SARS-CoV-2 Spike Protein Destabilizes Microvascular Homeostasis.

SARS-CoV-2 infection can cause compromised respiratory function and thrombotic events. SARS-CoV-2 binds to and mediates downregulation of angiotensin converting enzyme 2 (ACE2) on cells that it infects. Theoretically, diminished enzymatic activity of ACE2 may result in increased concentrations of pro-inflammatory molecules, angiotensin II, and Bradykinin, contributing to SARS-CoV-2 pathology. Using immunofluorescence microscopy of lung tissues from uninfected, and SARS-CoV-2 infected individuals, we find evidence that ACE2 is highly expressed in human pulmonary alveolar epithelial cells and significantly reduced along the alveolar lining of SARS-CoV-2 infected lungs. Ex vivo analyses of primary human cells, indicated that ACE2 is readily detected in pulmonary alveolar epithelial and aortic endothelial cells. Exposure of these cells to spike protein of SARS-CoV-2 was sufficient to reduce ACE2 expression. Moreover, exposure of endothelial cells to spike protein-induced dysfunction, caspase activation, and apoptosis. Exposure of endothelial cells to bradykinin caused calcium signaling and endothelial dysfunction (increased expression of von Willibrand Factor and decreased expression of Krüppel-like Factor 2) but did not adversely affect viability in primary human aortic endothelial cells. Computer-assisted analyses of molecules with potential to bind bradykinin receptor B2 (BKRB2), suggested a potential role for aspirin as a BK antagonist. When tested in our in vitro model, we found evidence that aspirin can blunt cell signaling and endothelial dysfunction caused by bradykinin in these cells. Interference with interactions of spike protein or bradykinin with endothelial cells may serve as an important strategy to stabilize microvascular homeostasis in COVID-19 disease. IMPORTANCE SARS-CoV-2 causes complex effects on microvascular homeostasis that potentially contribute to organ dysfunction and coagulopathies. SARS-CoV-2 binds to, and causes downregulation of angiotensin converting enzyme 2 (ACE2) on cells that it infects. It is thought that reduced ACE2 enzymatic activity can contribute to inflammation and pathology in the lung. Our studies add to this understanding by providing evidence that spike protein alone can mediate adverse effects on vascular cells. Understanding these mechanisms of pathogenesis may provide rationale for interventions that could limit microvascular events associated with SARS-CoV-2 infection.

Cytomegalovirus infection is associated with an increase in aortic stiffness in older men which may be mediated in part by CD4 memory T-cells.

Human Cytomegalovirus (CMV) infection is associated with atherosclerosis, higher cardiovascular disease (CVD) risk, and an increase in memory T-cells (Tmem). T-cells have also been implicated in CVD, independently of CMV infection. To better understand the CMV-associated CVD risk, we examined the association between CMV (IgG) serostatus and central aortic (carotid-to-femoral) pulse wave velocity (cfPWV), an early, independent predictor of CVD. We also investigated if such an association might be reflected by the distribution of Tmem and/or other T-cell subsets. Methods: Healthy older volunteers (60-93 years) underwent routine clinical and laboratory evaluation, including assessment of cfPWV in eligible participants. Flow-cytometry was used to assess proportions of memory T-cells, CD28null T-cells, and CMV-specific T-cells. The following associations were examined; CMV serostatus/cfPWV, CMV serostatus/proportion of Tmem, proportion of Tmem/cfPWV, CD28null T-cells/cfPWV, and CMV-specific T-cells/cfPWV. Linear regression models were used to adjust for age, sex, socioeconomic status, smoking, waist-to-hip ratio, cholesterol, and blood pressure as required. Results: Statistically significant positive associations were found (P-values for the fully adjusted models are given); CMV serostatus/cfPWV in men (P ≤ 0.01) but not in women, CMV serostatus/proportions of CD4 Tmem in men (P ≤ 0.05) but not in women; proportions of CD4 Tmem/cfPWV among CMV seropositive (CMV+) people (P ≤ 0.05) but not CMV seronegative (CMV-) people. Conclusion: CMV infection increases the CVD risk of older men by increasing cfPWV. This may be mediated in part by increased proportions of CD4 Tmem, higher numbers of which are found in CMV+ older people and more so among men than women. Given the high prevalence of CMV worldwide, our findings point to a significant global health issue. Novel strategies to mitigate the increased CVD risk associated with CMV may be required.

Jugular Vein Injection of High-Titer Lentiviral Vectors Does Not Transduce the Aorta-Brief Report.

OBJECTIVE: Efficient gene transfer to the vascular wall via intravenous vector injection would be useful for experimental vascular biology and gene therapy. Initial studies of lentiviral vector tropism suggested that intravenously injected vectors do not transduce murine vascular tissue; however, there are also reports of highly efficient aortic transduction after jugular vein injection of high-titer lentiviral vectors. We sought to reproduce these results. Approach and Results: We injected high-titer preparations of GFP (green fluorescent protein)-expressing lentiviral vector into jugular veins of 8 mice; 6 mice received vehicle only. Four days later, samples of aorta (thoracic and abdominal), liver, spleen, and other tissues were harvested and processed for quantitative polymerase chain reaction detection of vector DNA and immunohistochemical detection of GFP. Our vector DNA assay did not detect transduction of any of the 16 aortic segments. This finding excludes an aortic transduction efficiency of >0.02 vector copies per cell. In contrast, vector DNA was detected in all 8 spleen and liver extracts (median, 0.8 and 0.1 vector copies per cell, respectively; P<0.001 versus vehicle controls). Quantitative polymerase chain reaction signals from DNA extracted from heart, lung, kidney, skeletal muscle, and femoral artery did not differ from background polymerase chain reaction signals from DNA extracted from tissues of vehicle-injected mice (P≥0.7 for all). Immunohistochemistry revealed GFP in scattered cells in spleen and liver, not in aorta. CONCLUSIONS: Injection of high-titer lentiviral vectors via the jugular vein transduces cells in the spleen and liver but does not efficiently transduce the aorta. Graphic Abstract: A graphic abstract is available for this article.

Investigating the Association of Giant Cell Arteritis with Varicella Zoster Virus in Temporal Artery Biopsies or Ascending Aortic Resections.

OBJECTIVE: A variety of infectious agents, including varicella zoster virus (VZV), have been hypothesized to play a role in the pathogenesis of giant cell arteritis (GCA). The detectability of the virus in patients with GCA is debatable. To further investigate an association between GCA and VZV infection, 10 years of GCA cases were evaluated for VZV by immunohistochemistry (IHC). METHODS: All temporal artery biopsies and ascending aortic resections positive for GCA from 2007 to 2017 at Brigham and Women's Hospital were immunostained using a VZV antibody cocktail (SG1-1, SG1-SG4, NCP-1, and IE-62). RESULTS: Forty-one temporal artery biopsies and 47 ascending aortic resections positive for GCA were identified, all of which were found to be negative for VZV by IHC. Twelve temporal artery biopsies in this cohort were previously analyzed by unbiased metagenomics sequencing and were negative for VZV DNA. CONCLUSION: These results argue against a clinically relevant association between VZV infection and GCA, and support neither routine testing for VZV nor treatment with antiviral drugs.

Infectivity of Zika virus on primary cells support tree shrew as animal model.

Zika virus (ZIKV) is a mosquito-borne flavivirus that caused the public health emergency. Recently, we have proved a novel small animal tree shrew was susceptive to ZIKV infection and presented the most common rash symptoms as ZIKV patients. Here we further cultured the primary cells from different tissues of this animal to determine the tissue tropism of ZIKV infection in vitro. The results showed that the primary cells from tree shrew kidney, lung, liver, skin and aorta were permissive to ZIKV infection and could support viral replication by the detection of viral specific RNA intra- and extra-cells. In comparing, the skin fibroblast and vascular endothelial cells were highly permissive to ZIKV infection with high releasing of active virus particles in supernatants proved by its infectivity in established neonatal mouse model. The expressions of ZIKV envelop and nonstructural protein-1, and the effects and strong immune response of primary tree shrew cells were also detected followed by ZIKV infection. These findings provide powerful in vitro cell-level evidence to support tree shrew as animal model of ZIKV infection and may help to explain the rash manifestations in vivo.

The culture of primary duck endothelial cells for the study of avian influenza.

BACKGROUND: Endothelial cells play a major role in highly pathogenic avian influenza (HPAI) virus pathogenesis in gallinaceous poultry species (e.g. chicken, turkey and quail). Upon infection of gallinaceous poultry with HPAI viruses, endothelial cells throughout the body become rapidly infected, leading to systemic dissemination of the virus, disseminated intravascular coagulation, oedema and haemorrhaging. In contrast, the pathogenesis of HPAI viruses in most wild bird species (e.g. duck, goose and gull species) is not associated with endothelial tropism. Indeed, viral antigen is not found in the endothelial cells of most wild bird species following infection with HPAI viruses. This differential endothelial cell tropism in avian species is poorly understood, mainly due to the absence of appropriate cell culture systems. RESULTS: Here, we describe the isolation and purification of primary duck endothelial cells from the aorta or bone marrow of Pekin duck embryos. Cells were differentiated in the presence of vascular endothelial growth factor and, if needed, enriched via fluorescent-activated cell sorting based on the uptake of acetylated low-density lipoprotein. The expression of von Willebrand factor, a key marker of endothelial cells, was confirmed by polymerase chain reaction. Monocultures of duck endothelial cells, either derived from the aorta or the bone marrow, were susceptible to infection with an H5N1 HPAI virus but to a much lesser extent than chicken endothelial cells. CONCLUSIONS: The methods described herein to isolate and purify duck endothelial cells from the aorta or bone marrow could also be applied to obtain microvascular endothelial cells from other tissues and organs, such as the lung or the intestine, and represent a valuable tool to study the pathogenesis of avian viruses.

Murine Norovirus Infection Variably Alters Atherosclerosis in Mice Lacking Apolipoprotein E.

Macrophages play a key role in the development of atherosclerosis. Murine noroviruses (MNV) are highly prevalent in research mouse colonies and infect macrophages and dendritic cells. Our laboratory found that MNV4 infection in mice lacking the LDL receptor alters the development of atherosclerosis, potentially confounding research outcomes. Therefore, we investigated whether MNV4 likewise altered atherosclerosis in ApoE(-/-) mice. In the presence of oxidized LDL, MNV4 infection of ApoE(-/-) bone marrow-derived macrophages increased the gene expression of the inflammatory markers inducible nitric oxide synthase, monocyte chemoattractant protein 1, and IL6. In addition, proteins involved in cholesterol transport were altered in MNV4-infected ApoE -/- bone marrow-derived macrophages and consisted of increased CD36 and decreased ATP-binding cassette transporter A1. MNV4 infection of ApoE(-/-) mice at 12 wk of age (during the development of atherosclerosis) had a variable effect on atherosclerotic lesion size. In one study, MNV4 significantly increased atherosclerotic plaque area whereas in a second study, no effect was observed. Compared with controls, MNV4-infected mice had higher circulating Ly6C-positive monocytes, and viral RNA was detected in the aortas of some mice, suggesting potential mechanisms by which MNV4 alters disease progression. Plaque size did not differ when ApoE -/- mice were infected at 4 wk of age (early during disease development) or in ApoE -/- mice maintained on a high-fat, high-cholesterol diet. Therefore, these data show that MNV4 has the potential to exert a variable and unpredictable effect on atherosclerosis in ApoE(-/-) mice. We therefore propose that performing experiments in MNV-free mouse colonies is warranted.

Turnover Rate of NS3 Proteins Modulates Bluetongue Virus Replication Kinetics in a Host-Specific Manner.

UNLABELLED: Bluetongue virus (BTV) is an arbovirus transmitted to livestock by midges of the Culicoides family and is the etiological agent of a hemorrhagic disease in sheep and other ruminants. In mammalian cells, BTV particles are released primarily by virus-induced cell lysis, while in insect cells they bud from the plasma membrane and establish a persistent infection. BTV possesses a ten-segmented double-stranded RNA genome, and NS3 proteins are encoded by segment 10 (Seg-10). The viral nonstructural protein 3 (NS3) plays a key role in mediating BTV egress as well as in impeding the in vitro synthesis of type I interferon in mammalian cells. In this study, we asked whether genetically distant NS3 proteins can alter BTV-host interactions. Using a reverse genetics approach, we showed that, depending on the NS3 considered, BTV replication kinetics varied in mammals but not in insects. In particular, one of the NS3 proteins analyzed harbored a proline at position 24 that leads to its rapid intracellular decay in ovine but not in Culicoides cells and to the attenuation of BTV virulence in a mouse model of disease. Overall, our data reveal that the genetic variability of Seg-10/NS3 differentially modulates BTV replication kinetics in a host-specific manner and highlight the role of the host-specific variation in NS3 protein turnover rate. IMPORTANCE: BTV is the causative agent of a severe disease transmitted between ruminants by biting midges of Culicoides species. NS3, encoded by Seg-10 of the BTV genome, fulfills key roles in BTV infection. As Seg-10 sequences from various BTV strains display genetic variability, we assessed the impact of different Seg-10 and NS3 proteins on BTV infection and host interactions. In this study, we revealed that various Seg-10/NS3 proteins alter BTV replication kinetics in mammals but not in insects. Notably, we found that NS3 protein turnover may vary in ovine but not in Culicoides cells due to a single amino acid residue that, most likely, leads to rapid and host-dependent protein degradation. Overall, this study highlights that genetically distant BTV Seg-10/NS3 influence BTV biological properties in a host-specific manner and increases our understanding of how NS3 proteins contribute to the outcome of BTV infection.

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.

Extracellular but not cytosolic superoxide dismutase protects against oxidant-mediated endothelial dysfunction.

Superoxide (O2 (•-)) contributes to the development of cardiovascular disease. Generation of O2 (•-) occurs in both the intracellular and extracellular compartments. We hypothesized that the gene transfer of cytosolic superoxide dismutase (SOD1) or extracellular SOD (SOD3) to blood vessels would differentially protect against O2 (•-)-mediated endothelial-dependent dysfunction. Aortic ring segments from New Zealand rabbits were incubated with adenovirus (Ad) containing the gene for Escherichia coli ß-galactosidase, SOD1, or SOD3. Activity assays confirmed functional overexpression of both SOD3 and SOD1 isoforms in aorta 24 h following gene transfer. Histochemical staining for ß-galactosidase showed gene transfer occurred in the endothelium and adventitia. Next, vessels were prepared for measurement of isometric tension in Kreb's buffer containing xanthine. After precontraction with phenylephrine, xanthine oxidase impaired relaxation to the endothelium-dependent dilator acetylcholine (ACh, max relaxation 33±4% with XO vs. 64±3% without XO, p<0.05), whereas relaxation to the endothelium-independent dilator sodium nitroprusside was unaffected. In the presence of XO, maximal relaxation to ACh was improved in vessels incubated with AdSOD3 (55±2%, p<0.05 vs. control) but not AdSOD1 (34±4%). We conclude that adenoviral-mediated gene transfer of SOD3, but not SOD1, protects the aorta from xanthine/XO-mediated endothelial dysfunction. These data provide important insight into the location and enzymatic source of O2 (•-) production in vascular disease.