ACE2 IS INVOLVED IN SARS-COV-2 INDUCED ENDOTHELIAL CELL INFLAMMATION INDEPENDENT OF VIRAL REPLICATION

Objective: COVID-19 association with cardiovascular disease is thought to be due to endothelial cell inflammation. ACE2 interactions with SARS-CoV-2 spike protein S1 subunit is important to viral infection. Here we questioned whether SARS-CoV-2 induces vascular inflammation via ACE2 and whether this is related to viral infection. Design and Methods: Human microvascular endothelial cells (EC) were exposed to recombinant S1p (rS1p) 0.66 ug/mL for 10 min, 5 h and 24 h. Gene expression was assessed by RT-PCR and levels of IL6 and MCP1, as well as ACE2 activity, were assessed by ELISA. Expression of ICAM1 and PAI1 was assessed by immunoblotting. ACE2 activity was blocked by MLN4760 (ACE2 inhibitor) and siRNA. Viral infection was assessed by exposing Vero E6 (kidney epithelial cells;pos ctl) and EC to 105 pfu of SARS-CoV-2 where virus titre was measured by plaque assay. Results: rS1p increased IL6 mRNA (14.2 ± 2.1 vs. C:0.61 ± 0.03 2-ddCT) and levels (1221.2 ± 18.3 vs. C:22.77 ± 3.2 pg/mL);MCP1 mRNA (5.55 ± 0.62 vs. C:0.65 ± 0.04 2-ddCT) and levels (1110 ± 13.33 vs. C:876.9 ± 33.4 pg/mL);ICAM1 (17.7 ± 3.1 vs. C:3.9 ± 0.4 AU) and PAI1 (5.6 ± 0.7 vs. C: 2.9 ± 0.2), p < 0.05. MLN4760, but not rS1p, decreased ACE2 activity (367.4 ± 18 vs. C: 1011 ± 268 RFU, p < 0.05) and blocked rS1p effects on ICAM1 and PAI1. ACE2 siRNA blocked rS1p-induced IL6 release, ICAM1, and PAI1 responses as well as rS1p-induced NFkB activation. EC were not susceptible to SARS-CoV-2 infection, while the virus replicated well in Vero E6. Conclusion: rS1p induces an inflammatory response through ACE2 in endothelial cells;an effect that was independent of viral infection.

Spike Proteins Increase Endothelial Calcium via TRPV4

Introduction: Endothelial dysfunction plays a central role in the pathogenesis of acute respiratory distress syndrome (ARDS) with COVID-19. Transient receptor potential vanilloid 4 (TRPV4), a cation channel ubiquitously expressed, can regulate inflammatory cytokines that play key roles in in acute lung injury/ARDS. However, it is unknown whether spike proteins can affect TRPV4 activity and related Ca2+]signaling in pulmonary microvascular endothelial cells. Hypothesis: We hypothesized that spike protein causes activation of TRPV4 channels, resulting in increases in intracellular Ca2+], may lead to pulmonary endothelial dysfunction. Method(s): Intracellular Ca2+]concentrations in human lung microvascular endothelial cells (HLMECs) were measured by calcium imaging in the presence of SARS CoV-2 Spike protein S1, receptor-binding domain (RBD) of S1, or protein S2 with or without co-incubation of the selective TRPV4 antagonist (HC-067047). Result(s): The intracellular Ca2+]concentration of HLMECs was significantly increased when incubated with S1 (1, 10nM) or S1 RBD (1, 10nM) for 12, 24, 48 hours, relative to control or S2 (p<0.05, respectively, Fig. A, B). Co-incubation of HC-067047 (500nM) significantly attenuated Ca2+]intracellular influx upon treatment with S1 (10nM, 24 hours, p<0.05) or S1 RBD (10nM, 24 hours, p<0.05) (Fig. C). TRPV4 sensitive current density was significantly increased when incubated with S1 (10nM) or S1 RBD (10nM) for 24 hours (p<0.05 vs. control, respectively, Fig. D-G), whereas co-incubated with HC-067047 (500nM) significantly reversed the S1 (10nM, 24 hours, p<0.05) or S1 RBD (10nM, 24 hours, p<0.05) induced increases of TRPV4 sensitive current density (Fig. D-G). Conclusion(s): The of SARS CoV-2 Spike protein S1 and S1 RBD caused the activation of TRPV4 channels, resulting in increased intracellular Ca2+], may lead to pulmonary endothelial dysfunction. (Figure Presented).

Circulating Extracellular Vesicles Carrying Thromboinflammatory Proteins Promote Pulmonary Vascular Injury in Covid-19 Patients

It is increasingly recognized that moderate/severe coronavirus disease 2019 (COVID-19) disease is in part due to a dysregulated immune response in conjunction with increased thromboinflammation, coagulopathy, and vascular injury. In this study, we analyzed the cargo of extracellular vesicles (EVs) isolated from the plasma of patients with COVID-19 for the identification of potential biomarkers of disease severity and to explore their role in disease pathogenesis. Severe acute respiratory syndrome coronavirus 2-infected patients hospitalized at the University of Kansas Health System were enrolled in the COVID-19 In-patient Biorepository and blood samples were collected for the isolation of plasmaderived EVs. The patients were grouped based on the WHO Clinical Progression Scale score during hospitalization. Our results revealed enrichment of proinflammatory, procoagulation, and tissue-remodeling markers in circulating EVs, distinguishing symptomatic COVID-19 patients from uninfected controls and delineating patients with moderate disease from the critically ill. Among all proteins analyzed, the levels of proinflammatory DAMP: EN-RAGE (aka calgranulin C or S100A12) showed the strongest correlation with length of hospitalization and disease severity. In addition, tissue factor levels/activity linked to EVs appeared to distinguish patients with severe disease from those with a moderate disease but on supplemental oxygen. Alterations in EV cargo corresponded to enhanced apoptosis of primary human pulmonary microvascular endothelial cells and smooth muscle hyperplasia on exposure to EVs from COVID-19 patients. In conclusion, our findings suggest a pivotal role of EVs in the pathogenesis of acute COVID-19 disease. Whether these EV-mediated alterations continue leading to long-haul COVID including cardiopulmonary vascular complications is the focus of our ongoing studies.

Cell-based high throughput screening to identify FDA/ EMA approved drugs that inhibit the endothelial pro-thrombotic switch during severe COVID19 infection

Background: A major complication of COVID19 is severe endothelial injury with micro-and macro-thrombotic disease in the lung and other organs. Several studies have identified high levels of inflammatory cytokines ( cytokine storm ), powerful activators of the endothelium, in plasma of severe COVID19 patients;indeed, COVID19 plasma was shown to activate endothelial cells (EC) in vitro. A consequence of EC activation is loss of anti-coagulant function, with release of pro-thrombotic Von Willebrand Factor (VWF). High levels of plasma VWF in severe COVID19 patients indicate systemic endothelial activation and increased risk of thrombosis. Aim(s): To identify drugs that decrease endothelial activation and VWF release, which may have a therapeutic impact in COVID19 patients. Method(s): We established an in vitro model of endothelial activation driven by 6 cytokines selected because of their high levels in COVID19 plasma. Cells were treated with the 6-cytokine cocktail for 24 hr;endothelial activation was confirmed by a panel of markers including ICAM1, measured by RT-qPCR and immunofluorescence (IF). Result(s): The treatment induced release of VWF and increased VWF-platelet string formation in a platelet flow-based assay. To identify drugs that blocked cytokine-induced VWF release, a high-throughput screening was carried out in human umbilical vein EC (HUVEC);VWF and ICAM1 expression were detected by IF;DAPI was used as nuclear stain. High content imaging screen of 3049 drugs from FDA/EMA-approved drug libraries identified drugs able to decrease VWF release following cytokine treatment. Top hits from several therapeutic classes including anti-inflammatory, anti-viral and hormones were taken forward for validation. Two hits were confirmed to inhibit cytokine-induced VWF release and VWF-platelet string formation. Selected findings were validated in lung microvascular EC. Conclusion(s): This study identified candidate drugs that reduce the enhanced VWF release caused by the cytokine storm typical of severe COVID19;these may be beneficial in the treatment of the pro-thrombotic risk in COVID19 patients.

Rabeprazole is an agonist of HIF-1 and promotes vascular repair and resolution of inflammatory lung injury

Background: There are no effective treatments for inflammatory lung injury, including acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Severe COVID-19 patients commonly suffer from ARDS. The repositioning of existing drugs is one possible strategy for treatment of ALI/ARDS. We previously showed that vascular repair and resolution of inflammatory lung injury is dependent upon endothelial hypoxia-inducible factor 1 alpha (HIF1a) and forkhead box M1 (FoxM1). Aim(s): To identify a candidate agonist of HIF1a/FoxM1 signaling that could effectively treat ALI/ARDS. Method(s): FDA-approved drugs were screened using reporter assays, toxicity assays, and gene expression analyses in vitro. Inflammatory lung injury was assessed in a mouse model of lipopolysaccharide-induced endotoxemia. Result(s): We used high throughput screening of a library 1200 FDA-approved drugs, along with hypoxia-response element-driven luciferase reporter assays, cell toxicity assays, and molecular analyses of human lung microvascular endothelial cells, to identify candidate drugs that enhance HIF1 signaling in vitro. One of these drugs, Rabeprazole (Aciphex), caused dose-dependent increases in hypoxia-response element activity without increasing cell death. By treating wild type mice orally with Rabeprazole on 2 consecutive days after sepsis challenge, we were able to identify a dose of Rabeprazole that is well tolerated and enhances vascular repair and resolution of inflammatory lung injury. In timeline studies, we found that Rabeprazole treatment reduces lung vascular leakage, edema, and inflammatory cytokine expression during the repair phrase. We next used conditional knockout mice to show that Rabeprazole increases vascular repair and resolution of inflammatory lung injury through endothelial HIF1a and FoxM1. Rabeprazole-dependent decreases in lung vascular leakage, edema, and inflammatory cytokine expression were completely absent in the conditional HIF1a and FoxM1 knockout mice. Conclusion(s): Rabeprazole improves murine vascular repair and resolution of sepsis-induced inflammatory lung injury via endothelial HIF1a/FoxM1. This drug represents a promising candidate for repurposing to effectively treat ALI/ARDS.

Downregulation of thrombomodulin-thrombin-activated protein C pathway as a mechanism for SARS-CoV-2 induced endotheliopathy and microvascular thrombosis

Background: There is emerging evidence of microvascular thrombosis and thrombotic microangiopathy (TMA) induced by COVID-19, presumably from endothelial injury or endotheliopathy . Thrombomodulin (TM) is an endothelial glycoprotein that plays a crucial role as a natural anticoagulant, binding thrombin to activate protein C (PC). TM is shed from endothelial surface during injury. We hypothesize SARS-CoV-2 spike proteins cause direct microvascular endothelial injury, leading to TM shedding, decreased activation of PC, and consequently, microvascular thrombosis in COVID-19. Aim(s): To assess: 1) endothelial injury (by soluble TM [sTM] levels) in a cohort of critically-ill COVID-19 pediatric patients;2) endothelial injury (TM shedding) in vitro by SARS-CoV-2 spike proteins and the subsequent functional consequence in activated PC (APC) levels. Method(s): STM in plasma samples from SARS-CoV-2 positive patients admitted to Texas Children's Hospital Pediatric Intensive Care Unit (n = 34) and healthy controls (n = 38) were measured by ELISA. IRB approval and waiver of informed consent were obtained. In vitro, confluent glomerular microvascular endothelial cells (GMVECs) were incubated for 24 hours in the presence or absence (control) of purified SARS-CoV-2 spike proteins, S1 and S2. In some experiments, cell lysates were collected, and TM was measured by ELISA;in others, GMVECs were further supplemented with PC and thrombin for 1 hour, followed by supernatant collection for APC measurement by ELISA. Result(s): STM levels were significantly higher in the COVID-19 pediatric patients (p < 0.01) (Fig. 1). In vitro, surface bound TM (Fig 2a) and soluble APC (Fig 2b) were significantly lower in GMVECs after addition of spike proteins (p < 0.05). Conclusion(s): We provide evidence of endothelial injury in COVID-19 patients and demonstrate a potential pathway of SARS-CoV-2 induced thrombosis. Decreased surface-bound TM results in lower amount of thrombin-TM complex, hence lesser activation of PC, likely leading to a pro-thrombotic state. These findings in GMVECs could explain the vulnerability of kidneys to COVID-19-induced TMA.

The Impact of COVID-19 Spike Protein on Retinal Microvascular Environment

Purpose : Background: Despite being primarily a respiratory disease, COVID-19 can lead to non-respiratory complications, including myocardial infarction and acute ischemic stroke. Moreover, COVID-19 spike protein (SP) was reported in the retina of deceased patients with COVID-19. Retinal microvascular abnormalities as loss of microvasculature and distinct thinning of the microcapillaries were reported in patients who recovered from COVID-19. We are still in the midst of the COVID-19 pandemic, with more deaths and cases every day. Therefore investigating the impact of COVID-19 on the retinal neurovascular environment and the long-term effect of this virus on vision is of great interest. Purpose: To study the contribution of COVID-19 SP to retinal inflammation and vascular death. Methods : Methods: COVID-19 SP, a highly glycosylated protein that allows the virus to penetrate the cell and cause infection, was injected intravitreally in 6-8 weeks global h-ACE2 knock-in mice and wild-type mice. Mice were sacrificed after 14 days, then vascular cell death and inflammation were evaluated by the presence of acellular capillaries and the expression of inflammatory and apoptotic markers. To complement our in-vivo studies, Human Microvascular Endothelial Cells (HMEC) were treated with 100 nM COVID-19 SP for 48 hours. The expression of inflammatory and apoptotic markers was assessed by PCR western blot. Results : Results: Our results showed that HMEC exposed to COVID-19 SP for 48 hours displayed an increase in inflammatory and apoptotic markers expression including TNF-α, IL-1β, IL-6, and cleaved caspase-3 compared to control conditions. Additionally, COVID-19 SP enhanced the oxidative stress in HMEC, evident by the increase in nitro-tyrosine formation, superoxide dismutase, and NADPH oxidase complex 1 (NOX1 and NOX5) expression. The in-vivo findings came in agreement with our in-vitro studies. We found that intravitreal injection of the COVID-19 SP-induced 1) strong activation of the retinal glial cells, assessed by GFAP radial staining, and 2) increased vascular death, assessed by acellular capillaries formation 14 days after the injection. Conclusions : Conclusions: Our findings highlight the possible role of COVID-19 SP in inducing retinal inflammation and vascular death. Further studies are required to reveal the impact of COVID-19 SP on visual acuity and the possibility of causing visual impairment using various animal models.

Cigarette Smoke Regulates Endothelial ACE2 Expression in the Lung

Rationale: Angiotensin-converting enzyme 2 (ACE2) is a vasoactive enzyme involved in regulation of vascular tone and blood pressure by reducing angiotensin II and increasing ang(1-7). It is also implicated in the pathogenesis of coronaviruses including SARS-CoV-2. Epidemiological reports differ in implicating cigarette smoking as a risk factor for SARS-CoV-2 infection (COVID-19). Previous studies have been conflicting regarding the implications of cigarette smoke exposure on ACE2 signaling. We hypothesized that cigarette smoke exposure will increase ACE2 expression and impair endothelial cell function. Methods: Female 8-week-old A/J mice were randomly assigned to either air exposure or 48 minutes per day, 5 days per week of cigarette smoke exposure. Mainstream whole-body cigarette smoke exposure was delivered by the SCIREQ “InExpose” smoking system with standard 3R4F research cigarettes. Mouse were sacrificed at 1 and 12 weeks of smoke exposure, and lungs were homogenized and subjected to ACE2 ELISA (Abcam). To investigate the effect of smoking on ACE2 expression and endothelial barrier function, serum starved human pulmonary microvascular endothelial cells (PMVECs) were exposed to cigarette smoke extract (CSE). CSE was prepared at a concentration of 1 cigarette/5 ml in serumfree DMEM and quiescent PMVECs were treated with 1% CSE, 3% CSE or vehicle. Cells were processed for real-time RT-PCR and ELISA 4 hours later, assessment of apoptosis, or underwent TEER to assess endothelial cell barrier function. Results: Lung tissue ACE2 levels were significantly elevated following 1-week of cigarette smoke-exposure. This increase was accompanied by increased macrophage count in bronchoalveolar lavage. Interestingly, at 12-weeks of cigarette smoke-exposure, lung ACE2 was reduced by 15% response. Chronic cigarette smoke-exposure was accompanied by increased right ventricular systolic pressure and Fulton index. In PMVEC models, CSE dose-dependently increased ACE2 mRNA and protein expression. This was accompanied by altered EC barrier function and EC apoptosis. Conclusions: The dose and duration of cigarette smoke exposure affects ACE2 signaling, leading to altered apoptosis and endothelial cell barrier function. These findings have implications for SARS-CoV-2 pathogenesis as well as for furthering our understanding of the effects of smoking on vascular health.

COVID-19 spike-protein causes cerebrovascular rarefaction and deteriorates cognitive functions in a mouse model of humanized ACE2

COVID-19 pandemic has affected our health and economy. Clinical trials confirmed multiple neurological symptoms due to COVID-19, ranging from headaches, insomnia to stroke, and encephalopathy. More studies are required to unravel the cellular and molecular mechanisms to find a cure for these neurological symptoms. Here, we investigate the effect of COVID-19 spike protein (S-protein) on the cerebrovasculature and cognitive functions in two mouse models that express humanized ACE-2 (h ACE2), a receptor essential for cellular infection and COVID-19 internalization. We hypothesize that COVID-19 S-protein causes cognitive dysfunction via the deterioration of cerebrovascular functions. Methods: S-protein was either injected intravenously or directly into the hippocampus of K-18 (h ACE2 in epithelial cells) or global h-ACE2 knock-in (h ACE2 KI) mice or wild-type mice. Cognitive functions were assessed by Y-maze and Barnes maze. Cerebrovascular density was determined using confocal 3-D image reconstruction. Human brain microvascular endothelial cells (HBMVEC) were treated with S-protein and assessed for apoptosis and inflammatory markers using immunoblotting and RT-PCR. K-18 and h-ACE2 KI mice received intraocular injections of S-protein;retinas were evaluated for vascular cell death and inflammation. Results: S-protein injections caused significant deterioration in memory and learning function of K-18 and h-ACE2 KI mice but not in the wild-type mice (P<0.05). S-protein significantly increased inflammatory mediators, cytokine production, and apoptosis in the brains and HBMVECs (P<0.05). Significant cerebrovascular rarefaction was detected only in K-18 and h-ACE2 KI mice compared to wild-type mice (P<0.05). Retinal vascular cell death and inflammation were significantly increased after S-protein injection. (P<0.05) Conclusions: COVID-19 spike protein decreases cognitive function via increased endothelial cell inflammation, apoptosis, and cerebrovascular rarefaction. Humanized ACE2 animal models are excellent and reliable for investigating the neurological symptoms of COVID-19.

Eicosapentaenoic acid reduces pulmonary endothelial cell adhesion protein expression with IL-6

INTRODUCTION: Infectious agents, including SARSCoV- 2, cause pulmonary endothelial cell (EC) dysfunction that leads to acute respiratory distress syndrome (ARDS). EC dysfunction involves increased leukocyte recruitment and cell permeability mediated by various junctional proteins, integrins, and adhesion molecules. The omega-3 fatty acid eicosapentaenoic acid (EPA) and its metabolites modulate inflammation and vascular function. These actions of EPA may contribute to reduced cardiovascular events as reported in outcome trials such as REDUCE-IT. Currently, EPA is being tested in patients at risk for or with COVID-19. This study tested the effects of EPA on protein expression in human pulmonary ECs following challenge by the cytokine IL-6 to simulate conditions encountered in advanced viral infections. METHODS: Human lung microvascular endothelial cells (HMVEC-L) were post-treated with vehicle or EPA (40 μM) in 2% FBS after a 2 hr challenge with IL-6 at 12 ng/ml for 24 h. Proteomic analysis used LC/MS to assess relative expression levels of EC proteins. Only significant (p< 0.05) changes in protein expression between treatment groups >1-fold were analyzed. Specific pathway analysis was carried out using gene set enrichment analysis (GSEA). RESULTS: HMVEC-L treated with EPA following challenge with IL-6 showed significant changes in over 400 proteins compared with IL-6 treatment alone. EPA specifically diminished eleven proteins in the “integrin cell surface interactions” pathway. These pathways proteins included integrins alpha-V, alpha-6, and beta-1, along with PECAM-1, junction adhesion molecule C (JAM3), fibronectin, and ICAM- 2. CONCLUSIONS: EPA reduced expression of pulmonary endothelial adhesion and permeability proteins following IL-6 treatment. The ability of EPA to inhibit EC dysfunction and inflammation may have benefits for patients with or at risk for ARDS due to viruses such as SARS-CoV-2 or sepsis.

Eicosapentaenoic acid increases endothelial function and Heme oxygenase-1 in pulmonary inflammation

INTRODUCTION: Endothelial cell (EC) dysfunction results in reduced nitric oxide (NO) bioavailability leading to inflammation and increased susceptibility to infectious agents. Heme oxygenase-1 (HO-1) produces potent antioxidant and anti-inflammatory products including carbon monoxide. SARS-CoV-2 and influenza affect ECs in multiple vascular beds, including pulmonary tissue. The omega-3 fatty acid eicosapentaenoic acid (EPA) and its metabolites preserve EC function in a manner that may contribute to reduced incident cardiovascular events (REDUCE-IT). Currently, EPA is being tested in patients with or at risk for COVID-19. This study tested the effects of EPA on NO and peroxynitrite (ONOO-) release under conditions of inflammation using lipopolysaccharide (LPS) and the cytokine IL-6. We also measured expression of HO-1 after cell challenge with IL-6. METHODS: Human lung microvascular endothelial cells (HMVEC-L) were pretreated with vehicle or EPA (40 μM) in 2% FBS for 2 h, then challenged with either IL-6 (12 ng/ml) or LPS (200 ng/ml) for 24 h. Cells (including untreated controls) were stimulated with calcium ionophore to measure maximum production of NO and peroxynitrite (ONOO-) using tandem porphyrinic nanosensors. Proteomic analysis was performed using LC/MS to assess relative expression levels. Only significant (p< 0.05) changes in protein expression between treatment groups >1-fold were analyzed. RESULTS: HMVEC-L challenged with LPS and IL-6 showed a pronounced loss of NO release by 22% (p< 0.01) and 18% (p< 0.01), respectively, concomitant with an increase in ONOO- by 28% (p< 0.01) and 26% (p< 0.01), respectively. As a result, the [NO]/[ONOO-] ratio, a marker of eNOS coupling efficiency, decreased by 39% (p< 0.001) and 35% (p< 0.001) with LPS and IL-6, respectively. However, EPA increased this ratio by 39% (p< 0.01) in both LPS and IL-6 treated cells. EPA also caused a 5.7-fold (p = 4.4 × 10-38) increase in expression of HO-1 with IL-6. CONCLUSIONS: These findings indicate that EPA improves NO bioavailability and reduces nitroxidative stress in pulmonary ECs during inflammation with LPS or IL-6. These studies indicate a protective effect of EPA on pulmonary ECs that may reduce inflammatory activation during sepsis, influenza, or advanced COVID-19 that may mediate many aspects of multiorgan system failure.

Eicosapentaenoic acid favorably modulates protein expression in pulmonary endothelial inflammation

INTRODUCTION: SARS-CoV-2 and other viruses can cause endothelial cell (EC) dysfunction in multiple vascular beds, including pulmonary tissue. Infected patients may then develop acute respiratory distress syndrome (ARDS) and cardiovascular (CV) complications. The omega-3 fatty acid eicosapentaenoic acid (EPA) and its bioactive metabolites favorably modulate inflammation and EC function. These benefits of EPA may contribute to reduced CV events as reported in outcome trials (REDUCE-IT). Currently, EPA is being tested in patients with or at risk for COVID-19. This study tested the effects of either EPA pre- or post-treatment on global protein expression in human pulmonary ECs under conditions of inflammation using the cytokine IL-6 to simulate conditions of advanced viral infections. METHODS: Human lung microvascular endothelial cells (HMVEC-L) were pre-treated with either EPA (40 μM) or IL-6 (12 ng/mL) for 2 hr and then treated with IL-6 or EPA, respectively, for 24 hr in media with 2% FBS. Proteomic analysis was performed using LC/MS to assess relative protein expression levels. Only significant (p< 0.05) changes in protein expression between treatment groups >1-fold were analyzed. Expression of soluble intercellular adhesion molecule-1 (sICAM-1) was separately measured with immunochemistry. RESULTS: HMVEC-L pre- and post-treated with EPA during challenge with IL-6 showed significant changes in 100 (49/51 up/down) and 441 (229/212 up/down) proteins, respectively, compared with IL-6 treatment alone. Among the 31 proteins that were significantly modulated by both EPA pre- and post-treatment, thioredoxin reductase 1 increased relative to IL-6 alone, while matrix metalloproteinase 1 and fibronectin both decreased. Other proteins, such as hypoxia up-regulated protein 1, were differentially modulated by EPA relative to IL-6 (increased in pre-treatment, decreased in post-treatment). Finally, EPA significantly reduced sICAM- 1expression by 41% and 12% compared with IL-6 alone in the pre- and post-treatment models, respectively. CONCLUSIONS: These findings indicate that EPA favorably modulates the expression of multiple inflammatory and cytoprotective proteins during inflammation. These studies support a broad anti-inflammatory effect of EPA on pulmonary ECs that may have therapeutic implications for patients at risk for ARDS due to infectious agents including SARS-CoV-2 or other viruses.

Novel SARS-CoV-2 variants induce higher toxicity in cardiovascular cells

Objective: SARS-CoV-2 causes the coronavirus disease 2019 (COVID-19) and has spawned a global health crisis. Virus infection can lead to elevated markers of cardiac injury and inflammation associated with a higher risk of mortality. However, it is so far unclear whether cardiovascular damage is caused by direct virus infection or is mainly secondary due to inflammation. Recently, additional novel SARS-CoV-2 variants have emerged accounting for more than 70% of all cases in Germany. To what extend these variants differ from the original strain in their pathology remains to be elucidated. Here, we investigated the effect of the novel SARS-CoV-2 variants on cardiovascular cells. Results: To study whether cardiovascular cells are permissive for SARSCoV-2, we inoculated human iPS-derived cardiomyocytes and endothelial cells from five different origins, including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells, in vitro with SARS-CoV-2 isolates (G614 (original strain), B.1.1.7 (British variant), B.1.351 (South African variant) and P.1 (Brazilian variant)). While the original virus strain infected iPS-cardiomyocytes and induced cell toxicity 96h post infection (290±10 cells vs. 130±10 cells;p=0.00045), preliminary data suggest a more severe infection by the novel variants. To what extend the response to the novel variants differ from the original strain is currently investigated by phosphoproteom analysis. Of the five endothelial cells studied, only human coronary artery EC took up the original virus strain, without showing viral replication and cell toxicity. Spike protein was only detected in the perinuclear region and was co-localized with calnexin-positive endosomes, which was accompanied by elevated ER-stress marker genes, such as EDEM1 (1.5±0.2-fold change;p=0.04). Infection with the novel SARS-CoV-2 variants resulted in significant higher levels of viral spike compared to the current strain. Surprisingly, viral up-take was also seen in other endothelial cell types (e.g. HUVEC). Although no viral replication was observed (850±158 viral RNA copies at day 0 vs. 197±43 viral RNA copies at day 3;p=0.01), the British SARS-CoV-2 variant B.1.1.7 reduced endothelial cell numbers (0.63±0.03-fold change;p=0.0001). Conclusion: Endothelial cells and cardiomyocytes showed a distinct response to SARS-CoV-2. Whereas cardiomyocytes were permissively infected, endothelial cells took up the virus, but were resistant to viral replication. However, both cell types showed signs of increased toxicity induced by the British SARS-CoV-2 variant. These data suggest that cardiac complications observed in COVID-19 patients might at least in part be based on direct infection of cardiovascular cells. The more severe cytotoxic effects of the novel variants implicate that patients infected with the new variants should be even more closely monitored.