Lipid Metabolism in Vascular Smooth Muscle Cells Infuenced by HCMV Infection.

BACKGROUND: The present study was designed to observe the infection of human cytomegalovirus (HCMV) to human vascular smooth muscle cells (VSMCs), and the effect of viral infection on lipid metabolism in VSMCs. METHODS: The cytopathic effects were observed by inverted microscopy and viral infection were examined by electron microscopy and RT-PCR. The lipid metabolism related gene profiling of VSMCs after HCMV infection was assayed by cDNA assay and the abnormal expression of genes were validated by quantitative RT-PCR. The content of cholesterol in VSMCs after HCMV infection was assayed by cholesterol detection kit. RESULTS: VSMCs showed obvious cytopathic effects after HCMV infection. Intact viral particles could be detected in VSMCs using electron microscope. By use of RT-PCR technology, IE gene of HCMV could be amplified from VSMCs. The expression of cell lipid metabolism related gene profiling showed obvious disorders. The expression levels of HMG-CoA synthase and HMG-CoA reductase after infection increased significantly. The cellular cholesterol content (µmol/106 cells) was significantly higher than that of mock infected group at 72h post infection. CONCLUSION: HCMV can infect VSMCs and the infection can affect cellular lipid metabolism related gene expression, which get involved in the occurrence and development of atherosclerosis (AS).

Endothelial cell permeability during hantavirus infection involves factor XII-dependent increased activation of the kallikrein-kinin system.

Hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) are diseases caused by hantavirus infections and are characterized by vascular leakage due to alterations of the endothelial barrier. Hantavirus-infected endothelial cells (EC) display no overt cytopathology; consequently, pathogenesis models have focused either on the influx of immune cells and release of cytokines or on increased degradation of the adherens junction protein, vascular endothelial (VE)-cadherin, due to hantavirus-mediated hypersensitization of EC to vascular endothelial growth factor (VEGF). To examine endothelial leakage in a relevant in vitro system, we co-cultured endothelial and vascular smooth muscle cells (vSMC) to generate capillary blood vessel-like structures. In contrast to results obtained in monolayers of cultured EC, we found that despite viral replication in both cell types as well as the presence of VEGF, infected in vitro vessels neither lost integrity nor displayed evidence of VE-cadherin degradation. Here, we present evidence for a novel mechanism of hantavirus-induced vascular leakage involving activation of the plasma kallikrein-kinin system (KKS). We show that incubation of factor XII (FXII), prekallikrein (PK), and high molecular weight kininogen (HK) plasma proteins with hantavirus-infected EC results in increased cleavage of HK, higher enzymatic activities of FXIIa/kallikrein (KAL) and increased liberation of bradykinin (BK). Measuring cell permeability in real-time using electric cell-substrate impedance sensing (ECIS), we identified dramatic increases in endothelial cell permeability after KKS activation and liberation of BK. Furthermore, the alterations in permeability could be prevented using inhibitors that directly block BK binding, the activity of FXIIa, or the activity of KAL. Lastly, FXII binding and autoactivation is increased on the surface of hantavirus-infected EC. These data are the first to demonstrate KKS activation during hantavirus infection and could have profound implications for treatment of hantavirus infections.

Human cytomegalovirus induces upregulation of arginase II: possible implications for vasculopathies.

Both human cytomegalovirus (HCMV) and arginase II (ARG II) have been implicated in the pathogenesis of cardiovascular diseases. The effects of HCMV on ARG II are unknown. The aim of this study was to investigate the effects of HCMV on ARG II expression in endothelial and vascular smooth muscle cells (SMC) both in vitro and ex vivo. Endothelial and SMC were infected with either HCMV or UV-irradiated HCMV. Expression of ARG II, endothelial or inducible nitric oxide synthase (eNOS and iNOS, respectively) and viral immediate early (IE) was quantified using quantitative PCR. Ganciclovir and short interfering RNA were used to determine the viral gene mediating the effects on ARG II. Detection of viral antigens and ARG II expression was performed by immunofluorescence or immunohistochemistry. HCMV infection increased both ARG II mRNA and protein levels in the examined cells; this effect was mediated by the HCMV IE2-p86 protein. The upregulation of ARG II was accompanied by a downregulation of eNOS but an induction of iNOS in HCMV-infected endothelial cells. Both eNOS and iNOS expressions were induced in HCMV-infected SMC. ARG II was abundantly expressed in endothelial cells, foam cells and SMC and was importantly significantly upregulated in HCMV-immunoreactive human carotid atherosclerotic plaques. HCMV IE2-p86 mediates ARG II upregulation in vitro and ARG II is co-expressed with HCMV antigens in human carotid atherosclerotic plaques. We speculate that HCMV may contribute to endothelial dysfunction via ARG II induction and reduced eNOS production.

Pseudotyping the adenovirus serotype 5 capsid with both the fibre and penton of serotype 35 enhances vascular smooth muscle cell transduction.

Ex vivo gene therapy during coronary artery bypass grafting (CABG) holds great potential to prevent excessive smooth muscle cell (SMC) proliferation, neointima formation and graft failure. The most successful preclinical strategies to date have utilised vectors based on the species C adenovirus, Ad5, which engages the Coxsackie and Adenovirus receptor (CAR) as its primary attachment receptor. Profiling receptors on human SMCs demonstrated the absence of CAR but substantial expression of the species B receptor CD46. We performed transduction experiments using Ad5 and the CD46-utilising adenovirus Ad35, and found Ad35 significantly more efficient at transducing SMCs. To evaluate whether transduction could be further augmented, we evaluated chimeric CD46-utilising Ad5/Ad35 vectors comprising the Ad5 capsid pseudotyped with the Ad35 fibre alone (Ad5/F35) or in combination with the Ad35 penton (Ad5/F35/P35). In human smooth muscle cells (hSMCs), Ad5/F35/P35 mediated significantly higher levels of transduction than either parental vector or Ad5/F35. Ex vivo transduction experiments using mouse aortas from CD46 transgenics demonstrated that Ad5/F35/P35 was significantly more efficient at transducing SMCs than the other vectors tested. Finally, ex vivo transduction and immunofluorescent colocalisation experiments using human tissue from CABG procedures confirmed the preclinical potential of Ad5/F35/P35 as an efficient vector for vascular transduction during CABG.

Viral Toll Like Receptor activation of pulmonary vascular smooth muscle cells results in endothelin-1 generation; relevance to pathogenesis of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare but fatal condition in which raised pulmonary vascular resistance leads to right heart failure and death. Endothelin-1 is a potent endogenous vasoconstrictor, which is considered to be central to many of the events that lead to PAH, and is an important therapeutic target in the treatment of the condition. In many cases of PAH, the aetiology is unknown but inflammation is increasingly thought to play an important role and viruses have been implicated in the development of disease. The Toll Like Receptors (TLRs) play a key role in innate immune responses by initiating specific anti-bacterial and anti-viral defences in recognition of signature molecular motifs on the surface of invading pathogens. In this study, we set out to examine the expression of bacterial and viral TLRs in human pulmonary artery smooth muscle cells and to establish whether their activation could be relevant to PAH. We found that the viral TLR3 and bacterial TLRs 4 and 6 were most abundantly expressed in human pulmonary artery smooth muscle cells. Using specific TLR ligands, we found that activation of TLRs 3 and 4 resulted in IL-8 release by human pulmonary artery smooth muscle cells but that only TLR3 stimulation resulted in IP10 and endothelin-1 release. These data suggest that human pulmonary artery smooth muscle cells express significant levels of viral TLR3 and respond to its activation by releasing endothelin-1. This may have importance in understanding the association between viruses and the development of PAH.

SARS-CoV-2 spike protein-mediated cell signaling in lung vascular cells.

Currently, the world is suffering from the pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses angiotensin-converting enzyme 2 (ACE2) as a receptor to enter the host cells. So far, 60 million people have been infected with SARS-CoV-2, and 1.4 million people have died because of COVID-19 worldwide, causing serious health, economical, and sociological problems. However, the mechanism of the effect of SARS-CoV-2 on human host cells has not been defined. The present study reports that the SARS-CoV-2 spike protein alone without the rest of the viral components is sufficient to elicit cell signaling in lung vascular cells. The treatment of human pulmonary artery smooth muscle cells or human pulmonary artery endothelial cells with recombinant SARS-CoV-2 spike protein S1 subunit (Val16 - Gln690) at 10 ng/ml (0.13 nM) caused an activation of MEK phosphorylation. The activation kinetics was transient with a peak at 10 min. The recombinant protein that contains only the ACE2 receptor-binding domain of the SARS-CoV-2 spike protein S1 subunit (Arg319 - Phe541), on the other hand, did not cause this activation. Consistent with the activation of cell growth signaling in lung vascular cells by the SARS-CoV-2 spike protein, pulmonary vascular walls were found to be thickened in COVID-19 patients. Thus, SARS-CoV-2 spike protein-mediated cell growth signaling may participate in adverse cardiovascular/pulmonary outcomes, and this mechanism may provide new therapeutic targets to combat COVID-19.

Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product.

Vascular smooth muscle cell (SMC) proliferation in response to injury is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis after balloon angioplasty. The retinoblastoma gene product (Rb) is present in the unphosphorylated and active form in quiescent primary arterial SMCs, but is rapidly inactivated by phosphorylation in response to growth factor stimulation in vitro. A replication-defective adenovirus encoding a nonphosphorylatable, constitutively active form of Rb was constructed. Infection of cultured primary rat aortic SMCs with this virus inhibited growth factor-stimulated cell proliferation in vitro. Localized arterial infection with the virus at the time of balloon angioplasty significantly reduced SMC proliferation and neointima formation in both the rat carotid and porcine femoral artery models of restenosis. These results demonstrate the role of Rb in regulating vascular SMC proliferation and suggest a gene therapy approach for vascular proliferative disorders associated with arterial injury.

Requirement for generation of H2O2 for platelet-derived growth factor signal transduction.

Stimulation of rat vascular smooth muscle cells (VSMCs) by platelet-derived growth factor (PDGF) transiently increased the intracellular concentration of hydrogen peroxide (H2O2). This increase could be blunted by increasing the intracellular concentration of the scavenging enzyme catalase or by the chemical antioxidant N-acetylcysteine. The response of VSMCs to PDGF, which includes tyrosine phosphorylation, mitogen-activated protein kinase stimulation, DNA synthesis, and chemotaxis, was inhibited when the growth factor-stimulated rise in H2O2 concentration was blocked. These results suggest that H2O2 may act as a signal-transducing molecule, and they suggest a potential mechanism for the cardioprotective effects of antioxidants.

EV71 induces VCAM-1 expression via PDGF receptor, PI3-K/Akt, p38 MAPK, JNK and NF-kappaB in vascular smooth muscle cells.

Enterovirus 71 (EV71) is a widespread virus that causes severe and fatal diseases in patients, including circulation failure. The mechanisms underlying EV71-initiated intracellular signaling pathways to influence host cell functions remain unknown. In this study, we identified a requirement for PDGFR, PI3-K/Akt, p38 MAPK, JNK, and NF-kappaB in the regulation of VCAM-1 expression by rat vascular smooth muscle cells (VSMCs) in response to viral infection. EV71 induced VCAM-1 expression in a time- and viral concentration-dependent manner. Infection of VSMCs with EV71 stimulated VCAM-1 expression and phosphorylation of PDGFR, Akt, and p38 MAPK which were attenuated by AG1296, wortmannin, and SB202190, respectively. The phosphorylation of JNK stimulated by EV71 was not detected under present conditions. In contrast, JNK inhibitor SP600125 inhibited EV71-induced VCAM-1 expression. Furthermore, VCAM-1 expression induced by EV71 was significantly attenuated by a selective NF-kappaB inhibitor (helenalin). Consistently, EV71-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha as well as VCAM-1 mRNA expression was blocked by helenalin, AG1296, SB202190, SP600125, wortmannin, and LY294002. Moreover, the involvement of p38 MAPK, PI3-K/Akt, and NF-kappaB in EV71-induced VCAM-1 expression was reveled by that transfection with dominant negative plasmids of p38 MAPK, p85, Akt, NIK, IKK-alpha, and IKK-beta attenuated these responses. These findings suggest that in VSMCs, EV71-induced VCAM-1 expression was mediated through activation of PDGFR, PI3-K/Akt, p38 MAPK, JNK, and NF-kappaB pathways.

The expression of human brain vascular smooth muscle cell AT receptor after the UL83 gene of HCMV inhibition by small interfering RNAs.

OBJECTIVES: To explore the role of angiotensin receptors (AT1 and AT2) and human cytomegalovirus (HCMV) infection in atherosclerosis, and to observe effect of HCMV UL83 gene on angiotensin receptor AT receptors. METHODS: Cell model of RNA interference (RNAi) HCMV UL83 gene was set up by transfection RNAi into HCMV infected human cerebral artery vascular smooth muscle cells cultured in vitro by application of transfection technique and adoption of RT-PCR, etc. Effect of HCMV on gene expression of AT1 and AT2 receptors in human cerebral artery vascular smooth muscle cells cultured in vitro under the action of Ganciclovir (GCV), AT1 and AT2 receptor antagonists by immunofluorescence technique, RT-PCR and protein electrophoresis. RESULTS: The duplication and protein expression of HCMV UL83 gene mRNA was successfully interfered using RNAi technique. After silencing HCMV UL83 gene, gene expression of AT1 receptor was down-regulated while that of AT2 was up-regulated. AT1 and AT2 receptor antagonists and GCV, under certain condition, could inhibit the activation on AT1 and AT2 receptor in human cerebral artery vascular smooth muscle cells induced by HCMV infection. CONCLUSION: Cell model of HCMV infected UL83 silence gene was successfully set up in human cerebral artery vascular smooth muscle cells cultured in vitro. HCMV UL83 might play a role by activating AT1 receptor and inhibiting expression of AT2 receptor.

Downregulation of the α1- and ß1-subunit of sGC in Arterial Smooth Muscle Cells of OPSCC Is HPV-Independent.

The nitric oxide (NO)-sensitive soluble guanylyl cyclase (sGC) is a heterodimeric enzyme with an α and ß subunit. NO binds to heme of the ß1-subunit of sGC, activates the enzyme in the reduced heme iron state in vascular smooth muscle cells (VSMCs), and generates cGMP-inducing vasodilatation and suppression of VSMC proliferation. In the complex tumor milieu with higher levels of reactive oxygen species (ROS), sGC heme iron may become oxidized and insensitive to NO. To change sGC from an NO-insensitive to NO-sensitive state or NO-independent manner, protein expression of sGC in VSMC is required. Whether sGCα1ß1 exists at the protein level in arterial VSMCs of oropharyngeal squamous cell carcinoma (OPSCC) is unknown. In addition, whether differences in the genetic profile between human papillomavirus (HPV)-positive and HPV-negative OPSCC contributes to the regulation of sGCα1ß1 is unclear. Therefore, we compared the effects of HPV-positive and HPV-negative OPSCC on the expression of sGCα1ß1 in arterial VSMCs from tumor-free and tumor-containing regions of human tissue sections using quantitative immunohistochemistry. In comparison to the tumor-free region, we found a decrease in expression of both α1- and ß1-subunits in the arterial VSMC layer of the tumor-containing areas. The OPSCC-induced significant downregulation of the α1- and ß1-subunits of sGC in arterial VSMC was HPV-independent. We conclude that the response of sGC to NO in tumor arterial VSMCs may be impaired by oxidation of the heme of the ß1-subunit, and thus, α1- and ß1-subunits of sGC could be targeted to degradation under oxidative stress in OPSCC in an HPV-independent manner. The degradation of sGCα1ß1 in VSMCs may result in increased proliferation of VSMCs, promoting tumor arteriogenesis in OPSCC. This can be interrupted by preserving the active heterodimer sGCα1ß1 in arterial VSMCs.

Targeted gene expression analyses and immunohistology suggest a pro-proliferative state in tricuspid aortic valve-, and senescence and viral infections in bicuspid aortic valve-associated thoracic aortic aneurysms.

BACKGROUND AND AIMS: Despite the potential life-threatening consequences of thoracic aortic aneurysms (TAAs), the pathogenesis of these diseases is still poorly understood. While some aspects of TAA formation have been elucidated, the role of vascular smooth muscle cells (SMCs) in both bicuspid aortic valve (BAV)-associated and degenerative tricuspid aortic valve (TAV)-associated TAAs has not yet been fully unravelled. Thus, this work was aimed at uncovering processes in SMC biology that may contribute to TAA formation. METHODS: Using isolated SMCs and tissue samples from TAAs linked to BAV syndrome, TAV-associated degenerative TAAs and control aortas, we performed targeted mRNA expression profile analyses and conducted immunohistological analyses on aortic wall tissue sections. RESULTS: While SMC expression profiles and tissue analyses in TAV-TAAs clearly point toward a pro-proliferative state of the aortic media SMCs, BAV-TAA SMCs and tissue provide evidence for DNA damage, DNA damage response signalling as well as profound TLR-3 signalling. CONCLUSIONS: The data presented in this study emphasizes the importance of SMCs in TAA development. Furthermore, our results provide evidence that the state of SMCs in the BAV-TAA (senescent) and TAV-TAA (pro-proliferative) differs significantly. For the first time, we also present findings that may argue for the occurrence of a viral infection in BAV-TAA SMCs.

Mechanism of rhinovirus-induced changes in airway smooth muscle responsiveness.

An important interplay exists between specific viral respiratory infections and altered airway responsiveness in the development and exacerbations of asthma. However, the mechanistic basis of this interplay remains to be identified. This study addressed the hypothesis that rhinovirus (RV), the most common viral respiratory pathogen associated with acute asthma attacks, directly affects airway smooth muscle (ASM) to produce proasthmatic changes in receptor-coupled ASM responsiveness. Isolated rabbit and human ASM tissue and cultured ASM cells were inoculated with human RV (serotype 16) or adenovirus, each for 6 or 24 h. In contrast to adenovirus, which had no effect, inoculation of ASM tissue with RV induced heightened ASM tissue constrictor responsiveness to acetylcholine and attenuated the dose-dependent relaxation of ASM to beta-adrenoceptor stimulation with isoproterenol. These RV-induced changes in ASM responsiveness were largely prevented by pretreating the tissues with pertussis toxin or with a monoclonal blocking antibody to intercellular adhesion molecule-1 (ICAM-1), the principal endogenous receptor for most RVs. In extended studies, we found that the RV-induced changes in ASM responsiveness were associated with diminished cAMP accumulation in response to dose-dependent administration of isoproterenol, and this effect was accompanied by autologously upregulated expression of the Gi protein subtype, Gialpha3, in the ASM. Finally, in separate experiments, we found that the RV-induced effects on ASM responsiveness were also accompanied by autologously induced upregulated mRNA and cell surface protein expression of ICAM-1. Taken together, these findings provide new evidence that RV directly induces proasthmatic phenotypic changes in ASM responsiveness, that this effect is triggered by binding of RV to its ICAM-1 receptor in ASM, and that this binding is associated with the induced endogenously upregulated expression of ICAM-1 and enhanced expression and activation of Gi protein in the RV-infected ASM.

Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty.

Vascular smooth muscle cell (VSMC) proliferation after arterial injury is important in the pathogenesis of a number of vascular proliferative disorders, including atherosclerosis and restenosis after balloon angioplasty. Thus, a better understanding of the molecular mechanisms underlying VSMC proliferation in response to arterial injury would have important therapeutic implications for patients with atherosclerotic vascular disease. The p21 protein is a negative regulator of mammalian cell cycle progression that functions both by inhibiting cyclin dependent kinases (CDKs) required for the initiation of S phase, and by binding to and inhibiting the DNA polymerase delta co-factor, proliferating cell nuclear antigen (PCNA). In this report, we show that adenovirus-mediated over-expression of human p21 inhibits growth factor-stimulated VSMC proliferation in vitro by efficiently arresting VSMCs in the G1 phase of the cell cycle. This p21-associated cell cycle arrest is associated both with significant inhibition of the phosphorylation of the retinoblastoma gene product (Rb) and with the formation of complexes between p21 and PCNA in VSMCs. In addition, we demonstrate that localized arterial infection with a p21-encoding adenovirus at the time of balloon angioplasty significantly reduced neointimal hyperplasia in the rat carotid artery model of restenosis. Taken together, these studies demonstrate the important role of p21 in regulating Rb phosphorylation and cell cycle progression in VSMC, and suggest a novel cytostatic gene therapy approach for restenosis and related vascular proliferative disorders.

Pertussis toxin-sensitive G proteins as mediators of the signal transduction pathways activated by cytomegalovirus infection of smooth muscle cells.

We demonstrated recently that the arachidonic acid (AA) cascade is involved in cytomegalovirus (CMV)-induced generation of reactive oxygen species (ROS) and the activation of nuclear factor (NF)-kappaB in human smooth muscle cells (SMCs). Since AA release from neutrophils is mediated by pertussis toxin (PTx)-sensitive guanine nucleotide-binding (G) proteins, we hypothesized by analogy that CMV stimulates ROS generation in SMCs and ultimately activates NF-kappaB via a PTx-sensitive G protein-coupled pathway. Our first test of this hypothesis demonstrated that PTx blocked AA release induced by CMV infection of SMCs, as well as blocked the terminal products of this reaction, ROS generation and NF-kappaB activation. More proximal components of the pathway were then examined. CMV infection increased phosphorylation and activity of cytosolic phospholipase A2 (cPLA2), an enzyme causing AA release; these effects were inhibited by PTx. CMV infection activated mitogen-activated protein (MAP) kinase, a key enzyme for cPLA2 phosphorylation, an effect also inhibited by PTx. Finally, inhibition of MAP kinase kinase (MAPKK), which phosphorylates and thereby activates MAP kinase, inhibited CMV-induced ROS generation. These data demonstrate that a PTx-sensitive G protein-dependent signaling pathway mediates cellular effects of CMV infection of SMCs. The downstream events include phosphorylation and activation of MAP kinase by MAPKK and subsequent phosphorylation and activation of cPLA2 (with its translocation to cell membranes), followed by stimulation of the AA cascade, which generates intracellular ROS and thereby activates NF-kappaB.

Human Cytomegalovirus-Encoded Receptor US28 Is Expressed in Renal Allografts and Facilitates Viral Spreading In Vitro.

BACKGROUND: Renal transplantation is the preferred treatment for patients with end-stage renal disease. Human cytomegalovirus (HCMV) activation is associated with decreased renal graft function and survival. Human cytomegalovirus encodes several immune modulatory proteins, including the G protein-coupled receptor US28, which scavenges human chemokines and modulates intracellular signaling. METHODS: Our aim was to identify the expression and localization of US28 in renal allograft biopsies by immunohistochemistry and determine its role in viral spreading in vitro. RESULTS: Immunohistochemistry revealed US28 in 31 of 34 renal transplant biopsies from HCMV-seropositive donors. Expression was independent of HCMV viremia or IgG serostatus. US28 was predominantly expressed in the cytoplasm of vascular smooth muscle cells (VSMCs) and tubular epithelial cells, with a median positivity of 20% and 40%, respectively. Also, US28-positive cells were present within arterial neointima. In contrast to US28, HCMV-encoded immediate early antigen was detected in less than 5% of VSMCs, tubular epithelial cells, interstitial endothelium, interstitial inflammatory infiltrates, and glomerular cells.Primary VSMCs were infected with green fluorescent protein-tagged wild type or US28-deficient HCMV. The viral spreading of US28-deficient HCMV, via culture medium or cell-to-cell transmission, was significantly impeded as shown by green fluorescent protein (ie, infected) cell quantification and quantitative real-time polymerase chain reaction. Additionally, the number and size of foci was smaller. CONCLUSIONS: In summary, HCMV-encoded US28 was detected in renal allografts from HCMV-positive donors independent of viremia and serostatus. Also, US28 facilitates HCMV spreading in VSMCs in vitro. Because the vasculature is affected in chronic renal transplant dysfunction, US28 may provide a potential target for therapeutic intervention.

Adenovirus-mediated transfer of human placental ectonucleoside triphosphate diphosphohydrolase to vascular smooth muscle cells suppresses platelet aggregation in vitro and arterial thrombus formation in vivo.

BACKGROUND: Platelet-rich thrombus formation is a critical event in the onset of cardiovascular disease. Because ADP plays a significant role in platelet aggregation, its metabolism is important in the regulation of platelet activation and recruitment. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) is a key enzyme involved in vascular ADP metabolism. We recently isolated 2 isoforms of E-NTPDase from the human placenta. The present study examined whether these isoforms suppress platelet aggregation and thrombus formation after adenovirus-mediated gene transfer to vascular smooth muscle cells (SMCs). METHODS AND RESULTS: We constructed adenovirus vectors expressing human placental E-NTPDase isoforms I (AdPlac I) and II (AdPlac II) or bacterial beta-galactosidase (AdLacZ). Vascular SMCs infected with AdPlac I expressed significant NTPDase activity and inhibited the platelet aggregation induced by ADP and collagen in vitro. In contrast, SMCs infected with AdPlac II and AdLacZ did not exert antiplatelet effects. To investigate the antithrombotic and antiproliferative effects of placental E-NTPDase isoform I in vivo, we generated thrombosis in rat carotid arteries by systemically administered rose Bengal and transluminal green light 5 days after gene transfer and examined neointimal growth 3 weeks after thrombus formation. Blood flow in AdLacZ-infected arteries rapidly deteriorated and vanished within 96+/-18 seconds of occlusive thrombus formation. In contrast, blood flow in AdPlac I-infected arteries was preserved for at least 10 minutes during irradiation. In addition, thrombus formation and subsequent neointimal growth were obviously suppressed. CONCLUSIONS: The local expression of placental E-NTPDase in injured arteries might prevent arterial thrombosis and subsequent neointimal growth.

HCMV infection of human vascular smooth muscle cells leads to enhanced expression of functionally intact PDGF beta-receptor.

BACKGROUND: Cytomegaloviruses have been shown to promote atherogenesis in animal models. In humans, several epidemiological and clinical studies suggest involvement of the human cytomegalovirus (HCMV) in the development of atherosclerosis. HCMV is suspected to be associated with an enhanced restenosis rate and the occurrence of vasculopathies after solid organ transplantation. However, knowledge about the cellular and molecular bases of these findings is very limited. METHODS AND RESULTS: Human coronary artery smooth muscle cells (HCASMC) were successfully infected with HCMV in vitro. Infection of HCASMC with all HCMV strains analyzed resulted in a substantial upregulation of the beta-receptor of platelet-derived growth factor (PDGFR-beta) expression as demonstrated by immunohistochemistry, immunofluorescence, FACS, and Western blot analysis. The amount of PDGFR-beta protein present in HCASMC rapidly increased after 12 h of infection and this difference persisted for 72 h post-infection. We showed by quantitative FACS analysis that the extent of PDGFR-beta upregulation differed significantly between the HCMV strains TB40E, Toledo, and AD169. The expression of insulin-like growth factor receptors as well as hepatocyte growth factor receptors, however, was down-modulated in HCMV-infected HCASMC. Most importantly, the HCMV-associated upregulation of PDGFR-beta protein resulted in functionally intact receptors. A significantly higher increase of proliferative activity following stimulation with PDGF-BB was observed in HCMV-infected HCASMC compared to the uninfected control. CONCLUSIONS: Our data suggest that HCMV directly activates the PDGF system, which could promote atherogenesis and restenosis by activation of smooth muscle cell proliferation and neointima formation.

Cytomegalovirus infection of rats increases the neointimal response to vascular injury without consistent evidence of direct infection of the vascular wall.

BACKGROUND: Previous studies suggest that infection may play a role in restenosis and atherogenesis; cytomegalovirus (CMV) is one of the implicated pathogens. To determine a potential causal role of CMV in these disease processes, we assessed whether CMV infection increases the neointimal response to injury of the rat carotid artery. METHODS AND RESULTS: Carotid injury was performed on 60 rats; immediately thereafter, 30 rats were infected with rat CMV, and the other 30 were mock-infected. Six weeks later, rats were euthanized, and the salivary glands, spleen, and carotid arteries were harvested. CMV infection was associated with significant exacerbation of the neointimal response to injury (neointimal to medial ratio 0.81+/-0. 59 versus 0.31+/-0.38 in CMV-infected versus control rats; P<0.0001). This occurred despite absence of infectious virus from vascular tissues and detection of CMV DNA by polymerase chain reaction in the injured artery only at day 3 after infection. Persistent distant infection, associated with systemic cytokine response, was evidenced by isolation of infectious virus from homogenates of both salivary glands and spleen and by higher serum levels of interleukin (IL)-2 and IL-4 (but not interferon-gamma and tumor necrosis factor-alpha) in infected versus noninfected rats. CONCLUSIONS: CMV infection of immunocompetent adult rats increases the neointimal response to vascular injury, suggesting that CMV may play a causal role in atherosclerosis/restenosis. Importantly, this CMV-induced response occurs even without the presence of virus in the vascular wall, suggesting that inflammatory and immune responses to infection of nonvascular tissues may contribute to the vascular response to injury.

Expression of tissue inhibitor of matrix metalloproteinases 1 by use of an adenoviral vector inhibits smooth muscle cell migration and reduces neointimal hyperplasia in the rat model of vascular balloon injury.

BACKGROUND: Cell migration is a major contributor to injury-induced neointimal hyperplasia and depends on alteration of the proteolytic balance within the arterial wall toward matrix breakdown. This is partly mediated by the matrix metalloproteinases (MMPs) and their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). METHODS AND RESULTS: An increase in expression of biologically active and immunoreactive TIMP-1 was seen in vitro after infection of rat smooth muscle cells (SMCs) with Av1.TIMP1 (an adenoviral vector containing the human TIMP1 cDNA). Infection of rat SMCs with Av1.TIMP1 reduced migration in vitro by 27% compared with control virus-infected cells (37.6+/-4.34 versus 51+/-5.01 cells per high-power field, P<0.05). The adenoviral vector was delivered to the injured rat carotid artery, and 4 days later, immunoreactive protein was identified and migration of SMCs reduced by 60% (5.2+/-0. 5 versus 12.8+/-1.5 cells per section, P<0.05, n=5). Neointimal area 14 days after injury showed a 30% reduction in the animals receiving the Av1.TIMP1 virus compared with controls (0.09+/-0.01 versus 0. 14+/-0.01 mm2, P=0.02, n=14). CONCLUSIONS: The response to arterial balloon injury involves MMP-dependent SMC migration and can be attenuated in vivo by the transmural expression of TIMP-1 by adenoviral gene transfer.