WNT3A induces a contractile and secretory phenotype in cultured vascular smooth muscle cells that is associated with increased gap junction communication.

Evidence suggests a role for Wnt signaling in vascular wound repair and remodeling events. Despite this, very little is known about the effect of Wnt ligands on the structure and function of vascular cells. In this study, we treated vascular smooth muscle cells with 250 ng/ml of recombinant Wnt3a for 72 h and observed changes in the cell phenotype. Our data suggest Wnt3a completely alters the phenotype of vascular smooth muscle cells. The Wnt3a-treated cells appeared larger and had increased formation of stress fibers. These cells also had increased expression of the smooth muscle contractile proteins, calponin and smooth muscle α-actin, and contracted a collagen lattice faster than control cells. The Wnt3a-treated smooth muscle cells displayed increased extracellular matrix synthesis, as measured by collagen I and III mRNA expression, along with increased expression of MMP2 and MMP9, but decreased TIMP2 levels. The Wnt3a-induced change in cell phenotype was associated with increased expression of the gap junction protein connexin 43. Consistent with this, Wnt3a-treated smooth muscle cells displayed enhanced intercellular communication, as measured by the scrape-loading dye transfer technique. The canonical Wnt antagonist, dickkopf-related protein 1, completely reversed the contractile protein and connexin 43 expression seen in the Wnt3a-treated cells, suggesting these changes were dependent on canonical Wnt signaling. Collectively, this data suggest Wnt3a promotes a contractile and secretory phenotype in vascular smooth muscle cells that is associated with increased gap junction communication.

Toll-like receptor 4-mediated activation of p38 mitogen-activated protein kinase is a determinant of respiratory virus entry and tropism.

Respiratory viruses exert a heavy toll of morbidity and mortality worldwide. Despite this burden there are few specific treatments available for respiratory virus infections. Since many viruses utilize host cell enzymatic machinery such as protein kinases for replication, we determined whether pharmacological inhibition of kinases could, in principle, be used as a broad antiviral strategy for common human respiratory virus infections. A panel of green fluorescent protein (GFP)-expressing recombinant respiratory viruses, including an isolate of H1N1 influenza virus (H1N1/Weiss/43), was used to represent a broad range of virus families responsible for common respiratory infections (Adenoviridae, Paramyxoviridae, Picornaviridae, and Orthomyxoviridae). Kinase inhibitors were screened in a high-throughput assay that detected virus infection in human airway epithelial cells (1HAEo-) using a fluorescent plate reader. Inhibition of p38 mitogen-activated protein kinase (MAPK) signaling was able to significantly inhibit replication by all viruses tested. Therefore, the pathways involved in virus-mediated p38 and extracellular signal-regulated kinase (ERK) MAPK activation were investigated using bronchial epithelial cells and primary fibroblasts derived from MyD88 knockout mouse lungs. Influenza virus, which activated p38 MAPK to approximately 10-fold-greater levels than did respiratory syncytial virus (RSV) in 1HAEo- cells, was internalized about 8-fold faster and more completely than RSV. We show for the first time that p38 MAPK is a determinant of virus infection that is dependent upon MyD88 expression and Toll-like receptor 4 (TLR4) ligation. Imaging of virus-TLR4 interactions showed significant clustering of TLR4 at the site of virus-cell interaction, triggering phosphorylation of downstream targets of p38 MAPK, suggesting the need for a signaling receptor to activate virus internalization.

Bosentan enhances viral load via endothelin-1 receptor type-A-mediated p38 mitogen-activated protein kinase activation while improving cardiac function during coxsackievirus-induced myocarditis.

Reduced cardiac output is one of the consequences of myocarditis. Bosentan, an endothelin-1 receptor (ET1R) antagonist, could be useful to reduce cardiac afterload, preserving cardiac output. In this study, we investigated the potential therapeutic use of bosentan in an animal model of viral myocarditis. Using a mouse model of coxsackievirus B3 (CVB3)-induced myocarditis, we demonstrated preserved ejection fraction (EF) and fractional shortening (FS) by treatment with bosentan (68+/-5.8% EF and 40+/-3.7% FS for treated versus 48+/-2.2% EF and 25+/-2.6% FS for controls; P=0.028). However, bosentan enhanced cardiac viral load (10.4+/-6.7% in the bosentan group versus 5.0+/-5.5% in control group; P=0.02), likely through enhancement of p38 mitogen-activated protein kinase (MAPK) phosphorylation (0.77+/-0.40% ATF2 activation in the bosentan group versus 0.03+/-0.02% in controls; P=0.0002), mediated by endothelin receptor type-A. We further demonstrate that a water soluble inhibitor of p38 MAPK, SB203580 HCl, is a potent inhibitor of virus replication in the heart (0.28% antisense viral genome stained area for 3 mg/kg dose versus 2.9% stained area for controls; P=0.01), attenuates CVB3-induced myocardial damage (blinded cardiac histopathologic scores of 1.8+/-1.6 and 2.05+/-1.2 for the 3 mg/kg and 10 mg/kg doses, respectively, versus 3.25+/-1.2 for the controls), and preserves cardiac function (69+/-3.5% EF for 3 mg/kg dose and 71+/-6.7% EF for 10 mg/kg dose versus 60+/-1.5% EF control; P=0.038 and P=0.045, as compared to control, respectively). Bosentan, a prescribed vasodilator, improves cardiac function but enhances viral load and myocarditis severity through ETRA mediated p38 MAPK activation; p38 MAPK is a desirable antiviral target. Caution must be exercised during treatment of suspected infectious myocarditis with supportive vasoactive remedies.

Ablation of matrix metalloproteinase-9 increases severity of viral myocarditis in mice.

BACKGROUND: Coxsackievirus B3 (CVB3) causes human myocarditis, which can result in cardiac damage, maladaptive remodeling, and heart failure. Matrix metalloproteinases (MMP)-8 and -9 have been identified in virus-infected myocardium, but their particular roles and underlying mechanisms of effect are unknown. For the first time, we examine the severity of CVB3-induced myocarditis in MMP-8-and MMP-9-deficient mice. METHODS AND RESULTS: CVB3-infected MMP-8 and MMP-9 knockout (KO) mice and corresponding wild-type (WT) mice were euthanized and harvested at 9 days after infection. Expression of MMP-2, -8, -12, and -13 and tissue inhibitors of MMPs was assessed by zymography or immunoblotting on harvested hearts, and in situ hybridization was performed to detect active infection. Infected MMP-9 KO mice had greater myocardial injury and foci of infection than WT mice despite similar pancreatic infection. Increased fibrosis (10.6+/-2.7% versus 7.1+/-2.6%, P=0.04), viral titer, as well as decreased cardiac output, were evident in MMP-9 KO compared with WT mice as assessed by picrosirius red staining, plaque assay, and echocardiography, respectively. Immune infiltration was also greatly increased in MMP-9 KO compared with WT mice (15.2+/-12.6% versus 2.0+/-3.0%, P<0.002). Myocardial interferon-beta1, interferon-gamma, interleukin-6, interleukin-10, and macrophage inflammatory protein-1alpha expression was elevated in MMP-9 KO mice as measured by quantitative real-time polymerase chain reaction and ELISA. In contrast, MMP-8 KO mice had the same degree of cardiac injury, fibrosis, and viral infection as their WT counterparts. CONCLUSIONS: During acute CVB3 infection, MMP-9 appears necessary to halt virus propagation in the heart, promote proper immune infiltration and remodeling, and preserve cardiac output.

IL-6 expression is correlated with increased T-cell proliferation and survival in the arterial wall in giant cell arteritis.

Giant cell arteritis (GCA) is the most common vasculitis in adults affecting large and medium-sized arteries. IL-6 and T cell accumulation within the arterial wall contribute to the pathogenesis of GCA, and blockade of IL-6 activity is efficacious in its treatment. We examined the relationship between levels of IL-6 expression and immunological processes that control the expansion of T cells in GCA-positive temporal artery biopsies. CD4 T cells accumulated in clusters within the media and deep intima of all GCA lesions. There was a significant positive correlation between the expression of IL-6 mRNA and increased frequency of proliferating CD4 T cells. The expansion of T cells can be inhibited by T regs but IL-6 expression was not correlated with differences in T reg accumulation. Increased IL-6 levels were also significantly correlated with lower frequencies of CD4 T cells undergoing apoptotic cell death. In conclusion, IL-6 may contribute to the accumulation of CD4 T cells in GCA by supporting their proliferation and survival within the arterial wall through mechanisms that are independent of effects on local T reg expansion.

1,25 Dihydroxyvitamin D3 Inhibits TGFß1-Mediated Primary Human Cardiac Myofibroblast Activation.

AIMS: Epidemiological and interventional studies have suggested a protective role for vitamin D in cardiovascular disease, and basic research has implicated vitamin D as a potential inhibitor of fibrosis in a number of organ systems; yet little is known regarding direct effects of vitamin D on human cardiac cells. Given the critical role of fibrotic responses in end stage cardiac disease, we examined the effect of active vitamin D treatment on fibrotic responses in primary human adult ventricular cardiac fibroblasts (HCF-av), and investigated the relationship between circulating vitamin D (25(OH)D3) and cardiac fibrosis in human myocardial samples. METHODS AND RESULTS: Interstitial cardiac fibrosis in end stage HF was evaluated by image analysis of picrosirius red stained myocardial sections. Serum 25(OH)D3 levels were assayed using mass spectrometry. Commercially available HCF-av were treated with transforming growth factor (TGF)ß1 to induce activation, in the presence or absence of active vitamin D (1,25(OH)2D3). Functional responses of fibroblasts were analyzed by in vitro collagen gel contraction assay. 1,25(OH)2D3 treatment significantly inhibited TGFß1-mediated cell contraction, and confocal imaging demonstrated reduced stress fiber formation in the presence of 1,25(OH)2D3. Treatment with 1,25(OH)2D3 reduced alpha-smooth muscle actin expression to control levels and inhibited SMAD2 phosphorylation. CONCLUSIONS: Our results demonstrate that active vitamin D can prevent TGFß1-mediated biochemical and functional pro-fibrotic changes in human primary cardiac fibroblasts. An inverse relationship between vitamin D status and cardiac fibrosis in end stage heart failure was observed. Collectively, our data support an inhibitory role for vitamin D in cardiac fibrosis.

Versican V1 Overexpression Induces a Myofibroblast-Like Phenotype in Cultured Fibroblasts.

BACKGROUND: Versican, a chondroitin sulphate proteoglycan, is one of the key components of the provisional extracellular matrix expressed after injury. The current study evaluated the hypothesis that a versican-rich matrix alters the phenotype of cultured fibroblasts. METHODS AND RESULTS: The full-length cDNA for the V1 isoform of human versican was cloned and the recombinant proteoglycan was expressed in murine fibroblasts. Versican expression induced a marked change in fibroblast phenotype. Functionally, the versican-expressing fibroblasts proliferated faster and displayed enhanced cell adhesion, but migrated slower than control cells. These changes in cell function were associated with greater N-cadherin and integrin ß1 expression, along with increased FAK phosphorylation. The versican-expressing fibroblasts also displayed expression of smooth muscle α-actin, a marker of myofibroblast differentiation. Consistent with this observation, the versican fibroblasts displayed increased synthetic activity, as measured by collagen III mRNA expression, as well as a greater capacity to contract a collagen lattice. These changes appear to be mediated, at least in part, by an increase in active TGF-ß signaling in the versican expressing fibroblasts, and this was measured by phosphorylation and nuclear accumulation of SMAD2. CONCLUSIONS: Collectively, these data indicate versican expression induces a myofibroblast-like phenotype in cultured fibroblasts.

A new transcriptional role for matrix metalloproteinase-12 in antiviral immunity.

Interferon-α (IFN-α) is essential for antiviral immunity, but in the absence of matrix metalloproteinase-12 (MMP-12) or IκBα (encoded by NFKBIA) we show that IFN-α is retained in the cytosol of virus-infected cells and is not secreted. Our findings suggest that activated IκBα mediates the export of IFN-α from virus-infected cells and that the inability of cells in Mmp12(-/-) but not wild-type mice to express IκBα and thus export IFN-α makes coxsackievirus type B3 infection lethal and renders respiratory syncytial virus more pathogenic. We show here that after macrophage secretion, MMP-12 is transported into virus-infected cells. In HeLa cells MMP-12 is also translocated to the nucleus, where it binds to the NFKBIA promoter, driving transcription. We also identified dual-regulated substrates that are repressed both by MMP-12 binding to the substrate's gene exons and by MMP-12-mediated cleavage of the substrate protein itself. Whereas intracellular MMP-12 mediates NFKBIA transcription, leading to IFN-α secretion and host protection, extracellular MMP-12 cleaves off the IFN-α receptor 2 binding site of systemic IFN-α, preventing an unchecked immune response. Consistent with an unexpected role for MMP-12 in clearing systemic IFN-α, treatment of coxsackievirus type B3-infected wild-type mice with a membrane-impermeable MMP-12 inhibitor elevates systemic IFN-α levels and reduces viral replication in pancreas while sparing intracellular MMP-12. These findings suggest that inhibiting extracellular MMP-12 could be a new avenue for the development of antiviral treatments.

An ERK-p38 subnetwork coordinates host cell apoptosis and necrosis during coxsackievirus B3 infection.

The host response to a virus is determined by intracellular signaling pathways that are modified during infection. These pathways converge as networks and produce interdependent phenotypes, making it difficult to link virus-induced signals and responses at a systems level. Coxsackievirus B3 (CVB3) infection induces death of cardiomyocytes, causing tissue damage and virus dissemination, through incompletely characterized host cell signaling networks. We built a statistical model that quantitatively predicts cardiomyocyte responses from time-dependent measurements of phosphorylation events modified by CVB3. Model analysis revealed that CVB3-stimulated cytotoxicity involves tight coupling between the host ERK and p38 MAPK pathways, which are generally thought to control distinct cellular responses. The kinase ERK5 requires p38 kinase activity and inhibits apoptosis caused by CVB3 infection. By contrast, p38 indirectly promotes apoptosis via ERK1/2 inhibition but directly causes CVB3-induced necrosis. Thus, the cellular events governing pathogenesis are revealed when virus-host programs are monitored systematically and deconvolved mathematically.

Cleavage of serum response factor mediated by enteroviral protease 2A contributes to impaired cardiac function.

Enteroviral infection can lead to dilated cardiomyopathy (DCM), which is a major cause of cardiovascular mortality worldwide. However, the pathogenetic mechanisms have not been fully elucidated. Serum response factor (SRF) is a cardiac-enriched transcription regulator controlling the expression of a variety of target genes, including those involved in the contractile apparatus and immediate early response, as well as microRNAs that silence the expression of cardiac regulatory factors. Knockout of SRF in the heart results in downregulation of cardiac contractile gene expression and development of DCM. The goal of this study is to understand the role of SRF in enterovirus-induced cardiac dysfunction and progression to DCM. Here we report that SRF is cleaved following enteroviral infection of mouse heart and cultured cardiomyocytes. This cleavage is accompanied by impaired cardiac function and downregulation of cardiac-specific contractile and regulatory genes. Further investigation by antibody epitope mapping and site-directed mutagenesis demonstrates that SRF cleavage occurs at the region of its transactivation domain through the action of virus-encoded protease 2A. Moreover, we demonstrate that cleavage of SRF dissociates its transactivation domain from DNA-binding domain, resulting in the disruption of SRF-mediated gene transactivation. In addition to loss of functional SRF, finally we report that the N-terminal fragment of SRF cleavage products can also act as a dominant-negative transcription factor, which likely competes with the native SRF for DNA binding. Our results suggest a mechanism by which virus infection impairs heart function and may offer a new therapeutic strategy to ameliorate myocardial damage and progression to DCM.

Wnt3a induces myofibroblast differentiation by upregulating TGF-ß signaling through SMAD2 in a ß-catenin-dependent manner.

Growing evidence suggests the Wnt family of secreted glycoproteins and their associated signaling pathways, linked to development, are recapitulated during wound repair and regeneration events. However, the role of the Wnt pathway in such settings remains unclear. In the current study, we treated mouse fibroblasts with 250 ng/mL of recombinant Wnt3a for 72 hours and examined its affect on cell morphology and function. Wnt3a induced a spindle-like morphology in fibroblasts characterized by the increased formation of stress fibres. Wnt3a decreased the proliferation of fibroblasts, but significantly increased cell migration as well as fibroblast-mediated contraction of a collagen lattice. Wnt3a significantly increased the expression of TGF-ß and its associated signaling through SMAD2. Consistent with this, we observed significantly increased smooth muscle α-actin expression and incorporation of this contractile protein into stress fibres following Wnt3a treatment. Knockdown of ß-catenin using siRNA reversed the Wnt3a-induced smooth muscle α-actin expression, suggesting these changes were dependent on canonical Wnt signaling through ß-catenin. Neutralization of TGF-ß with a blocking antibody significantly inhibited the Wnt3a-induced smooth muscle α-actin expression, indicating these changes were dependent on the increased TGF-ß signaling. Collectively, this data strongly suggests Wnt3a promotes the formation of a myofibroblast-like phenotype in cultured fibroblasts, in part, by upregulating TGF-ß signaling through SMAD2 in a ß-catenin-dependent mechanism. As myofibroblasts are critical regulators of wound healing responses, these findings may have important implications for our understanding of normal and aberrant injury and repair events.

Vascular endothelial growth factor increases human cardiac microvascular endothelial cell permeability to low-density lipoproteins.

BACKGROUND: Endothelial cell hyperpermeability is a proposed mechanism of increased lipid insudation into the vessel walls of allografts. Vascular endothelial growth factor (VEGF) is a potent inducer of vascular permeability and its expression is upregulated in human heart allografts. The goal of these experiments was to investigate the effects of VEGF on low-density lipoprotein (LDL) permeability through confluent monolayers of human cardiac microvascular endothelial cells (HCMEC) in vitro. METHODS: VEGF mRNA and protein expression was characterized in coronary arteries from cardiac allograft vasculopathy patients as compared with healthy controls using in situ hybridization and immunohistochemical staining of sub-adjacent sections. HCMEC were grown to confluence and treated with VEGF-A(121) or VEGF-A(165). Permeability of LDL in confluent endothelial monolayers was measured using fluorometry. Transendothelial electrical resistance (TER) measurements were used to indirectly measure the tight junctional status. Immunocytochemical staining was performed to visualize changes in CD31 and zonula occludens-1. RESULTS: We observed significant increases in VEGF expression within the superficial and deep intima and media of coronaries from allografts, as compared with controls. In vitro treatment with VEGF-A(121) and VEGF-A(165) significantly increased LDL passage through endothelial monolayers. We further showed that VEGF-A(121) and VEGF-A(165) caused significant decreases in TER at 2 to 4 hours post-treatment. Also, VEGF induced disruption of tight junctions, resulting in an increase in the intercellular gap formation. CONCLUSIONS: These results demonstrate that VEGF increases low-density lipoprotein permeability through endothelial monolayers, and this effect is correlated with VEGF-induced disruption of endothelial tight junctions resulting in the formation of intercellular gaps.

ERK MAP kinase-activated Arf6 trafficking directs coxsackievirus type B3 into an unproductive compartment during virus host-cell entry.

Clathrin- and caveolae-mediated endocytosis have been implicated in the productive entry of many viruses into host cells. ADP-ribosylation factor 6 (Arf6)-dependent endocytosis is another endocytosis pathway that traffics from the cell surface and it is the only Arf that traffics at the plasma membrane. However, little is known about Arf6-dependent trafficking during virus entry. This study showed that coxsackievirus type B3 (CVB3) associated with decay-accelerating factor in non-polarized HeLa cells can be redirected into non-productive compartments by Arf6-dependent internalization, thus restricting infection. Overexpression of wild-type (WT) and constitutively active (CA) Arf6 in HeLa cells resulted in a 2.3- and 3.6-fold decrease in infection, respectively. A dominant-negative inhibitor of Arf6 recovered restriction of infection by WT-Arf6 and CA-Arf6. RNA interference of endogenous Arf6 resulted in a 3.3-fold increase in CVB3 titre in HeLa cells. It was shown that coxsackie-adenovirus receptor (CAR) ligation by virus or CAR-specific antibody could activate extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase family and lead to Arf6-mediated viral restriction. In the absence of ERK activation, CVB3 internalization into early endosomes was inhibited and subsequent infection was reduced, but Arf6-mediated restriction was also abolished. In conclusion, receptor-mediated signalling enhances CVB3 entry whilst also activating non-productive pathways of virus entry; thus, virus infection is an equilibrium of productive and non-productive pathways of entry.

Regulation of the versican promoter by the beta-catenin-T-cell factor complex in vascular smooth muscle cells.

The proteoglycan versican is pro-atherogenic and central to vascular injury and repair events. We identified the signaling pathways and promoter elements involved in regulation of versican expression in vascular smooth muscle cells. Phosphatidylinositol 3-kinase inhibitor, LY294002, significantly decreased versican-luciferase (Luc) promoter activity and endogenous mRNA levels. We further examined the roles of protein kinase B and glycogen synthase kinase (GSK)-3beta, downstream effectors of phosphatidylinositol 3-kinase, in the regulation of versican transcription. Co-transfection of dominant negative and constitutively active protein kinase B constructs with a versican-Luc construct decreased and increased promoter activity, respectively. Inhibition of GSK-3beta activity by LiCl augmented accumulation of beta-catenin and caused induction of versican-Luc activity as well as versican mRNA levels. Beta-catenin has no DNA binding domain, therefore it cannot directly induce transcription of the versican promoter. Software analysis of the versican promoter revealed two potential binding sites for T-cell factors (TCFs), proteins that confer transcriptional activation of beta-catenin. Electrophoretic mobility shift and supershift assays revealed specific binding of human TCF-4 and beta-catenin to oligonucleotides corresponding to a potential TCF binding site in the versican promoter. In addition to binding assays, we directly assessed the dependence of versican promoter activity on TCF binding sites. Site-directed mutagenesis of the TCF site located -492 bp relative to the transcription start site markedly diminished versican-Luc activity. Co-transfection of TCF-4 with versican-Luc did not increase promoter activity, but addition of beta-catenin and TCF-4 significantly stimulated basal versican promoter activity. Our findings suggest that versican transcription is predominantly mediated by the GSK-3beta pathway via the beta-catenin-TCF transcription factor complex in smooth muscle cells, wherein such regulation contributes to the normal or aberrant formation of provisional matrix in vascular injury and repair events.

Coxsackievirus B3 replication is reduced by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway.

Coxsackievirus B3 (CVB3) is the most common human pathogen for viral myocarditis. We have previously shown that the signaling protein p21(ras) GTPase-activating protein (RasGAP) is cleaved and that mitogen-activated protein kinases (MAPKs) ERK1/2 are activated in the late phase of CVB3 infection. However, the role of intracellular signaling pathways in CVB3-mediated myocarditis and the relative advantages of such pathways to host or virus remain largely unclear. In this study we extended our prior studies by examining the interaction between CVB3 replication and intracellular signaling pathways in HeLa cells. We observed that CVB3 infection induced a biphasic activation of ERK1/2, early transient activation versus late sustained activation, which were regulated by different mechanisms. Infection by UV-irradiated, inactivated virus capable of receptor binding and endocytosis triggered early ERK1/2 activation, but was insufficient to trigger late ERK1/2 activation. By using a general caspase inhibitor (zVAD.fmk) we further demonstrated that late ERK1/2 activation was not a result of CVB3-mediated caspase cleavage. Treatment of cells with U0126, a selective inhibitor of MAPK kinase (MEK), significantly inhibited CVB3 progeny release and decreased virus protein production. Furthermore, inhibition of ERK1/2 activation circumvented CVB3-induced apoptosis and viral protease-mediated RasGAP cleavage. Taken together, these data suggest that ERK1/2 activation is important for CVB3 replication and contributes to virus-mediated changes in host cells. Our findings demonstrate coxsackievirus takeover of a particular host signaling mechanism and uncover a prospective approach to stymie virus spread and preserve myocardial integrity.