNA interference

To selectively silence each isoform of p38 MAPK and

NF-jB p65, RNA interference was performed. Briefly,

cells were seeded at a cell density of 1 9 105 cells/ml. The

next day, cells were transfected with siRNA targeting

individual p38 MAPK isoforms, p65 or with non-targeting

control siRNA (final concentration of siRNA was 50 nM)

in OptiMEM I (Invitrogen, Carlsbad, CA) using Lipofect-

amine2000 (Invitrogen). After 8 h of incubation, media

were changed to complete EBM. Cell lysates were extracted

on day 4 and the efficacy of RNA interference was assessed

by immunoblot.

Collagen gel contraction assay

Native type I collagen gels were prepared by mixing a

solution of rat tail tendon collagen (approximately 3 mg/ml),

complete EBM and cell suspension so that the final

mixture resulted in 0.75 mg/ml collagen and 3 9 105

cells/ml. A 500 ll aliquot of the resulting solution was

then cast into each well of a 24-well culture plate and

allowed to polymerize. After gelation was completed, in

about 20 min at room temperature, the gels were gently

released into 60-mm dishes (three gels in each dish)

containing 5 ml of 1:1 EBM with or without p38 inhibi-

tors (5 lM) and/or IL-1b (0.5 ng/ml) and incubated at

37�C for 4 days. Gel size was measured with the Optomax

V image analyzer (Optomax, Burlington, MA). Data are

expressed as the percentage of gel area compared with the

original gel size.

Wound closure assay

Cells were pretreated with or without p38 inhibitors (5 lM)

and/or IL-1b (0.5 ng/ml) in a 1:1 EBM for 2 days. Fixed

width linear wounds were then created using a cell scraper

(Corning Inc., Corning, NY), and the remaining cells were

incubated in a 1:4 mixture complete EBM to EBM-2 for

16 h in the presence or absence of p38 inhibitors (5 lM)

and/or IL-1b (0.5 ng/ml). Cells were then fixed, stained

and photographed. To quantify cell migration, a grid was

overlaid on each image at 0 and 16 h after wounding using

Photoshop Elements 2.0 software (Adobe Systems Inc.,

San Jose, CA) to make 1,064 tiny squares. The number of

squares that contained migrated cells was counted. Three

fields for each condition were quantified. Data are pre-

sented as percentage of squares occupied by migrated cells

after wounding.

Profiling of DNA content by flow cytometry

Nearly confluent cells were treated with or without p38

inhibitors (5 lM) in EBM-2 only (serum and growth factor

free medium) for 2 days. Both attached and floating cells

were collected and fixed with cold 70% ethanol in PBS

at 4�C for 30 min. Cells were then pelleted by centrifu-

gation and re-suspended in the staining solution (50 lg

propidium iodide, 100 lg RNase A in 1 ml PBS). After 1 h

staining at 4�C, flow cytometric DNA content profiling was

performed.

Statistical analysis

Data are expressed as mean ± standard deviation (SD). An

unpaired two-tailed Student’s t test was used for single

comparisons. For group test, one-way ANOVA followed

by Tukey’s test was used with the PRISM4 program.

p values \0.05 were considered as statistically significant.

Results

Suppression of p38 results in endothelial cell

morphologic change into spindle-shaped fibroblast-like

cells

Under normal culture conditions, HPAECs grow as a

monolayer of polygonal shaped cells. In the presence of the

pro-inflammatory cytokine IL-1b, HPAECs undergo spin-

dle-shaped morphologic alteration (Fig. 1a) along with

activation of p38 MAPK (Fig. 1b, c). We next investigated

if pharmacologic p38 inhibitors could block the cell mor-

phologic change induced by IL-1b. Surprisingly, inhibition

of p38 MAPK by p38 inhibitors (SB203580 or SB220025)

p38 and tissue repair on endothelial cells 235

123

per se induced cell morphologic alteration when added

alone and further augmented the inflammatory cytokine-

induced change (Fig. 2a). This was evidenced by micro-

photography of the cells, measurement of the cell length

(Fig. 2a), and by the polymerization of F-actin (Fig. 2b).

Next, we examined if p38 inhibitors can reduce endothelial

cell markers and/or induce mesenchymal cell markers.

However, the endothelial cell marker, VE–cadherin, was

not significantly altered by p38 inhibitor (data not shown).

Furthermore, none of the tested mesenchymal cell markers

including N-cadherin, vimentin, or fibronectin expression

was affected by p38 inhibitors (data not shown). Similar to

HPAEC, the p38 inhibitor-induced spindle-shaped mor-

phologic alteration was also observed in human dermal

microvascular endothelial cells (data not shown). In addi-

tion, the effect of the p38 inhibitors on endothelial cell

morphologic change was reversible following removal of

the reagents (data not shown).

To further confirm the effect of the pharmacologic

inhibitors of p38 and to identify the p38 isoforms that

modulate HPAEC morphology, siRNAs targeting individ-

ual isoforms of p38a, b, c or d were transfected into the

cells. As shown in Fig. 3a, the siRNA targeting each p38

isoform could significantly and specifically silence their

respective target. Suppression of p38a, c or d resulted in

cell morphologic change (Fig. 3b) and elongation in cell

length (Fig. 3b, p \ 0.05 compared to control siRNA).

Polymerization of F-actin was altered only by suppression

of p38c or d (Fig. 3c). In contrast, p38b-siRNA did not

affect cell morphology and cell length (p [ 0.05), or

F-actin polymerization (Fig. 3b, c). Although the inhibition

of p38a led to a slight decrease in VE–cadherin expression,

suppression of any one of the p38 isoforms did not alter

expression of N-cadherin or vimentin (data not shown).

Inhibition of p38 prevents cell death from deprivation

of serum and growth factors

HPAECs were cultured to sub-confluence in complete

EBM. When the cells were subsequently cultured in basal

medium without serum and growth factors, the majority of

the cells detached and were lost within 4 days (Con-

trol ? DMSO panel, Fig. 4a). An average of 22.5 ± 5.3%

of the cells survived after 4 days under this condition

(Fig. 4a insert). In the presence of either SB203580 or

SB220025, however, a significantly higher number of cells

survived (53.4 ± 14.3% or 43.8 ± 9.3%, respectively,

p \ 0.05, both comparisons) and the majority of these

surviving cells underwent morphologic change (Fig. 4a).

Interestingly, the elongated fibroblast-like appearance was

also observed in the few surviving cells under the control

condition (Fig. 4a). Furthermore, the protective effect of

p38 inhibitors on cell survival was also observed in the

presence of IL-1b (Fig. 4a); that is, nearly all of the cells

survived in the presence of p38 inhibitors plus IL-1b (81.7 ±

8.1% SB203580 plus IL-1b; 75.2 ± 2.5% SB220025 plus

IL-1b vs. 57.1 ± 7.5% of IL-1b alone, p \ 0.01, both

comparisons).

Fig. 1 Effect of IL-1b on cell

morphology and activation of

p38 MAPK in HPAECs. a Cell

morphology. Human pulmonary

artery endothelial cells

(HPAECs) were treated with

2 ng/ml of IL-1b for 2 days.

Cells were then stained and

photographed under microscope.

Scale bar 200 lm. b Time-

dependent p38 activation. Total

cell proteins were extracted after

0, 20, 40, 60 min of cytokine

treatment (2 ng/ml), and then

immunoblotted for

phosphorylated-p38 MAPK and

b-actin. c Semi-quantification of

phosphorylated p-38 versus

b-actin. Vertical axis density

versus control; horizontal axistime of treatment. Data

presented is an average of three

separate experiments. *p \ 0.05

compared to control by one way

ANOVA followed by Tukey’s

correction

236 N. Kanaji et al.

123

Similar to the effect of pharmacologic p38 inhibitors,

RNA interference targeting p38 isoforms also prevented

cells from death induced by serum and growth factor

withdrawal (Fig. 4b). Although all of the four siRNAs

targeting individual isoforms showed a protective effect on

cell survival compared to control siRNA (surviving cells:

15.9 ± 4.1%), the p38a-siRNA had a strongest effect

(95.4 ± 9.4%, p \ 0.01) and others had a more modest but

statistically significant effect (48.2 ± 7.7% b-siRNA, p \0.01; 40.5 ± 6.8% c-siRNA, p \ 0.01; 53.2 ± 4.4%

d-siRNA, p \ 0.01).

To evaluate if the cells were undergoing apoptosis or

necrosis following deprivation of growth factors, DNA

content profiling by FACS analysis was conducted. Con-

sistent with the microscopic observation and cell number

count, DNA content profiling assay demonstrated that an

average of 26.4 ± 3.2% of the cells underwent apoptosis

after 2 days culture in the basal medium only (DMSO

panel, Fig. 4c). This apoptosis was significantly blocked by

either SB203580 or SB220025 (16.6 ± 2.0% or 13.3 ±

3.2%, respectively, p \ 0.05, Fig. 4c).

Inhibition of p38 modulates tissue repair functions

To investigate if inhibition of p38 MAPK alters tissue

repair functions in HPAECs, the gel contraction assay (an

in vitro model of remodeling of extracellular matrix) and

the wound closure assay (an in vitro wound healing model)

were performed. Under control conditions, normal HPAECs

were able to contract native type I collagen gels when the

cells were cast into the gels. Gel size decreased gradually

and reached 75.2 ± 1.5% of initial size on day 4 (Fig. 5a).

SB203580 and SB220025 augmented collagen gel con-

traction by HPAECs (69.6 ± 1.7% and 60.7 ± 2.6% of

initial size on day 4, respectively, p \ 0.05, Fig. 5a).

The inflammatory cytokine IL-1b also augmented gel

contraction mediated by HPAEC (67.1 ± 2.5%, p \ 0.05,

Fig. 5a). The cytokine effect was further augmented in the

Fig. 2 Effect of pharmacologic

p38 inhibitors on cell

morphology and F-actin

cytoskeletal arrangement. a Cell

morphology and cell length.

Cells were treated with or

without p38 inhibitors

(SB203580 or SB220025,

5 lM) in the presence or

absence of IL-1b (0.5 ng/ml) for

2 days. Cells were then fixed,

stained and photographed. The

cell length was measured as

described in the methods

(inserts). Values are

means ± SD of three separate

experiments. *p \ 0.05

compared to DMSO without

IL-1b. **p \ 0.05 compared to

DMSO with IL-1b. b F-actin

polymerization. F-actin was

visualized by FITC–phalloidin

as described in the methods

p38 and tissue repair on endothelial cells 237

123

P38

p38

p38

P38

-actin

NT siRNA targeting p38

Control siRNA p38 -siRNA p38 -siRNA

p38 -siRNA p38 -siRNA

Cell length 100% 129.7±9.6% * 99.1±2.9%

141.7±12.5% * 128.8±9.6% *

Control siRNA p38 -siRNA p38 -siRNA

p38 -siRNA p38 -siRNA

A

C

B

Fig. 3 Effect of siRNAs

targeting individual p38

isoforms on cell morphology

and F-actin polymerization.

a Suppression of p38 isoforms

by siRNA. Sub-confluent

HPAECs were transfected with

siRNAs using Lipofectamine

2000 described in the method.

On day 4, total proteins were

extracted and immunoblotted

for each p38 MAPK isoform.

Five micrograms of total protein

was loaded and b-actin was

blotted as a loading control.

b Cell morphology. On day 0,

cells were transfected with

siRNAs. On day 4, cells were

fixed, stained and photographed.

Cell length was measured as

described in the methods

(inserts). Values are

means ± SD of three separate

experiments. *p \ 0.05

compared to control siRNA.

c F-actin polymerization. After

4 days of transfection of

siRNAs, F-actin was visualized

by FITC–phalloidin

238 N. Kanaji et al.

123

control siRNA p38 -siRNA p38 -siRNA

p38 -siRNA p38 -siRNA

day 0 day 3 (serum and growth factor free)

control siRNA

100% Survived cells 15.9±4.1% 95.4±9.4% * 48.2±7.7% *

40.5±6.8% * 53.2±4.4% *

A

B

C

Fig. 4 Effect of p38

suppression on cell survival

following withdrawal of serum

and growth factors. a Effect of

pharmacologic p38 inhibitors on

cell survival. HPAECs were

cultured until sub-confluent

with complete medium. The

cells were then treated with

either p38 inhibitors or IL-1b(0.5 ng/ml) or both in a basal

medium without serum or any

growth factors. After 4 days,

attached cells were fixed,

stained and photographed. Cell

number was counted (inserts).

Values are means ± SD of

three separate experiments.

*p \ 0.05 compared to DMSO

without cytokine. **p \ 0.05

compared to DMSO with IL-1balone. b Effect of siRNAs

targeting p38 isoforms on cell

survival. After transfection with

siRNAs, cells were fed with a

basal medium containing no

serum or growth factors. After

3 days, attached cells were

fixed, stained and photographed.

Cell number was counted

(inserts). Values are

means ± SD of three separate

experiments. *p \ 0.01

compared to control siRNA.

c Evaluation of apoptosis by

DNA content profiling. Cells

were treated with or without p38

inhibitors for 2 days in a basal

medium. Both floating and

attached cells were harvested

and used for DNA content

profiling. Values are

means ± SD of three separate

experiments. *p \ 0.05

compared to DMSO

p38 and tissue repair on endothelial cells 239

123

presence of either of the p38 inhibitors, SB203580 or

SB220025 (61.0 ± 2.0% or 47.6 ± 3.2%, respectively,

p \ 0.05, Fig. 5a). Augmentation of collagen gel contrac-

tion by p38 inhibitors was further confirmed by siRNAs.

While the cells transfected with control siRNA contracted

the gels to 75.8 ± 3.0% of initial size on day 4, either

p38a- or d-siRNA led to augmentation of gel contrac-

tion (54.8 ± 7.9% or 53.0 ± 5.0%, respectively, p \ 0.05,

Fig. 5b). In contrast, p38b- and c-siRNAs did not affect gel

contraction (p [ 0.05).

Next, we evaluated wound closure in the presence or

absence of p38 inhibitors. Under control conditions, nearly

half (46.0 ± 4.0%) of the wounded area was covered by

the migrated cells (Control ? DMSO, Fig. 5c). Interest-

ingly, the two p38 inhibitors had opposite effects on wound

closure; that is, SB203580 stimulated wound closure

(66.5 ± 2.6%, p \ 0.05), and SB220025 inhibited wound

closure (29.5 ± 4.4%, p \ 0.05, Fig. 5c). Furthermore,

SB203580 augmented the cytokine effect on wound

closure (70.3 ± 4.2% of IL-1b alone vs. 94.1 ± 0.9% of

IL-1b ? SB203580, p \ 0.05), while SB220025 signifi-

cantly blocked the effect of cytokine-augmented wound

closure (49.7 ± 7.9% of IL-1b ? SB220025, p \ 0.05,

Fig. 5c). In addition, we examined the wound closure assay

after RNA interference (Fig. 5d). Interestingly, transfection

with p38a-siRNA resulted in greater wound closure com-

pared to control siRNA (71.9 ± 9.6% vs. 48.4 ± 4.5%,

p \ 0.05), while suppression of other p38 isoforms slightly

but significantly inhibited wound closure (p38b-siRNA;

35.9 ±

3.3%, p38c-siRNA; 32.3 ± 6.5%, and p38d-siRNA; 31.2 ±

6.9%, p \ 0.05). Similar to the disparate effects of the p38

inhibitors on wound closure, SB203580 increased while

SB220025 decreased trans-endothelial electrical resistance

measured with electric cell-substrate impedance sensing

(data not shown).

Interaction between NF-jB and p38 MAPK signaling

pathways

Since NF-jB is a major signal transduction mediator of

the effect of IL-1b in a variety of cells including endo-

thelial cells, the role of NF-jB p65 activation in p38

inhibitor-induced cell morphologic alteration was exam-

ined. To accomplish this, first, effect of p38 inhibitor

(SB203580) on p65 phosphorylaton and p38 phosphory-

lation was evaluated by immunoblot. As shown in Fig. 6a,

SB203580 appeared to block IL-1b-induced p38 phos-

phorylation. Surprisingly, however, SB203580 further

enhanced IL-1b-induced p65 phosphorylation. Second, the

NF-jB signaling pathway was blocked by RNA interfer-

ence targeting NF-jB p65 followed by treatment with p38

inhibitors and/or cytokines. While suppression of p65 by

siRNA (Fig. 6b) prevented cell morphologic change in

response to IL-1b, it did not affect the morphologic

change in response to p38 inhibitor (SB203580) as shown

by microphotography (Fig. 6c) and measurement of cell

length (Fig. 6c insert), indicating p38 inhibitor-induced

HPAEC morphologic change is not mediated by p65

subunit of NF-jB.

Discussion

The present study demonstrates that IL-1b induces p38

phosphorylation and alteration of cell morphology.

Suppression of p38 MAPK, however, does not block the

IL-1b effect, but enhances IL-1b-induced cell morphologic

change. Furthermore, p38 suppression per se induces an

elongated and spindle-shaped cell morphologic change

along with the organization of the F-actin cytoskeleton.

Moreover, inhibition of p38 MAPK per se also prevents

HPAECs from undergoing apoptosis induced by depriva-

tion of serum and growth factors. In addition, suppression

of p38a or d increases contraction of type I collagen gels by

the HPAECs. Suppression of p38a increases wound clo-

sure, while suppression of p38b, c or d decreases it. Finally,

both NF-jB p65 and p38 are activated by IL-1b, but sup-

pression of p65 does not alter either p38 activation or the

Fig. 5 Effect of p38 suppression on gel contraction and cell migration.

a Effect of pharmacologic p38 inhibitors on collagen gel contraction

mediated by HPAECs. Cells were cast into collagen gels and treated

with or without p38 inhibitors (5 lM) and/or IL-1b (0.5 ng/ml) for

4 days. Gel size was measured with an image analyzer on day 4.

Vertical axis gel size expressed as percent of initial size (%). Horizontalaxis treatment with or without IL-1b. Open bars DMSO only; hatchedbars SB203580; solid bars SB220025. Data presented are means ± SD

of three separate experiments. *p \ 0.05 compared to DMSO without

cytokines. **p \ 0.05 compared to DMSO with same cytokine

treatment. b Effect of siRNAs targeting p38 isoforms on collagen gel

contraction. After transfection with siRNAs, the cells were cast into

collagen gels (day 0) and allowed to contract for 4 days. Gel size was

measured with an image analyzer. Vertical axis gel size expressed as

percent of initial size (%) on day 4. Horizontal axis siRNA treatment.

Data presented are means ± SD of three separate experiments.

*p \ 0.05 compared to control siRNA. c Effect of pharmacologic

p38 inhibitors on wound closure. HPAECs were pre-treated with or

without p38 inhibitors (5 lM) and/or IL-1b (0.5 ng/ml) for 2 days. The

cell layer was then wounded with a cell scraper followed by incubation

with same p38 inhibitors with or without IL-1b for additional 16 h.

Wound repair was quantified by counting the number of migrated cells

as described in the method (inserts). Data presented are means ± SD of

three separate experiments. *p \ 0.05 compared to DMSO without

cytokine. **p \ 0.05 compared to DMSO with IL-1b treatment.

d Effect of siRNAs targeting p38 isoforms on wound closure. After

3 days of transfection with siRNAs, the cells were wounded and

allowed to repair for 16 h. Wound repair was quantified as described

above (inserts). Data presented are means ± SD of three separate

experiments. *p \ 0.05 compared to control siRNA

c

240 N. Kanaji et al.

123

effect of p38 inhibitors on HPAECs, suggesting the effect

of p38 on HPAECs is NF-jB independent.

Pulmonary vascular endothelial cells have been reported

to be involved in the pathogenesis of inflammatory lung

diseases including chronic obstructive pulmonary disease

and pulmonary hypertension [16]. Inflammatory cytokines

such as IL-1b can alter endothelial cell functions such

as cell migration and susceptibility to apoptosis [17, 18].

p38 and tissue repair on endothelial cells 241

123

The current study provides new mechanistic insight into

IL-1b modulation of endothelial cell function. Specifically,

p38 MAPKs negatively modulate IL-1b effects on endo-

thelial cells. In addition, p38 MAPKs alone can regulate

endothelial cell functions. The four isoforms (a, b, c and d)

of p38 MAPKs share only about 60% amino acid sequence

identity, suggesting they may have highly diverse functions

[4, 19]. In the current study, we not only confirmed the

expression of all four p38 isoforms in HPAECs, but also

determined that the isoforms play different roles in regu-

lating HPAEC morphology, survival, and cell functions

related to tissue repair.

MAPKs are associated with cell differentiation [4]. In

this content, several reports have shown a positive role

of p38 MAPK in epithelial cells undergoing epithelial-

mesenchymal transition (EMT), that is, a p38 inhibitor

(SB203580, SB202190 or 2-(4-chlorophenyl)-4-(4-fluo-

rophenyl)-5-pyridin-4-yl-1,2-dihydropyrazol-3-one) or a

dominant negative-p38a blocked transforming growth

factor-b1-induced EMT in several types of epithelial cells

[20–22]. In contrast to these reports, our findings are dif-

ferent in several respects. First, the p38 inhibitors not only

augmented inflammatory cytokine-induced endothelial cell

morphologic change, but also these inhibitors per se

induced endothelial cells to undergo morphologic change

from typical polygonal-shaped endothelial cells into spin-

dle-shaped fibroblast-like cells. Second, none of the tested

mesenchymal markers including vimentin, N-cadherin or

fibronectin was altered by the p38 inhibitors in HPAECs.

These results suggest that the p38 signal pathway plays a

different role in endothelial cells compared to epithelial

cells. We can not conclude that the changes observed by

inhibition of p38 are consistent with endothelial-mesen-

chymal transition (EndMT). However, the current study

demonstrates that HPAECs lacking p38 MAPK undergo

morphologic change to become more contractile and are

A B

C

Fig. 6 Role of p65 in mediating IL-1b- or SB203580-induced cell

morphologic change. a Effect of IL-1b and SB203580 on p65 and p38

phosphorylation. Cells pretreated with SB203580 (5 lM) for 30 min

followed by stimulation with IL-1b (2 ng/ml, 20 min). Whole cell

lysate was immunoblotted for phosphorylated p65 or p38, and b-actin.

C control; S SB203580. b Immunoblot of p65 following siRNA

transfection. Cells were transfected with either a control siRNA or a

siRNA targeting NF-jB-p65 using lipofectamine 2000 as described in

the methods. Whole cell lysates were subjected for immunoblot of

phosphorylated p65 and b-actin. c Effect of p65 suppression on cell

morphologic change in response to p38 inhibitor or IL-1b stimulation.

Following siRNA transfection, the cells were treated with or without

SB203580 (5 lM) and/or IL-1b (2 ng/ml) for 2 days. The cells were

then stained and photographed. Cell length was measured as

described (inserts). Values are means ± SD of three separate

experiments. *p \ 0.05 compared to cells transfected with control

siRNA and treated with DMSO only. **p \ 0.05 compared to the

cells transfected with control siRNA and treated with IL-1b.#p \ 0.05 compared to the cells transfected with siRNA targeting

p65 and treated with DMSO only

242 N. Kanaji et al.

123

resistant to apoptosis. In addition, suppression of p38

MAPK further enhanced the augmentative effect of IL-1bon these functions. These results suggest that endothelial

cells can contribute to tissue repair whether or not the

criteria for EndMT are met.

Besides regulating cell differentiation, p38 MAPK is also

involved in modulating cell survival and death. There is

increasing evidence showing that inhibition of p38 MAPK

protects endothelial cells from apoptosis [23, 24]. Consis-

tent with these reports, the current study also demonstrated

that HPAEC survival is negatively regulated by the p38

signal pathway. Interestingly, it has been also reported that

activation of p38b promoted cell survival, whereas activa-

tion of p38a resulted in cell death in cardiac myocytes,

Jurkat cells and HeLa uterine cervical adenocarcinoma cells

[25–27]. Similarly, the anti-apoptotic effect of heme oxy-

genase-1 was dependent on the degradation of p38a and on

the expression of p38b in human umbilical vein and bovine

aortic endothelial cells [28]. These results suggest that p38aand p38b have opposite effects in controlling cell viability

of certain cell types, i.e., p38a is pro-apoptotic, whereas

p38b is anti-apoptotic. Unlike these reported data, the

current study demonstrated that inhibition of any one of the

p38 isoforms including p38b prevented HPAECs from

apoptosis and inhibition of p38a had the strongest effect.

These findings suggest the role of p38b isoform on cell

survival might vary among cell types.

Both cell morphologic change and cell viability are key

features of tissue repair following injury. Thus, we further

determined the role of p38 MAPK in regulating endothelial

cell tissue repair functions by evaluating collagen gel

contraction and wound closure. We demonstrated that the

pharmacologic p38 inhibitors significantly augmented

collagen gel contraction mediated by HPAECs, which is

opposite to the result Meyer-Ter-Vehn et al. [29] reported

using fibroblasts, that is, suppression of p38 MAPK

inhibited collagen gel contraction mediated by fibroblasts.

In our study, the effect of pharmacologic inhibitors on

collagen gel contraction was confirmed by RNA interference.

Suppression of p38a or d by siRNA also resulted in aug-

mentation of collagen gel contraction mediated by HPAEC.

Inhibition of p38c, however, did not alter gel contraction in

spite of its ability to induce fibroblast-like cell morphology,

suggesting that cell morphologic alteration is not always

parallel to contraction of collagen gels.

Several studies have reported that cell migration was

blocked by inhibition of p38 MAPK in epithelial, mesen-

chymal and endothelial cells [12, 20, 30, 31]. In the current

study, the two pharmacologic p38 inhibitors showed

opposite effect on endothelial wound closure, that is,

SB203580 enhanced endothelial wound closure while

SB220025 inhibited wound closure. SB203580 is a pyridyl

imidazole compound, and it inhibits p38a and b isoforms,

but not p38c or d [4, 19]. In the current study, only p38asuppression by siRNA resulted in enhancement of wound

closure, while suppression of the other three isoforms

slightly but significantly decreased wound closure. This

result indicates that p38a negatively, while other p38 iso-

forms positively modulates endothelial cell-mediated

wound closure. It has not been reported which p38 iso-

forms SB220025, a pyrimidinyl imidazole compound,

inhibits. Our data suggest that the inhibitory effect of

SB220025 may be more potent on p38d than on p38a.

IL-1b is known to activate the p38 signaling pathway,

which is also confirmed in the current study. Interestingly,

however, the current study demonstrated that suppression

of p38 by either pharmacologic inhibitors or specific siR-

NAs could dramatically enhance IL-1b effect on endothelial

cells, that is, alteration of morphology, resistance to

apoptosis and enhancement of collagen gel contraction.

This suggests that p38 activation could serve as an internal

feedback inhibitory role following IL-1b stimulation. To

our knowledge, these are novel findings for the function

of p38 in endothelial cells. In addition, SB203580, an

inhibitor of p38 phosphorylation in response to IL-1b stimu-

lation, augments IL-1b-induced p65 phosphorylation.

Furthermore, the suppression of NF-jB p65 did not affect

SB203580 or SB220025 induced endothelial cell morpho-

logic change while it did block the effect of IL-1b. Thus,

we propose that both p38 MAPK and NF-jB p65 mediate

signal transduction of IL-1b in endothelial cells and that

p38 MAPK negatively while NF-jB p65 positively medi-

ates the responses of endothelial cells evaluated in the

current study.

In the current study, we have demonstrated that p38

suppression by either a pharmacologic inhibitor or a siRNA

could not only lead to HAPEC morphologic change under

normal culture condition, but also result in cell survival in

the absence of growth factors. While p38 seems negatively

regulate cellular functions in HPAECs, the downstream

mechanism of p38 on cell morphologic change and cell

survival, however, remains to be further defined.

In summary, suppression of p38 MAPK induces

HPAECs to undergo a morphologic change from a polyg-

onal-shape into spindle-shaped fibroblast-like cells. The

cells in which p38 MAPK is inhibited are resistant to

apoptosis in response to serum and growth factor depri-

vation, and are more robust in contracting collagen gels.

IL-1b activates p38 MAPK but inhibition of p38 MAPK

further augments IL-1b-induced morphologic change, cell

survival, and gel contraction. Inhibition of p38a enhances

wound closure, while inhibition of other isoforms decrea-

ses it. These findings suggest that p38 MAPK may

modulate tissue remodeling mediated by endothelial cells,

especially in an inflammatory milieu where inflammatory

cytokine IL-1b is present.

p38 and tissue repair on endothelial cells 243

123

Acknowledgments The authors thank Ms. Lillian Richards for the

excellent secretarial support of this manuscript. Funding: Larson

Endowment, University of Nebraska Medical Center and LB506.

References

1. Saklatvala J, Davis W, Guesdon F. Interleukin 1 (il1) and tumour

necrosis factor (tnf) signal transduction. Philos Trans R Soc Lond

B Biol Sci. 1996;351(1336):151–7.

2. Chaudhuri V, Zhou L, Karasek M. Inflammatory cytokines

induce the transformation of human dermal microvascular

endothelial cells into myofibroblasts: a potential role in skin

fibrogenesis. J Cutan Pathol. 2007;34(2):146–53.

3. Kanaji N, Sat T, Wang XQ, Kim M, Nakanishi M, Li YJ, Basma

H, Patil A, Michalski J, Nelson AJ, Sun J, Liu X, Rennard SI

Interleukin-1beta induces endothelial-mesenchymal transition via

the nf-kappa b pathway. Am J Respir Crit Care Med.

2009;179:A2343.

4. Raman M, Chen W, Cobb MH. Differential regulation and

properties of mapks. Oncogene. 2007;26(22):3100–12.

5. Rossa C, Ehmann K, Liu M, Patil C, Kirkwood KL. Mkk3/6–p38

mapk signaling is required for il-1beta and tnf-alpha-induced

rankl expression in bone marrow stromal cells. J Interferon

Cytokine Res. 2006;26(10):719–29.

6. Chopra P, Kanoje V, Semwal A, Ray A. Therapeutic potential of

inhaled p38 mitogen-activated protein kinase inhibitors for

inflammatory pulmonary diseases. Expert Opin Investig Drugs.

2008;17(10):1411–25.

7. Duan W, Chan JH, McKay K, Crosby JR, Choo HH, Leung BP,

Karras JG, Wong WS. Inhaled p38alpha mitogen-activated pro-

tein kinase antisense oligonucleotide attenuates asthma in mice.

Am J Respir Crit Care Med. 2005;171(6):571–8.

8. Raia V, Maiuri L, Ciacci C, Ricciardelli I, Vacca L, Auricchio S,

Cimmino M, Cavaliere M, Nardone M, Cesaro A, et al. Inhibition

of p38 mitogen activated protein kinase controls airway inflam-

mation in cystic fibrosis. Thorax. 2005;60(9):773–80.

9. Renda T, Baraldo S, Pelaia G, Bazzan E, Turato G, Papi A,

Maestrelli P, Maselli R, Vatrella A, Fabbri LM, et al. Increased

activation of p38 mapk in copd. Eur Respir J. 2008;31(1):62–9.

10. Yoshida K, Kuwano K, Hagimoto N, Watanabe K, Matsuba T,

Fujita M, Inoshima I, Hara N. Map kinase activation and apop-

tosis in lung tissues from patients with idiopathic pulmonary

fibrosis. J Pathol. 2002;198(3):388–96.

11. Schett G, Zwerina J, Firestein G. The p38 mitogen-activated

protein kinase (mapk) pathway in rheumatoid arthritis. Ann

Rheum Dis. 2008;67(7):909–16.

12. Sumioka T, Ikeda K, Okada Y, Yamanaka O, Kitano A, Saika S.

Inhibitory effect of blocking tgf-beta/smad signal on injury-

induced fibrosis of corneal endothelium. Mol Vis. 2008;14:

2272–81.

13. Hui L, Bakiri L, Mairhorfer A, Schweifer N, Haslinger C, Kenner

L, Komnenovic V, Scheuch H, Beug H, Wagner EF. P38alpha

suppresses normal and cancer cell proliferation by antagonizing

the jnk-c-jun pathway. Nat Genet. 2007;39(6):741–9.

14. Hale KK, Trollinger D, Rihanek M, Manthey CL. Differential

expression and activation of p38 mitogen-activated protein kinase

alpha, beta, gamma, and delta in inflammatory cell lineages.

J Immunol. 1999;162(7):4246–52.

15. Korb A, Tohidast-Akrad M, Cetin E, Axmann R, Smolen J,

Schett G. Differential tissue expression and activation of p38

mapk alpha, beta, gamma, and delta isoforms in rheumatoid

arthritis. Arthr Rheum. 2006;54(9):2745–56.

16. Peinado VI, Pizarro S, Barbera JA. Pulmonary vascular

involvement in copd. Chest. 2008;134(4):808–14.

17. Carmi Y, Voronov E, Dotan S, Lahat N, Rahat MA, Fogel M,

Huszar M, White MR, Dinarello CA, Apte RN. The role of

macrophage-derived il-1 in induction and maintenance of angi-

ogenesis. J Immunol. 2009;183(7):4705–14.

18. Madge LA, Pober JS. A phosphatidylinositol 3-kinase/akt path-

way, activated by tumor necrosis factor or interleukin-1, inhibits

apoptosis but does not activate nfkappab in human endothelial

cells. J Biol Chem. 2000;275(20):15458–65.

19. Goedert M, Cuenda A, Craxton M, Jakes R, Cohen P. Activation

of the novel stress-activated protein kinase sapk4 by cytokines

and cellular stresses is mediated by skk3 (mkk6); comparison of

its substrate specificity with that of other sap kinases. EMBO J.

1997;16(12):3563–71.

20. Bakin AV, Rinehart C, Tomlinson AK, Arteaga CL. P38 mito-

gen-activated protein kinase is required for tgfbeta-mediated

fibroblastic transdifferentiation and cell migration. J Cell Sci.

2002;115(Pt 15):3193–206.

21. Bates RC, Mercurio AM. Tumor necrosis factor-alpha stimulates

the epithelial-to-mesenchymal transition of human colonic orga-

noids. Mol Biol Cell. 2003;14(5):1790–800.

22. Rhyu DY, Yang Y, Ha H, Lee GT, Song JS, Uh ST, Lee HB.

Role of reactive oxygen species in tgf-beta1-induced mitogen-

activated protein kinase activation and epithelial-mesenchymal

transition in renal tubular epithelial cells. J Am Soc Nephrol.

2005;16(3):667–75.

23. Jung YS, Jeong EM, Park EK, Kim YM, Sohn S, Lee SH, Baik

EJ, Moon CH. Cadmium induces apoptotic cell death through p38

mapk in brain microvessel endothelial cells. Eur J Pharmacol.

2008;578(1):11–8.

24. Moriue T, Igarashi J, Yoneda K, Nakai K, Kosaka H, Kubota Y.

Sphingosine 1-phosphate attenuates h2o2-induced apoptosis in

endothelial cells. Biochem Biophys Res Commun. 2008;368(4):

852–7.

25. Nemoto S, Xiang J, Huang S, Lin A. Induction of apoptosis by

sb202190 through inhibition of p38beta mitogen-activated pro-

tein kinase. J Biol Chem. 1998;273(26):16415–20.

26. Tourian L Jr, Zhao H, Srikant CB. P38alpha, but not p38beta,

inhibits the phosphorylation and presence of c-flips in disc to

potentiate fas-mediated caspase-8 activation and type i apoptotic

signaling. J Cell Sci. 2004;117(Pt 26):6459–71.

27. Wang Y, Huang S, Sah VP, Ross J Jr, Brown JH, Han J, Chien

KR. Cardiac muscle cell hypertrophy and apoptosis induced by

distinct members of the p38 mitogen-activated protein kinase

family. J Biol Chem. 1998;273(4):2161–8.

28. Silva G, Cunha A, Gregoire IP, Seldon MP, Soares MP. The

antiapoptotic effect of heme oxygenase-1 in endothelial cells

involves the degradation of p38 alpha mapk isoform. J Immunol.

2006;177(3):1894–903.

29. Meyer-Ter-Vehn T, Gebhardt S, Sebald W, Buttmann M, Grehn

F, Schlunck G, Knaus P. P38 inhibitors prevent tgf-beta-induced

myofibroblast transdifferentiation in human tenon fibroblasts.

Invest Ophthalmol Vis Sci. 2006;47(4):1500–9.

30. Jung JW, Hwang SY, Hwang JS, Oh ES, Park S, Han IO. Ionising

radiation induces changes associated with epithelial-mesenchy-

mal transdifferentiation and increased cell motility of a549 lung

epithelial cells. Eur J Cancer. 2007;43(7):1214–24.

31. Yamanaka O, Saika S, Ohnishi Y, Kim-Mitsuyama S, Kamaraju

AK, Ikeda K. Inhibition of p38map kinase suppresses fibrogenic

reaction in conjunctiva in mice. Mol Vis. 2007;13:1730–9.

244 N. Kanaji et al.

123