ABSTRACT
The role of the renin-angiotensin system (RAS) in coronavirus disease 2019 (COVID-19) has received much attention, because the angiotensin-converting enzyme 2 (ACE2) has been identified as the main receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has been speculated that, in COVID-19, RAS dysregulation in favor of angiotensin II (Ang-II)-mediated signaling might result in severe tissue inflammation and lung injury. Likewise, the role of a pre-existing therapy with angiotensin-converting enzyme 1 inhibitors (ACEi) or Ang- II type 1 receptor blockers (ARBs) in COVID-19 is largely unclear. We evaluated the effects of the ACEi enalapril (ENA) and the ARB losartan (LOS) on SARS-CoV-2 infection in human ex vivo-cultured, precision-cut lung slices (PCLS) obtained from normal human lung tissue. The PCLS were pretreated for 5 days with vehicle, LOS or ENA (300 muM), followed by mock infection or infection with SARS-CoV-2 and (continued) incubation with vehicle, LOS or ENA for 1 or 2 days. Thereafter, PCLS were harvested for analysis of viral replication, inflammatory responses, endoplasmic reticulum (ER) stress and apoptosis pathways. Both LOS and ENA significantly reduced viral replication in PCLS, with ENA being more potent. LOS was more efficient than ENA in reducing the expression of IL1B, CCL2, CXCL2 and TNFA, but not of IL6, whereas ENA preferentially caused a reduction of IL6 and CCL2 in SARS-CoV-2-infected PCLS. Furthermore, ENA, but not LOS, significantly decreased the expression of viral entry factors, ACE2 and transmembrane serine protease 2 (TMPRSS2), in infected PCLS, both of which were found to be robustly induced upon SARS-CoV-2 infection. Importantly, LOS or ENA did not exert apoptosis or other cytotoxic effects. Renin-angiotensin system-antagonizing drugs do not seem to exert detrimental effects during SARS-CoV-2 infection.On the contrary, in an ex vivo model of human PCLS, such treatment was found to dampen SARS-CoV-2 infection and consecutive inflammation.
ABSTRACT
Background: COVID-19 is characterized by emergency hematopoiesis with a dysregulated myeloid compartment, comprising proinflammatory and immunosuppressive immune cells. Preexisting cardiovascular disease (CVD) is a major risk factor for severe and fatal COVID-19 outcomes. Individuals with atherosclerosis are known to have a proinflammatory immune cell phenotype. However, the mechanisms of how CVD causes worse outcomes during SARS-Cov2 infection remain unknown. Purpose: To investigate the mechanisms of how immune cells link atherosclerosis to worse COVID-19 outcomes Methods: Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) derived from hospitalized SARS-Cov2 infected patients in an uncomplicated phase of the disease not requiring intensive-care treatment with (n=5) and without (n=6) preexisting atherosclerosis was performed. Results: Baseline characteristics between the two groups were similar (atherosclerosis vs. no atherosclerosis: mean age 75 vs. 70 years, oxygen requirement 2.2 vs. 3.2 l/min, CRP 10.7 vs. 6.6 mg/dl, IL-6 61.6 vs. 60.6 pg/ml, all p>0.05). In accordance with previous COVID-19 scRNA-seq studies, we found low-density neutrophils, immature neutrophils, neutrophil like plasmablasts and mostly classical monocytes in the myeloid compartment. Low-density neutrophils from patients with atherosclerosis demonstrated an increased expression of proinflammatory (IL18R1 fold change (fc) = 3.3, IL18RAP fc=1.9, HMGB2 fc=1.8, S100A12 fc=1.7, TLR2 fc=1.5, S100A9 fc=1.4 C3AR1 fc=1.8, TLR4 fc= 1.4, all adjusted p-values <1.3x10–98) and immunosuppressive genes (IL1R2 fc=2.6, ARG1 fc=1.7, ANXA1 fc= 1.6, all adjusted p-values <4.1x10–67). Interestingly, we found an enrichment of proinflammatory COVID-19 specific neutrophil like plasmablasts in patients with atherosclerosis (p=0.049) with an increased expression of inflammatory genes (S100A12 fc=2.5, S100A9 fc=2.5, S100A8 fc=1.8, HMGB2 fc=2.8, IL18R1 fc=3.9 S100A10 fc=2, all adjusted p-values <1.1x10–54). In accordance, monocytes from patients with atherosclerosis showed an enrichment of inflammatory (S100A9 fc=1.6, NEAT1 fc=1.8, C3AR1 fc= 1.5, TLR2 fc= 1.5, IL13RA1 q=1.3, CCR2 fc=1.2, all p-values <1.3x10–60) and immunomodulatory genes (IL1R2 fc=3.5, CD163 fc=2.2, all adjusted p-values <2.7x10–87). Conclusions: Our data show for the first time that patients with atherosclerosis have a dysregulated myeloid immune response already in the uncomplicated phase of SARS-CoV-2 infection. Upregulated genes and cell populations found in this study have previously been associated with severe COVID-19. Therefore, the enhanced inflammatory response may contribute to the worse outcome of patients with CVD and might be addressed by antiinflammatory drugs. Further efforts are needed to understand how atherosclerosis may control chromatin accessibility to predispose for an enhanced inflammatory response. Funding Acknowledgement: Type of funding sources: Foundation. Main funding source(s): German Heart FoundationFigure 1
ABSTRACT
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.