Human Cytomegalovirus Infection Of Endothelial Cells Research Articles

Differential mRNA Expressions in HCMV infected HUVECs*

ObjectiveThe aim was to identify the gene expressions of human cytomegalovirus (HCMV)-infected human umbilical vein endothelial cells (HUVECs) and to study its possible pathogenic mechanism on atherosclerosis using microarray technology. MethodsThe gene expression differences in HCMV AD169 strain-infected HUVECs were studied by the microarray technology to explore the potential molecular mechanism of HCMV infection. The qPCRs were performed to verify the transcriptome results. ResultsA total of 2,583 differentially expressed genes, including 407 down-regulated genes and 2,176 up-regulated genes, were detected by the systematic bioinformatics analysis. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the significantly differentially expressed genes were mainly involved in regulating protein kinase activity, inflammatory response, ubiquitination, protein phosphorylation, cell metabolism, and exosomes, among which 12 genes had significant changes and were screened by protein-protein interaction (PPI) analysis and verified by qPCR. The experimental qPCR results were consistent with the microarray results. ConclusionThe GO and KEGG analyses revealed that the regulation of protein kinase activity, inflammatory response, ubiquitination, protein phosphorylation, and cell metabolism played important roles in the process of endothelial cell infection. Furthermore, 12 genes were involved in the process of HCMV infection of endothelial cells and contributed to the current understanding of the infection and pathogenic mechanisms of atherosclerosis.

MicroRNA-217 Promotes Angiogenesis of Human Cytomegalovirus-Infected Endothelial Cells through Downregulation of SIRT1 and FOXO3A

Human cytomegalovirus(HCMV) infection has been shown to contribute to vascular disease through the induction of angiogenesis. However, the role of microRNA in angiogenesis induced by HCMV infection remains unclear. The present study was thus designed to explore the potential effect of miR-1217 on angiogenesis and to disclose the underlying mechanism in endothelial cells. We found that HCMV infection of endothelial cells(ECs) enhanced expression of miR-217 and reduced SIRT1 and FOXO3A protein level in 24 hours post infection(hpi). Transfection of miR-217 inhibitor not only depressed cellular migration and tube formation induced by HCMV infection, but also enhanced SIRT1 and FOXO3A protein expression. Additionally, luciferase assay confirmed that miR-217 directly targeted FOXO3A mRNA 3`UTR. Furthermore, pretreatment with resveratrol depressed motility and tube formation of HCMV-infected ECs, which could be reversed by SIRT1 siRNA. Similarly, delivery of FOXO3A overexpression lentivirus suppressed proliferative rate, migration and tube formation of HCMV-infected ECs, which reversed by transfection of FOXO3A siRNA. In summary, HCMV infection of endothelial cells induces angiogenesis by both of miR-217/SIRT1 and miR-217/FOXO3A axis.

MiR-199a-5p promotes migration and tube formation of human cytomegalovirus-infected endothelial cells through downregulation of SIRT1 and eNOS

Human cytomegalovirus (HCMV) infection has been shown to contribute to vascular disease through the induction of angiogenesis. However, the role of microRNA in angiogenesis induced by HCMV infection remains unclear. The present study was thus designed to explore the potential effect of miR-199a-5p on angiogenesis and to investigate the underlying mechanism in endothelial cells. We found that HCMV infection of endothelial cells (ECs) enhanced expression of miR-199a-5p and reduced the SIRT1 protein level at 24h postinfection (hpi). Transfection with miR-199a-5p mimics significantly suppressed SIRT1 protein expression and promoted cellular migration and tube formation induced by HCMV infection, which could be reversed by transfection with an miR-199a-5p inhibitor. Furthermore, pretreatment with resveratrol depressed motility and tube formation of HCMV-infected ECs, which could be reversed by SIRT1 siRNA. Finally, overexpression of miR-199a-5p decreased the level of eNOS modulated by SIRT1, an effect repressed by transfection with an miR-199a-5p inhibitor. In summary, HCMV infection of endothelial cells upregulates miR-199a-5p expression and enhances cell migration and tube formation through downregulation of SIRT1/eNOS by miR-199a-5p.

Infection of Vascular Endothelial Cells with Human Cytomegalovirus under Fluid Shear Stress Reveals Preferential Entry and Spread of Virus in Flow Conditions Simulating Atheroprone Regions of the Artery

Atherosclerosis is a major pathogenic factor in cardiovascular diseases, which are the leading cause of mortality in developed countries. While risk factors for atherosclerosis tend to be systemic, the distribution of atherosclerotic plaques within the vasculature is preferentially located at branch points and curves where blood flow is disturbed and shear stress is low. It is now widely accepted that hemodynamic factors can modulate endothelial gene expression and function and influence the pathophysiological changes associated with atherosclerosis. Human cytomegalovirus (HCMV), a ubiquitous pathogen, has long been proposed as a risk factor for atherosclerosis. To date, the role of HCMV in atherogenesis has been explored only in static conditions, and it is not known how HCMV infection is influenced by the physiological context of flow. In this study, we utilized a parallel-plate flow system to simulate the effects of shear stresses in different regions of the vasculature in vitro. We found that endothelial cells cultured under low shear stress, which simulates the flow condition of atheroprone regions in vivo, are more permissive to HCMV infection than cells experiencing high shear stress or static conditions. Cells exposed to low shear stress show increased entry of HCMV compared to cells exposed to high shear stress or static conditions. Viral structural gene expression, viral titers, and viral spread are also enhanced in endothelial cells exposed to low shear stress. These results suggest that hemodynamic factors modulate HCMV infection of endothelial cells, thus providing new insights into the induction/acceleration of atherosclerosis by HCMV.

Valproic acid interferes with antiviral treatment in human cytomegalovirus-infected endothelial cells

The endothelium represents a natural site of human cytomegalovirus (HCMV) infection involved in viral spreading and persistence. Moreover, HCMV infection of endothelial cells has been associated with different pathological conditions of the cardiovascular system. Here, the influence of the antiepileptic drug valproic acid (VPA) was investigated on HCMV replication in human umbilical vein endothelial cells alone or in combination with the antiviral drugs ganciclovir, cidofovir or foscarnet. HCMV replication was observed by immunostaining for viral antigens and by virus yield assay. Protein expression and phosphorylation were examined by western blot. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction assay. Therapeutic VPA concentrations (< or =1 mM) increased HCMV immediate early antigen, late antigen, and viral titres of different endotheliotropic and non-endotheliotropic HCMV strains in a concentration- and time-dependent manner up to 30-fold. Moreover, VPA impaired the antiviral activity of the anti-HCMV drugs ganciclovir, cidofovir, and foscarnet. VPA inhibits histone deacetylases (HDAC) and induces HDAC-independently extracellular signal-regulated kinases 1/2 (ERK 1/2) phosphorylation in endothelial cells. Both effects observed, HCMV stimulation and interference with antiviral drugs, depend on HDAC inhibition but not on ERK 1/2 phosphorylation. These findings suggest to carefully monitor the frequency of HCMV reactivation in cardiovascular patients treated with VPA (or other HDAC inhibitors) in comparison to control individuals.

Targeting the NF-κB pathway through pharmacological inhibition of IKK2 prevents human cytomegalovirus replication and virus-induced inflammatory response in infected endothelial cells

Endothelial cells are important reservoirs for human cytomegalovirus (HCMV) replication, dissemination and persistence. HCMV infection of endothelial cells has been associated with a proinflammatory response characterized by an increased expression of chemokines and adhesion molecules and modulation of angiogenesis. Many of the host proinflammatory genes augmented in HCMV-infected endothelial cells are regulated, at least in part, by the NF-κB pathway. HCMV is a potent activator of NF-κB through the IKK-IκB signaling axis. To explore whether inhibition of HCMV-induced NF-κB activation may interfere with the onset of virus-associated inflammatory response, we measured the effects of the specific IKK2 inhibitor AS602868 on the expression of a panel of proinflammatory genes in HUVEC cells infected with a clinical isolate. Treatment of infected HUVEC with AS602868 was shown to impair HCMV-induced NF-κB activity, IE gene expression, viral replication and to prevent HCMV-induced upregulation of ICAM-1, IL-8, RANTES, IP-10, I-TAC and COX-2 gene expression. Consistent with these results, HCMV-mediated upregulation of another NF-κB-dependent gene, the plasminogen inhibitor type-1, a regulatory factor of endothelial proliferation and angiogenesis, was abrogated by AS602868. These results suggest that inhibition of HCMV-induced IKK-NF-κB activation may be of interest to limit the virus-induced inflammatory response of infected endothelial cells.

Human cytomegalovirus tropism for endothelial cells: not all endothelial cells are created equal.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that persists for the life of the host following initial infection. Genome analysis indicates that mammalian CMVs have cospeciated with their respective host over the last 80 million years (39). This prolonged period of coevolution has resulted in a high level of coadaptation between virus and host. The study of CMV, a virus that is exquisitely adapted for persistence within the host, is beginning to reveal strategies critical for virus survival. Although all herpesviruses persist for the life span of the host, recent findings suggest that HCMV has a unique replication strategy for maintenance within the host, wherein the virus establishes sites of persistent active replication even in the presence of high levels of preexisting HCMV-specific immunity. A number of cell types, including myeloid lineage cells, smooth muscle cells, and endothelial cells (ECs), appear to be critical as sites of HCMV persistent replication and latency. HCMV infections of myeloid lineage and of smooth muscle cells have been the focuses of previous reviews (see references 32 and 66). This review will focus on HCMV infection of ECs and the role of this cell type in virus persistence and latency. We will describe a “genomic island” of three genes that are essential for HCMV EC tropism and discuss mechanisms by which the products of these genes mediate HCMV infection in ECs.

Pathogenesis of human cytomegalovirus infection and cellular targets

In acquired immunodeficiency syndrome patients with human cytomegalovirus (HCMV), disseminated infection, and end-organ disease, autopsy findings show a generalized HCMV infection of endothelial cells. On the other hand, immunocompromised transplanted patients show presence of virus and virus products in peripheral blood leukocytes (PBL), when affected by a disseminated HCMV infection. All diagnostic assays are based on the detection of virus and viral components in PBL or whole blood, including polymorphonuclear leukocytes and monocytes. The interplay between endothelial cells and leukocytes represents the pathogenetic basis for all clinical syndromes originating during disseminated HCMV infections and is the trigger for the transmission of HCMV from mother to fetus during primary infections of pregnant women. The two biologic properties of endothelial cell tropism and leukocyte (polymorphonuclear- and monocyte-) tropism are shared by all recent clinical HCMV isolates, whereas they are missing in laboratory-adapted strains. The potential role of HCMV in the pathogenesis of atherosclerosis both in the immunocompetent (after angioplasty) and the heart transplant patient is receiving support from seroepidemiologic findings, in vivo animal models, in vitro data, and also some clinical observations. The interaction of endothelial cells and leukocytes with subsequent spreading of infection to smooth muscle cells may be a major pathogenetic mechanism at the basis of this important vascular disease.

The Effects of Intravenous Anesthetics on Human Cytomegalovirus Infection

Background: Reactivation of human cytomegalovirus (HCMV) from latency is a frequent complication of organ transplantation, and the molecular mechanism by which this occurs is unknown. Previous studies have shown that allogenaic transplantation combined with immunosuppression may be required to achieve complete reactivation in vivo and many anesthetics have wide range immunomodulatory properties. HCMV infection of endothelial cells plays an important role in the establishment of latency and persistence, which appears critical for the maintenance of HCMV within the host. Methods: We compared the effects of intravenous anaesthetics (propofol, thiopental, and ketamine) on the susceptability of endothelial cells to HCMV infection by indirect immunofluorescent assay at 48 hour postinfection and we also have investigated the time course of luciferase gene expression in human umbilical vein endothelial cell (HUVEC) infected with recombinant HCMV. Results: Treatment with anesthetics after HCMV strain Towne inoculation did not increase HUVEC susceptibility to HCMV infection by indirect immunofluorescent assay. Treatment of HUVEC with propofol, thiopental, and ketamine after the recombinant virus inoculation had no significant effects on the level of the late genes expression. Conclusions: Intravenous anesthetics (propofol, thiopental, and ketamine) did not increase the susceptability of endothelial cells to HCMV infection at plasma concentrations. Further studies are required to evaluate higher anesthetic concentration which may increases the susceptability of HUVEC to HCMV infection without cell destruction.