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.

Computational pathology reveals unique spatial patterns of immune response in H&E images from COVID-19 autopsies: preliminary findings.

Purpose: We used computerized image analysis and machine learning approaches to characterize spatial arrangement features of the immune response from digitized autopsied H&E tissue images of the lung in coronavirus disease 2019 (COVID-19) patients. Additionally, we applied our approach to tease out potential morphometric differences from autopsies of patients who succumbed to COVID-19 versus H1N1. Approach: H&E lung whole slide images from autopsy specimens of nine COVID-19 and two H1N1 patients were computationally interrogated. 606 image patches ( ∼ 55 per patient) of 1024 × 882 pixels were extracted from the 11 autopsied patient studies. A watershed-based segmentation approach in conjunction with a machine learning classifier was employed to identify two types of nuclei families: lymphocytes and non-lymphocytes (i.e., other nucleated cells such as pneumocytes, macrophages, and neutrophils). Based off the proximity of the individual nuclei, clusters for each nuclei family were constructed. For each of the resulting clusters, a series of quantitative measurements relating to architecture and density of nuclei clusters were calculated. A receiver operating characteristics-based feature selection method, violin plots, and the t-distributed stochastic neighbor embedding algorithm were employed to study differences in immune patterns. Results: In COVID-19, the immune response consistently showed multiple small-size lymphocyte clusters, suggesting that lymphocyte response is rather modest, possibly due to lymphocytopenia. In H1N1, we found larger lymphocyte clusters that were proximal to large clusters of non-lymphocytes, a possible reflection of increased prevalence of macrophages and other immune cells. Conclusion: Our study shows the potential of computational pathology to uncover immune response features that may not be obvious by routine histopathology visual inspection.

Molecular Detection of SARS-CoV-2 Infection in FFPE Samples and Histopathologic Findings in Fatal SARS-CoV-2 Cases.

OBJECTIVES: To report methods and findings of 2 autopsies with molecular evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive individuals. METHODS: Postmortem examination was completed following Centers for Disease Control and Prevention public guidelines. Numerous formalin-fixed paraffin-embedded (FFPE) tissue types from each case were surveyed for SARS-CoV-2 RNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR). SARS-CoV-2 viral genome was sequenced by next-generation sequencing (NGS) from FFPE lung tissue blocks. RESULTS: Postmortem examinations revealed diffuse alveolar damage, while no viral-associated hepatic, cardiac, or renal damage was observed. Viral RNA was detected in lungs, bronchi, lymph nodes, and spleen in both cases using qRT-PCR method. RNA sequencing using NGS in case 1 revealed mutations most consistent with Western European Clade A2a with ORF1a L3606F mutation. CONCLUSIONS: SARS-CoV-2 testing and viral sequencing can be performed from FFPE tissue. Detection and sequencing of SARS-CoV-2 in combination with morphological findings from postmortem tissue examination can aid in gaining a better understanding of the virus's pathophysiologic effects on human health.