Acute neutrophilic vasculitis (leukocytoclasia) in 36 COVID-19 autopsy brains.

BACKGROUND: Hypercytokinemia, the renin-angiotensin system, hypoxia, immune dysregulation, and vasculopathy with evidence of immune-related damage are implicated in brain morbidity in COVID-19 along with a wide variety of genomic and environmental influences. There is relatively little evidence of direct SARS-CoV-2 brain infection in COVID-19 patients. METHODS: Brain histopathology of 36 consecutive autopsies of patients who were RT-PCR positive for SARS-CoV-2 was studied along with findings from contemporary and pre-pandemic historical control groups. Immunostaining for serum and blood cell proteins and for complement components was employed. Microcirculatory wall complement deposition in the COVID-19 cohort was compared to historical control cases. Comparisons also included other relevant clinicopathological and microcirculatory findings in the COVID-19 cohort and control groups. RESULTS: The COVID-19 cohort and both the contemporary and historical control groups had the same rate of hypertension, diabetes mellitus, and obesity. The COVID-19 cohort had varying amounts of acute neutrophilic vasculitis with leukocytoclasia in the microcirculation of the brain in all cases. Prominent vascular neutrophilic transmural migration was found in several cases and 25 cases had acute perivasculitis. Paravascular microhemorrhages and petechial hemorrhages (small brain parenchymal hemorrhages) had a slight tendency to be more numerous in cohort cases that displayed less acute neutrophilic vasculitis. Tissue burden of acute neutrophilic vasculitis with leukocytoclasia was the same in control cases as a group, while it was significantly higher in COVID-19 cases. Both the tissue burden of acute neutrophilic vasculitis and the activation of complement components, including membrane attack complex, were significantly higher in microcirculatory channels in COVID-19 cohort brains than in historical controls. CONCLUSIONS: Acute neutrophilic vasculitis with leukocytoclasia, acute perivasculitis, and associated paravascular blood extravasation into brain parenchyma constitute the first phase of an immune-related, acute small-vessel inflammatory condition often termed type 3 hypersensitivity vasculitis or leukocytoclastic vasculitis. There is a higher tissue burden of acute neutrophilic vasculitis and an increased level of activated complement components in microcirculatory walls in COVID-19 cases than in pre-pandemic control cases. These findings are consistent with a more extensive small-vessel immune-related vasculitis in COVID-19 cases than in control cases. The pathway(s) and mechanism for these findings are speculative.

A case-control autopsy series of liver pathology associated with novel coronavirus disease (COVID-19).

BACKGROUND: Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for coronavirus disease 2019 (COVID-19) is most well-known for causing pulmonary injury, a significant proportion of patients experience hepatic dysfunction. The mechanism by which SARS-CoV2 causes liver injury is not fully understood. The goal of this study was to describe the hepatic pathology in a large cohort of deceased patients with COVID-19 as compared to a control group of deceased patients without COVID-19. METHODS: Consented autopsy cases at two institutions were searched for documentation of COVID-19 as a contributing cause of death. A group of consecutive consented autopsy cases during the same period, negative for SARS-CoV-2 infection, was used as a control group. The autopsy report and electronic medical records were reviewed for relevant clinicopathologic information. H&E-stained liver sections from both groups were examined for pertinent histologic features. Select cases underwent immunohistochemical staining for CD 68 and ACE2 and droplet digital polymerase chain reaction (ddPCR) assay for evaluation of SARS-CoV2 RNA. RESULTS: 48 COVID-19 positive patients (median age 73, M:F 3:1) and 40 COVID-19 negative control patients (median age 67.5, M:F 1.4:1) were included in the study. The COVID-19 positive group was significantly older and had a lower rate of alcoholism and malignancy, but there was no difference in other comorbidities. The COVID-19 positive group was more likely to have received steroids (75.6 % vs. 36.1 %, p < 0.001). Hepatic vascular changes were seen in a minority (10.6 %) of COVID-19 positive cases. When all patients were included, there were no significant histopathologic differences between groups, but when patients with chronic alcoholism were excluded, the COVID-19 positive group was significantly more likely to have steatosis (80.9 % vs. 50.0 %, p = 0.004) and lobular inflammation (45.7 % vs. 20.7 %, p = 0.03). Testing for viral RNA by ddPCR identified 2 of the 18 (11.1 %) COVID-19 positive cases to have SARS-CoV-2 RNA detected within the liver FFPE tissue. CONCLUSIONS: The most significant findings in the liver of COVID-19 positive patients were mild lobular inflammation and steatosis. The high rate of steroid therapy in this population may be a possible source of steatosis. Hepatic vascular alterations were only identified in a minority of patients and did not appear to play a predominant role in COVID-19 mediated hepatic injury. Low incidence of SARS-CoV-2 RNA positivity in liver tissue in our cohort suggests hepatic injury in the setting of COVID-19 may be secondary in nature.

Severe COVID-19 Is Characterized by an Impaired Type I Interferon Response and Elevated Levels of Arginase Producing Granulocytic Myeloid Derived Suppressor Cells.

COVID-19 ranges from asymptomatic in 35% of cases to severe in 20% of patients. Differences in the type and degree of inflammation appear to determine the severity of the disease. Recent reports show an increase in circulating monocytic-myeloid-derived suppressor cells (M-MDSC) in severe COVID 19 that deplete arginine but are not associated with respiratory complications. Our data shows that differences in the type, function and transcriptome of granulocytic-MDSC (G-MDSC) may in part explain the severity COVID-19, in particular the association with pulmonary complications. Large infiltrates by Arginase 1+ G-MDSC (Arg+G-MDSC), expressing NOX-1 and NOX-2 (important for production of reactive oxygen species) were found in the lungs of patients who died from COVID-19 complications. Increased circulating Arg+G-MDSC depleted arginine, which impaired T cell receptor and endothelial cell function. Transcriptomic signatures of G-MDSC from patients with different stages of COVID-19, revealed that asymptomatic patients had increased expression of pathways and genes associated with type I interferon (IFN), while patients with severe COVID-19 had increased expression of genes associated with arginase production, and granulocyte degranulation and function. These results suggest that asymptomatic patients develop a protective type I IFN response, while patients with severe COVID-19 have an increased inflammatory response that depletes arginine, impairs T cell and endothelial cell function, and causes extensive pulmonary damage. Therefore, inhibition of arginase-1 and/or replenishment of arginine may be important in preventing/treating severe COVID-19.

Posttraumatic Stress Disorder and Cardiovascular Disease: State of the Science, Knowledge Gaps, and Research Opportunities.

Posttraumatic stress disorder (PTSD) is characterized by a persistent maladaptive reaction after exposure to severe psychological trauma. Traumatic events that may precipitate PTSD include violent personal assaults, natural and human-made disasters, and exposure to military combat or warfare. There is a growing body of evidence for associations of PTSD with major risk factors for cardiovascular disease (CVD), such as hypertension and diabetes, as well as with major CVD outcomes, such as myocardial infarction and heart failure. However, it is unclear whether these associations are causal or confounded. Furthermore, the biological and behavioral mechanisms underlying these associations are poorly understood. Here, the available evidence on the association of PTSD with CVD from population, basic, and genomic research as well as from clinical and translational research are reviewed, seeking to identify major research gaps, barriers, and opportunities in knowledge acquisition and technology as well as research tools to support and accelerate critical research for near-term and longer-term translational research directions. Large-scale, well-designed prospective studies, capturing diverse and high-risk populations, are warranted that include uniform phenotyping of PTSD as well as broad assessment of biological and behavioral risk factors and CVD outcomes. Available evidence from functional brain imaging studies demonstrates that PTSD pathophysiology includes changes in specific anatomical brain regions and circuits, and studies of immune system function in individuals with PTSD suggest its association with enhanced immune inflammatory activity. However, establishment of animal models and human tissue biobanks is also warranted to elucidate the potential causal connection of PTSD-induced brain changes and/or inflammation with CVD pathophysiology. Emerging large-scale genome-wide association studies of PTSD will provide an opportunity to conduct mendelian randomization studies that test hypotheses regarding the presence, magnitude, and direction of causal associations between PTSD and CVD outcomes. By identifying research gaps in epidemiology and genomics, animal, and human translational research, opportunities to better justify and design future interventional trials are highlighted that may test whether treatment of PTSD or underlying neurobiological or immune dysregulation may improve or prevent CVD risk or outcomes.

SARS-CoV-2 Infects Endothelial Cells In Vivo and In Vitro.

SARS-CoV-2 infection can cause fatal inflammatory lung pathology, including thrombosis and increased pulmonary vascular permeability leading to edema and hemorrhage. In addition to the lung, cytokine storm-induced inflammatory cascade also affects other organs. SARS-CoV-2 infection-related vascular inflammation is characterized by endotheliopathy in the lung and other organs. Whether SARS-CoV-2 causes endotheliopathy by directly infecting endothelial cells is not known and is the focus of the present study. We observed 1) the co-localization of SARS-CoV-2 with the endothelial cell marker CD31 in the lungs of SARS-CoV-2-infected mice expressing hACE2 in the lung by intranasal delivery of adenovirus 5-hACE2 (Ad5-hACE2 mice) and non-human primates at both the protein and RNA levels, and 2) SARS-CoV-2 proteins in endothelial cells by immunogold labeling and electron microscopic analysis. We also detected the co-localization of SARS-CoV-2 with CD31 in autopsied lung tissue obtained from patients who died from severe COVID-19. Comparative analysis of RNA sequencing data of the lungs of infected Ad5-hACE2 and Ad5-empty (control) mice revealed upregulated KRAS signaling pathway, a well-known pathway for cellular activation and dysfunction. Further, we showed that SARS-CoV-2 directly infects mature mouse aortic endothelial cells (AoECs) that were activated by performing an aortic sprouting assay prior to exposure to SARS-CoV-2. This was demonstrated by co-localization of SARS-CoV-2 and CD34 by immunostaining and detection of viral particles in electron microscopic studies. Moreover, the activated AoECs became positive for ACE-2 but not quiescent AoECs. Together, our results indicate that in addition to pneumocytes, SARS-CoV-2 also directly infects mature vascular endothelial cells in vivo and ex vivo, which may contribute to cardiovascular complications in SARS-CoV-2 infection, including multipleorgan failure.

ACE2 chromogenic immunostaining protocol optimized for formalin-fixed paraffin-embedded human tissue sections.

Angiotensin-converting enzyme 2 (ACE2) is a key cellular entry factor for severe acute respiratory syndrome coronavirus 2. Hence, identifying cell types that express ACE2 is important for understanding the pathophysiology of coronavirus disease 2019. We performed extensive testing of multiple primary antibodies across various human tissue types. Here, we describe an optimized protocol for immunostaining of ACE2 in formalin-fixed paraffin-embedded human pancreas, small intestine, and kidney tissue sections obtained from organ donors and autopsies. For complete details on the use and execution of this protocol, please refer to Kusmartseva et al. (2020).

COVID-19 myocarditis: quantitative analysis of the inflammatory infiltrate and a proposed mechanism.

COVID-19 has a significant effect upon the cardiovascular system. While a number of different cardiovascular histopathologies have been described at post-mortem examination, the incidence of typical viral myocarditis in COVID-19 positive patients appears very low [1-3]. In this study, we further characterize and quantify the inflammatory cell infiltrate in a COVID-19 study cohort and compare the findings to both an age and disease matched control cohort and a cohort of patients diagnosed with typical inflammatory myocarditis. All study and control cohorts had 1 or more of the comorbidities most commonly associated with severe disease (hypertension, type II diabetes, obesity, or known cardiovascular disease). The results demonstrate a skewed distribution of the number of CD68+ cells in COVID-19 hearts, with upper quantiles showing a significant increase as compared to both matched control hearts, and those with myocarditis. In contrast, hearts from typical inflammatory myocarditis contained increased numbers of CD4+, and CD8+ cells compared to both COVID-19 and control cohorts. In conclusion, the presence of an increased number of CD68+ cells suggests that COVID-19 may incite a form of myocarditis different from typical viral myocarditis, and associated with diffusely infiltrative cells of monocytes/macrophage lineage.

Myocarditis is rare in COVID-19 autopsies: cardiovascular findings across 277 postmortem examinations.

The COVID-19 pandemic, the result of severe acute respiratory syndrome (SARS)-CoV-2, is a major cause of worldwide mortality with a significant cardiovascular component. While a number of different cardiovascular histopathologies have been reported at postmortem examination, their incidence is unknown, due to limited numbers of cases in any given study. A literature review was performed identifying 277 autopsied hearts across 22 separate publications of COVID-19 positive patients. The median age of the autopsy cohort was 75 and 97.6% had one or more comorbidities. Initial review of the data indicate that myocarditis was present in 20 hearts (7.2%); however, closer examination of additional reported information revealed that most cases were likely not functionally significant and the true prevalence of myocarditis is likely much lower (<2%). At least one acute, potentially COVID-19-related cardiovascular histopathologic finding, such as macro or microvascular thrombi, inflammation, or intraluminal megakaryocytes, was reported in 47.8% of cases. Significant differences in reporting of histopathologic findings occurred between studies indicating strong biases in observations and the need for more consistency in reporting. In conclusion, across 277 cases, COVID-19-related cardiac histopathological findings, are common, while myocarditis is rare.

Expression of SARS-CoV-2 Entry Factors in the Pancreas of Normal Organ Donors and Individuals with COVID-19.

Diabetes is associated with increased mortality from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Given literature suggesting a potential association between SARS-CoV-2 infection and diabetes induction, we examined pancreatic expression of angiotensin-converting enzyme 2 (ACE2), the key entry factor for SARS-CoV-2 infection. Specifically, we analyzed five public scRNA-seq pancreas datasets and performed fluorescence in situ hybridization, western blotting, and immunolocalization for ACE2 with extensive reagent validation on normal human pancreatic tissues across the lifespan, as well as those from coronavirus disease 2019 (COVID-19) cases. These in silico and ex vivo analyses demonstrated prominent expression of ACE2 in pancreatic ductal epithelium and microvasculature, but we found rare endocrine cell expression at the mRNA level. Pancreata from individuals with COVID-19 demonstrated multiple thrombotic lesions with SARS-CoV-2 nucleocapsid protein expression that was primarily limited to ducts. These results suggest SARS-CoV-2 infection of pancreatic endocrine cells, via ACE2, is an unlikely central pathogenic feature of COVID-19-related diabetes.

Pulmonary and cardiac pathology in African American patients with COVID-19: an autopsy series from New Orleans.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread rapidly across the USA, causing extensive morbidity and mortality, particularly in the African American community. Autopsy can considerably contribute to our understanding of many disease processes and could provide crucial information to guide management of patients with coronavirus disease 2019 (COVID-19). We report on the relevant cardiopulmonary findings in, to our knowledge, the first autopsy series of ten African American decedents, with the cause of death attributed to COVID-19. METHODS: Autopsies were performed on ten African American decedents aged 44-78 years with cause of death attributed to COVID-19, reflective of the dominant demographic of deaths following COVID-19 diagnosis in New Orleans. Autopsies were done with consent of the decedents' next of kin. Pulmonary and cardiac features were examined, with relevant immunostains to characterise the inflammatory response, and RNA labelling and electron microscopy on representative sections. FINDINGS: Important findings include the presence of thrombosis and microangiopathy in the small vessels and capillaries of the lungs, with associated haemorrhage, that significantly contributed to death. Features of diffuse alveolar damage, including hyaline membranes, were present, even in patients who had not been ventilated. Cardiac findings included individual cell necrosis without lymphocytic myocarditis. There was no evidence of secondary pulmonary infection by microorganisms. INTERPRETATION: We identify key pathological states, including thrombotic and microangiopathic pathology in the lungs, that contributed to death in patients with severe COVID-19 and decompensation in this demographic. Management of these patients should include treatment to target these pathological mechanisms. FUNDING: None.

Proteomic Architecture of Human Coronary and Aortic Atherosclerosis.

BACKGOUND: The inability to detect premature atherosclerosis significantly hinders implementation of personalized therapy to prevent coronary heart disease. A comprehensive understanding of arterial protein networks and how they change in early atherosclerosis could identify new biomarkers for disease detection and improved therapeutic targets. METHODS: Here we describe the human arterial proteome and proteomic features strongly associated with early atherosclerosis based on mass spectrometry analysis of coronary artery and aortic specimens from 100 autopsied young adults (200 arterial specimens). Convex analysis of mixtures, differential dependent network modeling, and bioinformatic analyses defined the composition, network rewiring, and likely regulatory features of the protein networks associated with early atherosclerosis and how they vary across 2 anatomic distributions. RESULTS: The data document significant differences in mitochondrial protein abundance between coronary and aortic samples (coronary>>aortic), and between atherosclerotic and normal tissues (atherosclerotic<

Bioinformatic Analysis of Coronary Disease Associated SNPs and Genes to Identify Proteins Potentially Involved in the Pathogenesis of Atherosclerosis.

Factors that contribute to the onset of atherosclerosis may be elucidated by bioinformatic techniques applied to multiple sources of genomic and proteomic data. The results of genome wide association studies, such as the CardioGramPlusC4D study, expression data, such as that available from expression quantitative trait loci (eQTL) databases, along with protein interaction and pathway data available in Ingenuity Pathway Analysis (IPA), constitute a substantial set of data amenable to bioinformatics analysis. This study used bioinformatic analyses of recent genome wide association data to identify a seed set of genes likely associated with atherosclerosis. The set was expanded to include protein interaction candidates to create a network of proteins possibly influencing the onset and progression of atherosclerosis. Local average connectivity (LAC), eigenvector centrality, and betweenness metrics were calculated for the interaction network to identify top gene and protein candidates for a better understanding of the atherosclerotic disease process. The top ranking genes included some known to be involved with cardiovascular disease (APOA1, APOA5, APOB, APOC1, APOC2, APOE, CDKN1A, CXCL12, SCARB1, SMARCA4 and TERT), and others that are less obvious and require further investigation (TP53, MYC, PPARG, YWHAQ, RB1, AR, ESR1, EGFR, UBC and YWHAZ). Collectively these data help define a more focused set of genes that likely play a pivotal role in the pathogenesis of atherosclerosis and are therefore natural targets for novel therapeutic interventions.

Pathobiology of Ischemic Heart Disease: Past, Present and Future.

This review provides a perspective on knowledge of ischemic heart disease (IHD) obtained from the contemporary era of research which began in the 1960s and has continued to the present day. Important discoveries have been made by basic and translational scientists and clinicians. Pathologists have contributed significantly to insights obtained from experimental studies and clinicopathological studies in humans. The review also provides a perspective for future directions in research in IHD aimed at increasing basic knowledge and developing additional therapeutic options for patients with IHD.

Novel therapeutic strategies for ischemic heart disease.

Despite significant advances in the physician's ability to initiate myocardial reperfusion and salvage heart tissue, ischemic heart disease remains one of the leading causes of death in the United States. Consequently, alternative therapeutic strategies have been intensively investigated, especially methods of enhancing the heart's resistance to ischemic cell death - so called "cardioprotective" interventions. However, although a great deal has been learned regarding the methods and mechanisms of cardioprotective interventions, an efficacious therapy has yet to be successfully implemented in the clinical arena. This review discusses the current understanding of cardioprotection in the context of ischemic heart disease pathophysiology, highlighting those elements of ischemic heart disease pathophysiology that have received less attention as potential targets of cardioprotective intervention.

Conditional knockout of myocyte focal adhesion kinase abrogates ischemic preconditioning in adult murine hearts.

BACKGROUND: Our laboratory has previously demonstrated the importance of a cytoskeletal-based survival signaling pathway using in vitro models of ischemia/reperfusion (IR). However, the importance of this pathway in mediating stress-elicited survival signaling in vivo is unknown. METHODS AND RESULTS: The essential cytoskeletal signaling pathway member focal adhesion kinase (FAK) was selectively deleted in adult cardiac myocytes using a tamoxifen-inducible Cre-Lox system (α-MHC-MerCreMer). Polymerase chain reaction (PCR) and Western blot were performed to confirm FAK knockout (KO). All mice were subjected to a 40-minute coronary occlusion followed by 24 hours of reperfusion. Ischemic preconditioning (IP) was performed using a standard protocol. Control groups included wild-type (WT) and tamoxifen-treated α-MHC-MerCreMer+/-/FAK(WT/WT) (experimental control) mice. Infarct size was expressed as a percentage of the risk region. In WT mice IP significantly enhanced the expression of activated/phosphorylated FAK by 36.3% compared to WT mice subjected to a sham experimental protocol (P ≤ 0.05; n = 6 hearts [sham], n = 4 hearts [IP]). IP significantly reduced infarct size in both WT and experimental control mice (43.7% versus 19.8%; P ≤ 0.001; 44.7% versus 17.5%; P ≤ 0.001, respectively). No difference in infarct size was observed between preconditioned FAK KO and nonpreconditioned controls (37.1% versus 43.7% versus 44.7%; FAK KO versus WT versus experimental control; P=NS). IP elicited a 67.2%/88.8% increase in activated phosphatidylinositol-3-kinase (PI3K) p85/activated Akt expression in WT mice, but failed to enhance the expression of either in preconditioned FAK KO mice. CONCLUSIONS: Our results indicate that FAK is an essential mediator of IP-elicited cardioprotection and provide further support for the hypothesis that cytoskeletal-based signaling is an important component of stress-elicited survival signaling.

Cardioprotection and myocardial reperfusion: pitfalls to clinical application.

With the advent of thrombolytic therapy and angioplasty, it has become possible to reduce myocardial infarct size through early reperfusion. Enormous effort has been expended to find therapies that can further reduce infarct size after early intervention. Animal studies have identified many cardioprotective pathways that have the potential to reduce infarct size if activated before the onset of ischemia. More recently, interventions effective at the onset of reperfusion have been described. Although basic research has identified many targets, most has been conducted in rodent models which may not be directly applicable to human disease and even promising agents have been disappointing in large-scale clinical trials. There are many potential explanations for this failure which is the subject of this review. Potential factors include (1) the variability inherent in the patient population, whereas animal studies usually use single sex homogeneous groups maintained on standard diets in carefully controlled environments; (2) the duration of ischemia is generally shorter in animal studies, resulting in potentially more salvageable myocardium than is often the case in patients; (3) that the animals are usually young without comorbidities, whereas the patient population is generally older and has significant comorbidities; (4) animals are not treated with medications a priori, whereas the patient population is often taking medications that may affect ischemic injury; and (5) animal studies may not involve thorough assessment of effects on organs other than the heart, whereas patients can experience adverse effects of treatment in other organs that can preclude clinical use.