Human cytomegalovirus inhibits Akt-mediated eNOS activation through upregulating PTEN (phosphatase...

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Cardiovascular Research

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Human cytomegalovirus inhibits Akt-mediated eNOS activation through

upregulating PTEN (phosphatase and tensin homolog deleted on

chromosome 10)

Ying H. Shen a,*, Lin Zhang, Budi Utama a, Jian Wang a, Yehua Gan a, Xinwen Wang a,

Jing Wang a, Li Chen a, Greg M. Vercellotti b, Joseph S. Coselli a,

Jawahar L. Mehta c, Xing Li Wang a,*

a Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Adult Cardiac Services, Texas Heart Institute,

St Luke’s Episcopal Hospital, Baylor College of Medicine, Houston Texas, United Statesb Departments of Medicine, University of Minneapolis, Minnesota, United States

c Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States

Received 17 August 2005; received in revised form 6 October 2005; accepted 21 October 2005

Available online 28 November 2005

Time for primary review 22 days

by guest on March 11, 2014

vascres.oxfordjournals.org/

Abstract

Objectives: Atherosclerosis is the leading cause of death in the United States, and human cytomegalovirus (HCMV) infection may play a

role in the development of this disease. Diminished expression and/or activity of endothelial nitric oxide synthase (eNOS) are an early event

in atherogenesis. In the current study, we investigated the effects of HCMV infection on eNOS activation in human aortic endothelial cells

(HAECs).

Methods and results: We found that HCMV inhibited eNOS phosphorylation/activation in HAECs. The signaling upstream of eNOS

involving Akt and PDK1 were also suppressed by the HCMV infection. Moreover, HCMV infection increased the expression of PTEN

(phosphatase and tensin homolog deleted on chromosome 10). Silencing PTEN expression with specific siRNA reversed the inhibitory

effects on eNOS activation in HCMV-infected cells indicating the involvement of PTEN in mediating HCMV’s inhibitory effects. Next we

observed that the activation of p38 MAPK stress signaling pathway mediates HCMV’s effects on PTEN up-regulation and eNOS

inactivation.

Conclusions: In summary, our findings suggest that inhibition of eNOS leading to endothelial dysfunction may be a basis of the pro-

atherogenic effects of HCMV. Importantly, upregulation of PTEN and activation of stress signal p38 MAPK are involved in HCMV’s

inhibitory effects on eNOS activation.

D 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved

Keywords: Cytomegalovirus; eNOS; Akt; PTEN; p38 MAPK

0008-6363/$ - see front matter D 2005 European Society of Cardiology. Publish

doi:10.1016/j.cardiores.2005.10.007

* Corresponding authors. MS NAB 2010, Baylor College of Medicine,

One Baylor Plaza, Houston, Texas 77030, United States. Tel.: +1 713 798

8406; fax: +1 713 798 1705.

E-mail addresses: [email protected]

(Y.H. Shen), [email protected] (X.L. Wang).

1. Introduction

There is increasing evidence that chronic infection with

certain organisms is associated with atherosclerosis [1].

Human cytomegalovirus (HCMV), a member of the

Herpesviridae family, is a widespread opportunistic patho-

gen that infects 50% to 90% of adult populations. In

addition to monocytes, endothelial cells presents a site of

latency for this virus [2–4]. Recent studies have indicated

69 (2006) 502 – 511

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that HCMV infection could be one of the causal factors for

atherosclerosis. Mouse CMV (MCMV) infection has been

shown to promote atherogenesis [5,6]. In humans, HCMV

DNA and antigens have been shown in atherosclerotic

lesions [7,8]. HCMV re-activation may be involved in the

pathogenesis of solid organ allograft rejection, and graft

atherosclerosis [9,10]. Existing HCMV infection appears to

be a strong independent risk factor for restenosis after

coronary atherectomy [11–13]. Although still far from

consistent, population studies have generally demonstrated

an association between HCMV seropositivity and the

increased risk of coronary atherosclerosis [2,3,14,15]. While

disturbed coagulation and inflammation could be involved

in HCMV-induced vasculogenesis, the mechanisms for

HCMV-induced vascular diseases are not well understood.

Understanding the mechanisms can lead to preventive and

therapeutic measures to eliminate this potentially curative

causal factor for vascular diseases.

Endothelium-derived NO, as an endogenous vasodilator,

prevents vascular inflammation and thrombus formation by

inhibiting platelet and leukocyte adherence. Reduced

eNOS expression and activity is a common feature in

various cardiovascular diseases including HCMV-seropos-

itive state [16]. It is, therefore, logical to hypothesize that

HCMV infection may promote atherogenesis by repressing

eNOS activation, resulting in endothelial dysfunction. In

this study, we examined this hypothesis, and explored

related signaling pathways. We found that HCMV sup-

presses Akt and eNOS activation, up-regulates PTEN, and

activates stress signaling p38 MAPK pathway. All these

signals could be related to the inhibitory effect of HCMV

infection in eNOS activity.

uest on March 11, 2014

2. Materials and methods

2.1. p38 MAPK siRNA and antibodies

PTEN siRNAwas obtained from Cell Signaling (Beverly,

MA). p38 MAPK siRNA and JNK siRNA were purchased

from Ambion (Austin, Texas). For Western blot analysis and

immunofluorescence staining, monoclonal and polyclonal

antibodies were obtained from Cell Signaling (Beverly,

MA) and anti-IE (HCMV immediate early gene) antibody

was purchased from Santa Cruz Biotechnology (Santa Cruz,

CA).

2.2. Cell culture

Primary human aortic endothelial cells (HAECs) were

obtained from Cell Applications (San Diego, CA) and

grown in EGM medium from Cambrex (East Rutherford,

NJ) containing basal media, 2% fetal bovine serum, growth

factors, cytokines, and supplements. Cells cultured up to

five passages were used in experiments. HAECs were

obtained in compliance with World Medical Association

Declaration of Helsinki in the Ethical Principles for Medical

Research Involving Human Subjects.

2.3. Infection of HAECs with HCMV

The VHL/E (a generous gift from Dr. Waldman) strain of

HCMV was used in the study [17]. Subconfluent HAEC

monolayers were infected with HCMV at multiplicity of

infection (MOI) 1 as described previously [18]. Supernatant

and cell fractions were harvested at various postinfection

(p.i.) times.

2.4. siRNAs and endothelial cell transfection

Silencing of PTEN and p38 MAPK gene expression in

primary aortic endothelial cells was achieved using the

siRNA technique. Transfection of HAECs was carried out

using LipofectAMINEi 2000 (Invitrogen, Carlsbad, Cal-

ifornia) according to the manufacturer’s instruction.

2.5. Western blot analysis

HAECs were collected from mock- and HCMV-

infected cells and washed with ice-cold PBS. Cells were

lysed in protein lysis buffer [20 mM Tris (pH 7.4), 150

mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5

mM sodium pyrophosphate, 1 mM h-glycerol phosphate,1 mM Na3VO4, 10 Ag/ml of each protease inhibitors

(aprotinin, leupeptin and pepstatin), and 1 mM phenyl-

methylsulfonyl fluoride] for 1 h on ice. Protein concen-

tration was measured by the Bradford method (Bio-Rad).

Fifteen Ag of protein per lane was separated by 10% or

12% SDS-polyacrylamide gels and transferred to PVDF

membranes. The membrane was blocked in 5% nonfat

powdered milk in TBST (50 mM Tris, pH 7.5, 150 mM

NaCl, 0.05% Tween 20). The membrane was incubated

with the primary antibody in 2% powdered milk in TBST,

washed extensively with TBST, and then incubated with

secondary anti-rabbit or anti-mouse horseradish peroxi-

dase-labeled antibody. Bands were visualized with ECL

(Amersham Biosciences, Piscataway, NJ) according to the

manufacturer’s instruction.

2.6. Real-time quantitative RT-PCR

Total RNA from treated cells was extracted with Trizol

(Invitrogen) according to the manufacturer’s protocol. The

mRNAs were reverse-transcribed into cDNAs with an

iScript cDNA synthesis kit (Bio-Rad) according to the

manufacturer’s instruction. Real-time PCR was performed

by using iCycler iQ real-time PCR detection system (Bio-

Rad). Primers were designed through Beacon Designer 2.0

software. The primers for human PTEN were, forward: 5V-CAAGATGATGTTTGAAACTATTCCAATG-3V and re-

verse: 5V-CCTTTAGCTGGCAGACCACAA-3V. The

mRNA levels were acquired from the value of threshold



Y.H. Shen et al. / Cardiovascular Research 69 (2006) 502–511504

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cycle (Ct) of the real-time PCR and normalized against the

house-keeping gene h-actin.

2.7. Immunofluorescence staining

For immunofluorescence assays, cells were grown on

glass coverslips, and infected. After infection, cells were

washed with PBS, fixed with 4% paraformaldehyde for 10

min, and permeabilized with 0.2% Triton X-100 for 5 min.

The coverslips were blocked with 1% BSA, incubated with

the primary antibody, washed extensively with PBS, and

then incubated with secondary anti-rabbit or anti-mouse

FITC or Texas Red-labeled antibody. One percent BSA in

PBS was used for blocking nonspecific binding sites and for

dilution of primary and secondary antibodies. The DNA dye

4V6VDiamidino-2-phenylindole dihydrochloride (DAPI) was

added at a concentration of 0.1 Ag/ml and incubated for 15

min to counterstain double-stranded DNA in nuclei. The

slides were examined with a Leica DMLS epifluorescent

microscope equipped with a Leica DC 100 digital camera

and the data was analyzed with Image-Pro Plus V4.5

software (Media Cybernetics, Inc).


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3. Results

3.1. HCMV inhibits eNOS activation

We infected HAECs with VHL/E, a clinical isolate

whose natural endothelial cytopathogenicity has been

preserved by propagation in endothelial cells. The eNOS

activation was then monitored in HCMV-infected cells by

measuring eNOS phosphorylation at ser 1177, which has

been shown to be involved in the activation of eNOS. As

shown in Fig. 1A, while the eNOS protein expression

remained unchanged, phosphorylated eNOS was dramati-

cally decreased in the infected cells. The inhibition started

as early as day 2 post-infection and was almost undetectable

by day 4 post-infection.

Insulin, which stimulates eNOS phosphorylation by

activating PI3K pathway, plays an important role in

regulating vascular function [19]. Therefore, we examined

whether HCMV infection impairs the insulin-stimulated

eNOS activation. HAECs, after infection with HCMV for 5

days, were stimulated with insulin for 30 min. As shown in

Fig. 1B, insulin stimulated eNOS phosphorylation in the

uninfected cells, but failed to stimulate eNOS phosphory-

lation in the infected cells.

3.2. HCMV inhibits Akt pathway

Since Akt activation enhances eNOS expression/activity

[20], we investigated whether HCMV infection inhibits

eNOS activation by suppressing Akt. Akt is activated by

phosphorylation at Ser-473 and Ser-308. We detected the

phosphorylation of Akt in HCMV-infected cells. As shown

in Fig. 2A, Akt phosphorylation at Ser-473 was inhibited by

HCMV infection in a time-dependent manner. However, the

total Akt level was not affected suggesting that HCMV

infection may block signaling upstream of Akt. Phosphoi-

nositide-dependent kinases PDKs (PDK1 and PDK2) are the

Akt upstream kinases, which directly phosphorylate Akt on

Thr-308 and Ser-473, respectively [21,22]. Accordingly, we

measured the phosphorylation of PDK1 in HCMV-infected

cells using anti-phospho-(Ser-241) PDK1 antibody. As

shown in Fig. 2B, PDK1 phosphorylation at Ser-241 was

inhibited by HCMV infection.

3.3. Up-regulation of the PTEN in HCMV-infected cells

Next we investigated the upstream molecules responsible

for the inhibition of eNOS activation. PTEN antagonizes

PI3K/Akt signaling by dephosphorylating PIP3 [23,24]. We,

therefore, examined whether PTEN is involved in the effects

of HCMV. We first measured PTEN activation in HCMV-

infected cells. Since PTEN can be activated by dephos-

phorylation at the C-terminal (Ser 380) [25–28], we

determined the PTEN phosphorylation at the Ser380/

Thr382/Thr383. As shown in Fig. 3A, HCMV infection

decreased PTEN phosphorylation at Ser380/Thr382/Thr383

indicating the activation of PTEN by HCMV infection. We

also showed that the PTEN protein was increased by

HCMV infection (Fig. 3A) and the increased expression

was specifically seen by immunofluorescence stains in the

HCMV-infected cells (Fig. 3B). Additionally, PTEN mRNA

levels in the infected cells were also significantly increased

(Fig. 3C) indicating that HCMV infection can induce PTEN

expression at mRNA level. Taken together, these data

indicate that HCMV infection can specifically induce PTEN

activation and expression.

3.4. The involvement of the PTEN in HCMV-induced

inhibition of eNOS phosphorylation and Akt activation

To examine whether the PTEN up-regulation was

involved in HCMV-induced inhibition of the Akt pathway

and eNOS activation, we blocked PTEN expression using

PTEN-specific siRNA. As shown in Fig. 4, HCMV

infection induced PTEN expression and reduced the

phosphorylation of Akt and eNOS (lane 2). PTEN siRNA

specifically inhibited the HCMV-induced PTEN upregula-

tion in a concentration-dependent manner (lane 3–5). As

PTEN expression was decreased, the inhibition of phos-

phorylation of Akt and eNOS was reversed, suggesting that

PTEN up-regulation is directly involved in HCMV-induced

inhibition of Akt signaling and eNOS activation.

3.5. HCMV infection leads to activation of p38 MAPK

pathway

To identify intracellular stress signaling pathways that

may mediate HCMV-induced inhibition of Akt signaling



Fig. 1. HCMV infection inhibited eNOS activation. (A) Reduced eNOS phosphorylation in HCMV-infected cells. HAECs were infected with VHL/E strain of

HCMV for indicated days and cell lysate was prepared. Phosphorylated eNOS was detected by Western blotting using anti-phospho (Ser-1177) eNOS antibody.

Membranes were stripped and reprobed for the total eNOS using anti-eNOS antibody. Representative blots and the meanTS.D. of phospho eNOS from five

independent experiments are shown. (B) Suppression of insulin-stimulated eNOS phosphorylation in HCMV-infected cells. Six days after infected with HCMV,

HAECs were starved in 0.5% FCS EBM for 6 h before stimulated with insulin 50 nM for 30 min. Cell lysate was prepared and the phosphorylation of eNOS at

the Ser-1177 and total eNOS were detected. Representative blots and the meanTS.D. of phospho eNOS from five independent experiments are shown.



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and eNOS activation, we examined the kinetics of p38

MAPK activation. As shown in Fig. 5A, exposure of

HAECs to HCMV stimulated p38 MAPK activity, whereas

JNK pathway, another MAPK kinase, remained unchanged

(data not shown). Next, we determined whether HCMV

infection could activate MKK3 and MKK6, two cellular

kinases that are known to phosphorylate p38 MAPK

[29,30]. MKK3 and MKK6 phosphorylation was activated

at late stage of the HCMV infection (Fig. 5B) suggesting

that increased MKK3/6 activity may account for p38 MAPK

activation at late stage of infection. The phosphorylation of

MLK3, an upstream kinase that can directly phosphorylate

MKK3/MKK6, was also increased in HCMV-infected cells

(Fig. 5B).

3.6. Role of p38 MAPK pathway in HCMV-induced eNOS

inhibition

To examine whether activation of p38 MAPK was

involved in HCMV-induced up-regulation of the PTEN



Fig. 2. HCMV infection inhibited Akt pathway. (A) Reduced Akt phosphorylation in HCMV infected cells. HAECs were infected with HCMV for different

time. Cell lysate was prepared. Phosphorylated Akt was detected by Western blotting using anti-phospho (Ser-473) Akt antibody. Membranes were stripped and

reprobed for the total Akt using anti-Akt antibody. Representative blots of three separate experiments are shown. (B) Reduced PDK1 phosphorylation in

HCMV infected cells. HAECs were infected with HCMV for different time. PDK1 phosphorylation was detected from the infected HAECs using anti-phospho

(Ser-241) PDK1 antibody. Total PDK1 was measured using anti-PDK1 antibody. Representative blots are shown.

Fig. 3. HCMV infection stimulated PTEN expression. (A) Increased PTEN activation and expression by HCMV infection. HAECs were infected with HCMV

and the cell lysate was prepared at different postinfection time. PTEN phosphorylation was determined by anti-phospho (Ser380/Thr382/Thr383) PTEN

antibody; and its expression was measured using anti-PTEN antibody. h-actin was measured as loading control. Representative blots from five independent

experiments are shown. (B) Increased PTEN expression in HCMV-infected cells. HCMV infected HAECs (day 6) were stained with anti-IE antibody-FITC

(green) and anti-PTEN antibody-Texas Red (red). Merged image shows co-localization. (C) HCMV infection increased PTEN mRNA. Total RNA from mock

and infected cells was extracted and the mRNAs were reverse-transcribed into cDNAs. PTEN mRNA levels were quantified by real-time PCR and normalized

to h-actin mRNA. Results are expressed as percentage of the control.




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Fig. 4. Involvement of PTEN in HCMV-induced Akt and eNOS inactivation. HAECs were infected with HCMV for 6 days before transfected with different

amounts of PTEN specific siRNA (25–150 nM). Twenty-four hours after transfection, total protein lysates from mock-infected and infected cells were

prepared and blotted with antibodies against PTEN, phosphorylated Akt and phosphorylated eNOS. h-actin was measured as loading control. Representative

blots of five separate experiments are shown.


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and inhibition of the insulin signaling, we suppressed p38

MAPK expression using p38 MAPK-specific siRNA. As

shown in Fig. 6, p38 MAPK expression was specifically

suppressed by p38 MAPK siRNA indicating that the siRNA

is efficient. Along with the reduction in p38 MAPK

expression, PTEN expression also fell, suggesting that p38

MAPK pathway is involved in HCMV-induced PTEN

expression. Furthermore, phosphorylation of Akt and eNOS

Fig. 5. HCMV infection activated p38 MAPK pathway. (A) Increase of phosphoryl

different times and cell lysate was prepared. Phosphorylated p38 MAPK were dete

antibody. The membrane was stripped and re-blotted with anti-p38 MAPK antib

separate experiments are shown. (B) Activation of p38 MAPK in HCMV-infected

FITC (green), anti-phospho (Thr-180/Tyr-182) p38 MAPK antibody-Texas Red

infection activated p38 MAPK pathway. The phosphorylation of MLK3 and MKK6

Ser-281) MLK3 antibody and anti-phospho (Ser-189/207) MKK3/MKK6 antibody

of five separate experiments are shown.

in HCMV-infected cells was completely reversed by the p38

MAPK siRNA treatment.

4. Discussion

In the present study, we have shown that HCMV

infection inhibits basal and insulin-stimulated eNOS phos-

ated p38 MAPK by HCMV infection. HAECs were infected with HCMV for

cted by Western blotting using anti-phospho (Thr-180/Tyr-182) p38 MAPK

ody. h-actin was measured as loading control. Representative blots of five

cells. HCMV-infected HAECs (day 6) were stained with anti-IE antibody-

(red) and DAPI (blue). Merged image shows co-localization. (C) HCMV

in mock and HCMV infected cells was detected by anti-phospho (Thr-277/

, respectively. h-actin was measured as loading control. Representative blots


ordjournals.org/



Fig. 7. Proposed pathway of the resistin-induced inhibition of eNOS

activation. HCMV induces activation of stress signaling MLK3/MKK3,6/

p38 MAPK pathway. The activated p38 MAPK can up-regulate the

expression of PTEN, which decreases PIP3 level that leads to reduced

activation of Akt and PDK1, hence inhibited eNOS activation.

Fig. 6. Involvement of p38 MAPK in HCMV-induced PTEN up-regulation and impairment of Akt and eNOS activation. HAECs were infected with HCMV for

6 days before transfected with different amount of p38 MAPK siRNA (10–150 nM). Twenty-four hours after transfection, total protein lysates from the treated

cells were prepared and blotted with antibodies against p38 MAPK, PTEN, phosphorylated Akt, Akt, phosphorylated eNOS, and eNOS. h-actin was measured

as loading control. Representative blots of five separate experiments are shown.




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phorylation and activation, which is likely to be the results

of the Akt inactivation through the up-regulation of PTEN

by the HCMV infection. Stress signal p38 MAPK appears to

be involved in the PTEN-triggered Akt and eNOS inacti-

vation in HCMV-infected cells. The significant eNOS

inhibition starts at 2 days post infection and persists to as

long as the experimental periods (8 days post infection). The

eNOS inactivation is accompanied by the simultaneous Akt

inactivation and p38 activation, which show a time-

dependent change in intensity and lags behind the changes

in eNOS. The proposed pathway by which HCMV infection

inhibits eNOS activation is shown in Fig. 7.

HCMV, a widespread opportunistic pathogen, causes

acute, latent, and chronic infections. Although the primary

infection may be asymptomatic in immunocompetent

individuals, the virus can cause a wide variety of severe

diseases in immunocompromised hosts. Involvement of

HCMV infection has been discovered in atherosclerosis

[14,16,31,32], thrombosis [33,34], allograft rejection

[35,36] and restenosis [37,38]. However, the precise

mechanisms of atherogenesis are not clear. Endothelium-

derived NO is an endogenous anti-atherogenic factor.

Deregulation of eNOS and subsequent decrease in NO

production are prominent features of various vascular

diseases and early events in atherogenesis.

Although it has been shown that HCMV-seropositive

individuals have impaired NO-dependent vasodilation [16],

little data is available about whether HCMV infection

interferes with eNOS activation. One recent study shows

that HCMV infection in human heart transplant recipients is

associated with high levels of endogenous inhibitor of NOS,

asymmetric dimethylarginine (ADMA).They found that

HCMV infection impairs dimethylarginine dimethylamino-

hydrolase (DDAH) activity that results in increased ADMA

[39]. Our results indicate that HCMV infection may also be

able to reduce NO production through inhibition of eNOS

phosphorylation/activation. As far as we are aware, our

study is the first to show the inhibition of eNOS activation

by HCMV infection. Since NO is a key molecule that plays






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critical role in regulating vascular function, we propose that

the reduction in eNOS activation as a result of HCMV

infection is a plausible link in accelerated atherogenesis in

HCMV-infected patients.

In this study, we also addressed the signal transduction

pathways for HCMV-induced inhibition of eNOS. As is

well-known, eNOS activity is regulated by post-translation-

al modifications through several signaling pathways, in-

cluding the ERK MAPK pathway [40], the AMP-activated

protein kinase (AMPK) pathway [41] and the Akt pathway.

Insulin induces eNOS activation through the IRS/PI3K/

PDK/Akt cascade, which plays a key role in insulin

signaling in target tissues. Our results show that HCMV

infection prevents eNOS activation by inhibiting Akt

pathway, although inhibition of other pathways by HCMV

infection cannot be ruled out. We have noted the report that

HCMV infection induces Akt phosphorylation in fibroblasts

[42]. While this discrepancy could be a cell specific

phenomenon, it could also result from differences in the

experimental conditions. It is also possible that HCMV

infection induces Akt phosphorylation in the early stages of

infection, and prolonged HCMV infection decreases Akt

phosphorylation. This dual effect was observed in the

occurrence of apoptosis in endothelial cells infected with

HCMV. In early stages of infection, HCMV was shown to

induce anti-apoptotic factors such as ERK MAPK and PI-

3K-signaling pathways that promotes cell survival [43,44];

however, in the later stages of infection, HCMV promoted

cell death [18]. Akt kinase, an established anti-apoptotic

factor, may be altered by HCMV infection to fit the needs of

cell regulation.

We further investigated the mechanisms for HCMV-

induced inhibition of the Akt signaling and eNOS activa-

tion. Our results show that HCMV infection can induce both

PTEN activation and expression. Importantly, silencing

PTEN by specific siRNA reversed the HCMV’s inhibitory

effects on Akt and eNOS activation, suggesting that the up-

regulated PTEN could be the mediator of these effects.

PTEN is a member of serine/threonine/tyrosine phosphatase

subfamily of protein phosphatases. It dephosphorylates

PtdIns (3,4,5)P3 into PtdIns(4,5)P2, and thus antagonizes

PI3K-dependent signaling pathways and Akt activation

[45]. PTEN has been shown to play critical role in

embryonic development, cell growth, apoptosis, differenti-

ation and migration [46]. PTEN is also widely expressed in

cardiomyocytes, fibroblasts, endothelial cells and vascular

smooth muscle cells [47], where it may modulate cell

survival/apoptosis, metabolism and function. Our finding

suggests that PTEN may be an important negative regulator

of the eNOS activation in vessel wall and be involved in the

development of endothelial dysfunction.

In searching for the mechanisms and pathways for

HCMV-induced PTEN upregulation and eNOS inhibition,

we found that HCMV infection activated stress signal p38

MAPK. To determine if HCMV infection also increased

activity of other MAPKs, we examined the activity of JNK

and found no evidence of significant increases for JNK

throughout the time course of the HCMV infection. This

observation indicates that the p38 MAPK activation is a

specific event. Inhibition of p38 MAPK fully reversed

HCMV-induced upregulation of PTEN and inhibition of Akt

and eNOS phosphorylation indicating the involvement of

p38 MAPK pathway in mediating HCMV’s effects. It is

known that p38 MAPK pathway plays an essential role in

regulating many cellular processes including inflammation,

cell differentiation, cell growth and death. Activation of p38

MAPK by extracellular stimuli such as bacterial pathogens

and cytokines triggers downstream responsive genes.

Several investigators have described the induction of the

p38 MAPK activation following HCMV infection [48,49].

Our observation provides important evidence for the cross

talk between stress signaling and Akt pathway, which leads

to eNOS inactivation in the HCMV-infected cells. Our

findings also indicate a novel mechanism that this cross talk

may be mediated by the PTEN upregulation.

In summary, we have demonstrated that HCMV infection

exerts an inhibitory effect on eNOS activation and Akt

signaling in endothelial cells. HCMV infection inhibits Akt

pathway by activating stress-response signal p38 MAPK

pathway, and up-regulating PTEN. HCMV-induced eNOS

inactivation may play an important role in endothelial

dysfunction and ensuing vascular diseases in those with

chronic or reactivated latent HCMV infection. The elucida-

tion of the molecular mechanisms leading to the inactivation

of Akt pathway and eNOS inhibition through PTEN and

stress signaling regulation will offer potential targets to

attenuate the HCMV-induced cytopathic effects, hence,

vascular diseases.

Acknowledgement

This project is supported by grants NIH/NHLBI R01-

HL071608 to XLW and AHA-TX 0565134Y to YHS.

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