Nipah Virus Efficiently Replicates in Human Smooth Muscle Cells without Cytopathic Effect.

Nipah virus (NiV) is a highly pathogenic zoonotic virus with a broad species tropism, originating in pteropid bats. Human outbreaks of NiV disease occur almost annually, often with high case-fatality rates. The specific events that lead to pathogenesis are not well defined, but the disease has both respiratory and encephalitic components, with relapsing encephalitis occurring in some cases more than a year after initial infection. Several cell types are targets of NiV, dictated by the expression of the ephrin-B2/3 ligand on the cell's outer membrane, which interact with the NiV surface proteins. Vascular endothelial cells (ECs) are major targets of infection. Cytopathic effects (CPE), characterized by syncytia formation and cell death, and an ensuing vasculitis, are a major feature of the disease. Smooth muscle cells (SMCs) of the tunica media that line small blood vessels are infected in humans and animal models of NiV disease, although pathology or histologic changes associated with antigen-positive SMCs have not been reported. To gain an understanding of the possible contributions that SMCs might have in the development of NiV disease, we investigated the susceptibility and potential cytopathogenic changes of human SMCs to NiV infection in vitro. SMCs were permissive for NiV infection and resulted in high titers and prolonged NiV production, despite a lack of cytopathogenicity, and in the absence of detectable ephrin-B2/3. These results indicate that SMC might be important contributors to disease by producing progeny NiV during an infection, without suffering cytopathogenic consequences.

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.

In vivo characterization of target cells for acute elephant endotheliotropic herpesvirus (EEHV) infection in Asian elephants (Elephas maximus).

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is a dangerous viral infectious disease in young Asian elephants. Despite hypotheses underlying pathogenesis of the disease, it is unclear which cell types the virus targets during acute or persistent infections. This study investigated the tissues and target cells permissive for EEHV infection and replication in vivo. Rabbit polyclonal antibodies against the non-structural proteins of EEHV, DNA polymerase (EEHV DNAPol), were generated and validated. These were used to examine EEHV infection and replication in various tissues of acute EEHV-HD cases and compared to an EEHV-negative control. The results indicated that viral antigens were distributed throughout the epithelia of the alimentary tract and salivary glands, endothelia and smooth muscle cells, and monocytic lineage cells of the EEHV-infected elephants. Moreover, EEHV DNAPol proteins were also found in the bone marrow cells of the EEHV1A-HD and EEHV1A/4-HD cases. This study demonstrated for the first time the target cells that favor in vivo EEHV replication during acute infection, providing a promising foundation for investigating EEHV propagation in vitro.

Dengue Virus Infection of Primary Human Smooth Muscle Cells.

Dengue virus (DENV) infection of humans is presently the most important arthropod-borne viral global threat, for which no suitable or reliable animal model exists. Reports addressing the effect of DENV on vascular components other than endothelial cells are lacking. Dengue virus infection of vascular smooth muscle cells, which play a physiological compensatory response to hypotension in arteries and arterioles, has not been characterized, thus precluding our understanding of the role of these vascular components in dengue pathogenesis. Therefore, we studied the permissiveness of primary human umbilical artery smooth muscle cells (HUASMC) to DENV 1-4 infection and compared with the infection in the previously reported primary human umbilical vein endothelial cells (HUVEC) and the classically used, non-transformed, and highly permissive Lilly Laboratories Cell-Monkey Kidney 2 cells. Our results show that HUASMC are susceptible and productive to infection with the four DENV serotypes, although to a lesser extent when compared with the other cell lines. This is the first report of DENV permissiveness in human smooth muscle cells, which might represent an unexplored pathophysiological contributor to the vascular collapse observed in severe human dengue infection.

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.

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.

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).

Rhinoviral stimuli, epithelial factors and ATP signalling contribute to bronchial smooth muscle production of IL-33.

BACKGROUND: Bronchial smooth muscle cells (BSMCs) from severe asthmatics have been shown to overexpress the Th2-driving and asthma-associated cytokine IL-33. However, little is known regarding factors involved in BSMC production of IL-33. Rhinovirus (RV) infections cause asthma exacerbations, which exhibit features of Th2-type inflammation. Here, we investigated the effects of epithelial-derived media and viral stimuli on IL-33 expression in human BSMCs. METHODS: Primary human BSMCs from healthy (n = 3) and asthmatic (n = 3) subjects were stimulated with conditioned media from primary human bronchial epithelial cells (BECs), double-stranded (ds)RNA, dsRNA/LyoVec, or infected with RV. BSMCs were also pretreated with the purinergic receptor antagonist suramin. IL-33 expression was analysed by RT-qPCR and western blot and ATP levels were determined in cell supernatants. RESULTS: RV infection and activation of TLR3 by dsRNA increased IL-33 mRNA and protein in healthy and asthmatic BSMCs. These effects were inhibited by dexamethasone. BSMC expression of IL-33 was also increased by stimulation of RIG-I-like receptors using dsRNA/LyoVec. Conditioned media from BECs induced BSMC expression of IL-33, which was further enhanced by dsRNA. BEC-derived medium and viral-stimulated BSMC supernatants exhibited elevated ATP levels. Blocking of purinergic signalling with suramin inhibited BSMC expression of IL-33 induced by dsRNA and BEC-derived medium. CONCLUSIONS: RV infection of BSMCs and activation of TLR3 and RIG-I-like receptors cause expression and production of IL-33. Epithelial-released factor(s) increase BSMC expression of IL-33 and exhibit positive interaction with dsRNA. Increased BSMC IL-33 associates with ATP release and is antagonised by suramin. We suggest that epithelial-derived factors contribute to baseline BSMC IL-33 production, which is further augmented by RV infection of BSMCs and stimulation of their pathogen-recognising receptors.

Evidence for the Use of Multiple Mechanisms by Herpes Simplex Virus-1 R7020 to Inhibit Intimal Hyperplasia.

Intimal hyperplasia (IH) is the primary cause of vein bypass graft failure. The smooth muscle cell (SMC) is a key element of IH as it phenotypically switches from a contractile to a synthetic state which can become pathological. R7020, which is an engineered strain of Herpes Simplex Virus-1, inhibits IH in animal models. Although it has many characteristics which make it a strong candidate for use as a prophylactic agent how it inhibits IH is not well understood. The objective of this study was to identify modes of action used by R7020 to function in blood vessels that may also contribute to its inhibition of IH. The cytopathic effect of R7020 on SMCs was determined in vitro and in a rabbit IH model. In vitro assays with R7020 infected SMCs were used to quantify the effect of dose on the release kinetics of the virus as well as the effects of R7020 on cell viability and the adhesion of peripheral blood mononuclear cells (PBMCs) to SMCs in the absence and presence of tumor necrosis factor alpha (TNF-α). The observed cytopathic effect, which included R7020 positive filopodia that extend from cell to cell and the formation of syncytia, suggests that R7020 remains cell associated after egress and spreads cell to cell instead of by diffusion through the extracellular fluid. This would allow the virus to rapidly infect vascular cells while evading the immune system. The directionality of the filopodia in vivo suggests that the virus preferentially travels from the media towards the intima targeting SMCs that would lead to IH. The formation of syncytia would inhibit SMC proliferation as incorporated cells are not able to multiply. It was also observed that R7020 induced the fusion of PBMCs with syncytia suggesting the virus may limit the effect of macrophages on IH. Furthermore, R7020 inhibited the proliferative effect of TNF-α, an inflammatory cytokine associated with increased IH. Thus, the results of this study suggest that R7020 inhibits IH through multiple mechanisms.

HIV-associated large-vessel vasculopathy: a review of the current and emerging clinicopathological spectrum in vascular surgical practice.

An established relationship exists between human immunodeficiency virus (HIV) and the vascular system, which is characterised by clinical expressions of aneurysmal and occlusive disease that emanate from a common pathological process. The exact pathogenesis is currently unknown; attempts to implicate opportunistic pathogens have been futile. Theories converge on leucocytoclastic vasculitis with the vaso vasora as the vasculopathic epicentre. It is thought that the virus itself or viral proteins trigger the release of inflammatory mediators that cause endothelial dysfunction and smooth muscle proliferation leading to vascular injury and thrombosis. The beneficial effects of highly active anti-retroviral therapy alter the natural history of the disease profile and promote longevity but are negated by cardiovascular complications. Atherosclerosis is an emerging challenge. Presently patients are managed by standard surgical protocols because of non-existent universal surgical interventional guidelines. Clinical response to treatment is variable and often compounded by complications of graft occlusion, sepsis and poor wound healing. The clinical, imaging and pathological observations position HIV-associated large-vessel vasculopathy as a unique entity. This review highlights the spectrum of HIV-associated large-vessel aneurysmal, occlusive and atherosclerotic disease in vascular surgical practice.

A high-affinity native human antibody neutralizes human cytomegalovirus infection of diverse cell types.

Human cytomegalovirus (HCMV) is the most common infection causing poor outcomes among transplant recipients. Maternal infection and transplacental transmission are major causes of permanent birth defects. Although no active vaccines to prevent HCMV infection have been approved, passive immunization with HCMV-specific immunoglobulin has shown promise in the treatment of both transplant and congenital indications. Antibodies targeting the viral glycoprotein B (gB) surface protein are known to neutralize HCMV infectivity, with high-affinity binding being a desirable trait, both to compete with low-affinity antibodies that promote the transmission of virus across the placenta and to displace nonneutralizing antibodies binding nearby epitopes. Using a miniaturized screening technology to characterize secreted IgG from single human B lymphocytes, 30 antibodies directed against gB were previously cloned. The most potent clone, TRL345, is described here. Its measured affinity was 1 pM for the highly conserved site I of the AD-2 epitope of gB. Strain-independent neutralization was confirmed for 15 primary HCMV clinical isolates. TRL345 prevented HCMV infection of placental fibroblasts, smooth muscle cells, endothelial cells, and epithelial cells, and it inhibited postinfection HCMV spread in epithelial cells. The potential utility for preventing congenital transmission is supported by the blockage of HCMV infection of placental cell types central to virus transmission to the fetus, including differentiating cytotrophoblasts, trophoblast progenitor cells, and placental fibroblasts. Further, TRL345 was effective at controlling an ex vivo infection of human placental anchoring villi. TRL345 has been utilized on a commercial scale and is a candidate for clinical evaluation.

Human cytomegalovirus immediate early protein 2 enhances myocardin-mediated survival of rat aortic smooth muscle cells.

Human cytomegalovirus (HCMV) may increase the incidence of restenosis and predispose to atherosclerosis. The lesions of restenosis and atherosclerosis often contain smooth muscle cells (SMCs) with high rates of proliferation and apoptosis. One of the immediate early (IE) gene products of HCMV-IE2 affects transcriptional activities of some cellular factors in SMCs, including myocardin. In this study, we studied the effects of IE2 and myocardin on PI3K pathway inducer wortmannin induced apoptosis in rat aortic SMCs. We show that the transcriptional activity of myocardin on Mcl-1 promoter is enhanced by co-expression of HCMV IE2 in rat aortic SMCs; and the expressions of mRNA and protein of antiapoptotic genes-Mcl-1 and Bcl-2 are upregulated by IE2 alone and co-transfection of myocardin and IE2, but decreased by myocardin-specific shRNA in rat aortic SMCs. We further demonstrate that co-expression of myocardin and HCMV IE2 declines apoptotic cell numbers and caspase-3 activities induced by serum starvation plus wortmannin in rat aortic SMCs. The results suggest that HCMV IE2 enhances myocardin-mediated survival of rat aortic SMCs under serum deprivation and PI3-kinase inhibition, partly via activation of Mcl-1's antiapoptosis effect. Our study connects HCMV IE2 to myocardin-induced transcriptional program for rat aortic SMCs survival and proliferation, involving in HCMV related restenosis and atherosclerosis.

Inhibition of phosphodiesterase 4 modulates cytokine induction from toll like receptor activated, but not rhinovirus infected, primary human airway smooth muscle.

BACKGROUND: Virus-induced exacerbations of Chronic Obstructive Pulmonary Disease (COPD) are a significant health burden and occur even in those receiving the best current therapies. Rhinovirus (RV) infections are responsible for half of all COPD exacerbations. The mechanism by which exacerbations occur remains undefined, however it is likely to be due to virus-induced inflammation. Given that phophodiesterase 4 (PDE4) inhibitors have an anti-inflammatory effect in patients with COPD they present a potential therapy prior to, and during, these exacerbations. METHODS: In the present study we investigated whether the PDE4 inhibitor piclamilast (10(-6) M) could alter RV or viral mimetic (5 µg/mL of imiquimod or poly I:C) induced inflammation and RV replication in primary human airway smooth muscle cells (ASMC) and bronchial epithelial cells (HBEC). The mediators IL-6, IL-8, prostaglandin E2 and cAMP production were assayed by ELISA and RV replication was assayed by viral titration. RESULTS: We found that in ASMCs the TLR3 agonist poly I:C induced IL-8 release was reduced while induced IL-6 release by the TLR7/8 agonist imiquimod was further increased by the presence of piclamilast. However, in RV infected ASMCs, virus replication and induced mediator release were unaltered by piclamilast, as was also found in HBECs. The novel findings of this study reveal that although PDE inhibitors may not influence RV-induced cytokine production in ASMCs and replication in either ASMCs or HBECs, they have the capacity to be anti-inflammatory during TLR activation by modulating the induction of these chemotactic cytokines. CONCLUSION: By extrapolating our in vitro findings to exacerbations of COPD in vivo this suggests that PDE4 inhibitors may have beneficial anti-inflammatory properties when patients are infected with bacteria or viruses other than RV.

Downregulation of bone morphogenetic protein receptor axis during HIV-1 and cocaine-mediated pulmonary smooth muscle hyperplasia: implications for HIV-related pulmonary arterial hypertension.

OBJECTIVE: Our previous findings support an additive effect of cocaine to HIV infection in the development of pulmonary arteriopathy through enhanced proliferation of human pulmonary smooth muscle cells. We now examined the role of antiproliferative bone morphogenetic protein receptor (BMPR) axis in HIV protein and cocaine-mediated pulmonary smooth muscle hyperplasia. APPROACH AND RESULTS: Stimulation of BMPR axis resulted in attenuation of synergistic increase in the proliferation of human pulmonary arterial smooth muscle cells in response to cocaine and HIV protein, transactivator of transcription (Tat). Interestingly, an increase in mRNA but decrease in protein levels of BMPR with correlated decrease in the activation of Sma- and MAD-related family protein 1/5/8 and Id1 gene expression was observed on combined treatment with cocaine and Tat when compared with the untreated cells at all time points tested. Although longer exposure to either cocaine or Tat alone also resulted in a significant decrease in the BMPR protein expression, the abrogation on combined treatment was still significantly more when compared with that of the monotreatments. Significant increase in mRNA but downmodulation of BMPR protein expression was also observed in the lung extracts from HIV-infected intravenous drug users (HIV+IVDU) when compared with that from HIV-infected non-IVDUs (HIV) or uninfected IVDUs (IVDU). Furthermore, significant decrease in BMPR protein expression was also observed in HIV or IVDUs when compared with normal controls that correlated with in vitro findings on chronic exposure to cocaine or HIV protein alone. CONCLUSIONS: Simultaneous exposure of pulmonary smooth muscle cells to viral protein(s) and cocaine exacerbates downregulation of BMPR axis that may result in enhanced pulmonary vasculature aberrations in HIV+IVDUs.

Rhinovirus and dsRNA induce RIG-I-like receptors and expression of interferon ß and λ1 in human bronchial smooth muscle cells.

Rhinovirus (RV) infections cause exacerbations and development of severe asthma highlighting the importance of antiviral interferon (IFN) defence by airway cells. Little is known about bronchial smooth muscle cell (BSMC) production of IFNs and whether BSMCs have dsRNA-sensing receptors besides TLR3. dsRNA is a rhinoviral replication intermediate and necrotic cell effect mimic that mediates innate immune responses in bronchial epithelial cells. We have explored dsRNA-evoked IFN-ß and IFN-λ1 production in human BSMCs and potential involvement of TLR3 and RIG-I-like receptors (RLRs). Primary BSMCs were stimulated with 0.1-10 µg/ml dsRNA, 0.1-1 µg/ml dsRNA in complex with the transfection agent LyoVec (dsRNA/LyoVec; selectively activating cytosolic RLRs) or infected with 0.05-0.5 MOI RV1B. Both dsRNA stimuli evoked early (3 h), concentration-dependent IFN-ß and IFN-λ1 mRNA expression, which with dsRNA/LyoVec was much greater, and with dsRNA was much less, after 24 h. The effects were inhibited by dexamethasone. Further, dsRNA and dsRNA/LyoVec concentration-dependently upregulated RIG-I and MDA5 mRNA and protein. dsRNA and particularly dsRNA/LyoVec caused IFN-ß and IFN-λ1 protein production (24 h). dsRNA- but not dsRNA/LyoVec-induced IFN expression was partly inhibited by chloroquine that suppresses endosomal TLR3 activation. RV1B dose-dependently increased BSMC expression of RIG-I, MDA5, IFN-ß, and IFN-λ1 mRNA. We suggest that BSMCs express functional RLRs and that both RLRs and TLR3 are involved in viral stimulus-induced BSMC expression of IFN-ß and IFN-λ1.

Characterising the mechanism of airway smooth muscle ß2 adrenoceptor desensitization by rhinovirus infected bronchial epithelial cells.

Rhinovirus (RV) infections account for approximately two thirds of all virus-induced asthma exacerbations and often result in an impaired response to ß2 agonist therapy. Using an in vitro model of RV infection, we investigated the mechanisms underlying RV-induced ß2 adrenoceptor desensitization in primary human airway smooth muscle cells (ASMC). RV infection of primary human bronchial epithelial cells (HBEC) for 24 hours produced conditioned medium that caused ß2 adrenoceptor desensitization on ASMCs without an effect on ASMCs viability. Less than 3 kDa size fractionation together with trypsin digestion of RV-induced conditioned medium did not prevent ß2 adrenoceptor desensitization, suggesting it could potentially be mediated by a small peptide or lipid. RV infection of BECs, ASMCs and fibroblasts produced prostaglandins, of which PGE2, PGF2α and PGI2 had the ability to cause ß2 adrenoceptor desensitization on ASMCs. RV-induced conditioned medium from HBECs depleted of PGE2 did not prevent ASMC ß2 adrenoceptor desensitization; however this medium induced PGE2 from ASMCs, suggesting that autocrine prostaglandin production may be responsible. Using inhibitors of cyclooxygenase and prostaglandin receptor antagonists, we found that ß2 adrenoceptor desensitization was mediated through ASMC derived COX-2 induced prostaglandins. Since ASMC prostaglandin production is unlikely to be caused by RV-induced epithelial derived proteins or lipids we next investigated activation of toll-like receptors (TLR) by viral RNA. The combination of TLR agonists poly I:C and imiquimod induced PGE2 and ß2 adrenoceptor desensitization on ASMC as did the RNA extracted from RV-induced conditioned medium. Viral RNA but not epithelial RNA caused ß2 adrenoceptor desensitization confirming that viral RNA and not endogenous human RNA was responsible. It was deduced that the mechanism by which ß2 adrenoceptor desensitization occurs was by pattern recognition receptor activation of COX-2 induced prostaglandins.

Modulating vascular intimal hyperplasia using HSV-1 mutant requires activated MEK.

Outcomes of cardiovascular procedures, such as angioplasty and stent or bypass grafting are limited by failure, predominantly caused by pathological smooth muscle cell (SMC) proliferation, known as intimal hyperplasia. Local delivery of a genetically engineered herpes simplex virus (HSV) is known to block vascular SMC proliferation while allowing for re-endothelialization. However, the mechanism this mutant virus uses to prevent SMC hyperplasia is unknown. The Ras signaling cascade is activated in SMCs undergoing hyperplasia leading to phosphorylation of the mitogen-activated protein kinase (MAPK). In this study we tested the hypothesis that MAPK kinase (MEK) activity is the molecular basis by which SMCs are susceptible to mutant HSV. We show that genetically engineered herpes simplex-1 viruses (HSV-1) can target proliferating SMCs. We demonstrate that the molecular basis of this HSV-1 anti-proliferative effect is MEK activation in SMCs. We demonstrate efficacy and practicality of the MEK-dependent HSV-1 for the treatment of intimal hyperplasia in a clinically relevant in vivo model. Important to this strategy is the ability to modulate the effects by controlling viral dose. These results propel genetically engineered HSV-1 therapy towards clinical evaluation in treatment of intimal hyperplasia.

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.

Adenovirus-mediated tissue factor pathway inhibitor gene transfer induces apoptosis by blocking the phosphorylation of JAK-2/STAT-3 pathway in vascular smooth muscle cells.

OBJECTIVE: In our previous study, we have demonstrated that tissue factor pathway inhibitor (TFPI) gene could induce vascular smooth muscle cell (VSMC) apoptosis. This study was conducted to investigate whether the overexpression of the TFPI gene can induce VSMC apoptosis by inhibiting JAK-2/STAT-3 pathway phosphorylation and thereby inhibiting the expression of such downstream targets as the apoptotic protein Bcl-2 and cell cycle protein cyclin D1. The effect of TFPI on the expression of survivin, a central molecule in cell survival, was also investigated. METHODS: Rat VSMCs were infected with recombinant adenovirus containing either the TFPI (Ad-TFPI) or LacZ (Ad-LacZ) gene or DMEM in vitro. TFPI expression was detected by ELISA. TUNEL staining and electron microscope were carried out to determine the apoptosis of VSMCs. The expression levels of JAK-2, p-JAK-2, STAT-3, p-STAT-3, cyclin D1, Bcl-2 and survivin were examined by western blot analysis. RESULTS: TFPI protein was detected in the TFPI group after gene transfer and the peak expression was at the 3rd day. At the 3rd, 5th and 7th days after gene transfer, the apoptotic rates by TUNEL assay in the TFPI group were 10.91 ± 1.66%, 13.46 ± 1.28% and 17.04 ± 1.95%, respectively, whereas those in the LacZ group were 3.28 ± 0.89%, 4.01 ± 0.72% and 4.89 ± 1.17%, respectively. We observed cell contraction, slight mitochondrial swelling, nuclear pyknosis and apoptotic body formation in TFPI-treated VSMCs using electron microscopy. JAK-2, p-JAK-2, STAT-3, p-STAT-3, cyclin D1 and Bcl-2, which are all involved in the JAK-2/STAT-3 pathway, were detected in the VSMCs on the 3rd, 5th and 7th days after gene transfer, which is consistent with previously demonstrated time points when VSMCs apoptosis occurred. The expression levels of p-JAK-2, p-STAT-3, cyclin D1 and Bcl-2 were significantly decreased over time in the TFPI group (each P<0.05) but not in the Ad-LacZ and DMEM groups. However, this attenuation of expression was not observed for JAK-2 and STAT-3 in any of the groups at any time points after gene transfer (each P>0.05). The expression level of survivin in the TFPI group also weakened significantly over time compared with the levels in the Ad-LacZ and DMEM groups (each P<0.05) at the 3rd, 5th and 7th days after gene transfer. CONCLUSION: The results demonstrated that TFPI played an apoptosis-inducing role in VSMCs in a manner that involves both the suppression of JAK-2/STAT-3 pathway phosphorylation and the down-regulation of survivin. Our data show for the first time that targeting the JAK-2/STAT-3 pathway and survivin by overexpressing TFPI may be a new avenue for the treatment of restenosis.

Evaluation of cellular determinants required for in vitro xenotropic murine leukemia virus-related virus entry into human prostate cancer and noncancerous cells.

The newly identified retrovirus-the xenotropic murine leukemia virus-related virus (XMRV)-has recently been shown to be strongly associated with familial prostate cancer in humans (A. Urisman et al., PLoS Pathog. 2:e25, 2006). While that study showed evidence of XMRV infection exclusively in the prostatic stromal fibroblasts, a recent study found XMRV protein antigens mainly in malignant prostate epithelial cells (R. Schlaberg et al., Proc. Natl. Acad. Sci. U. S. A. 106:16351-16356, 2009). To help elucidate the mechanisms behind XMRV infection, we show that prostatic fibroblast cells express Xpr1, a known receptor of XMRV, but its expression is absent in other cell lines of the prostate (i.e., epithelial and stromal smooth muscle cells). We also show that certain amino acid residues located within the predicted extracellular loop (ECL3 and ECL4) sequences of Xpr1 are required for efficient XMRV entry. Although we found strong evidence to support XMRV infection of prostatic fibroblast cell lines via Xpr1, we learned that XMRV was indeed capable of infecting cells that did not necessarily express Xpr1, such as those of the prostatic epithelial and smooth muscle origins. Further studies suggest that the expression of Xpr1 and certain genotypes of the RNASEL gene, which could restrict XMRV infection, may play important roles in defining XMRV tropisms in certain cell types. Collectively, our data reveal important cellular determinants required for XMRV entry into different human prostate cells in vitro, which may provide important insights into the possible role of XMRV as an etiologic agent in human prostate cancer.