key: cord-1054592-3l8u34ub authors: Magro, Cynthia M.; Mulvey, Justin; Kubiak, Jeffrey; Mikhail, Sheridan; Suster, David; Crowson, A. Neil; Laurence, Jeffrey; Nuovo, Gerard title: Severe COVID-19: A multifaceted viral vasculopathy syndrome date: 2020-10-13 journal: Ann Diagn Pathol DOI: 10.1016/j.anndiagpath.2020.151645 sha: 0a711e8c8d321432a76d785b13252ea27d024ea8 doc_id: 1054592 cord_uid: 3l8u34ub The objective of this study was to elucidate the pathophysiology that underlies severe COVID-19 by assessing the histopathology and the in situ detection of infectious SARS-CoV-2 and viral capsid proteins along with the cellular target(s) and host response from twelve autopsies. There were three key findings: 1) high copy infectious virus was limited mostly to the alveolar macrophages and endothelial cells of the septal capillaries; 2) viral spike protein without viral RNA localized to ACE2+ endothelial cells in microvessels that were most abundant in the subcutaneous fat and brain; 3) although both infectious virus and docked viral spike protein was associated with complement activation, only the endocytosed pseudovirions induced a marked up-regulation of the key COVID-19 associated proteins IL6, TNF alpha, IL1 beta, p38, IL8, and caspase 3. Importantly, this microvasculitis was associated with characteristic findings on hematoxylin and eosin examination that included endothelial degeneration and resultant basement membrane zone disruption and reduplication. It is concluded that serious COVID-19 infection has two distinct mechanisms: 1) a microangiopathy of pulmonary capillaries associated with a high infectious viral load where endothelial cell death releases pseudovirions into the circulation, and 2) the pseudovirions dock on ACE2+ endothelial cells most prevalent in the skin/subcutaneous fat and brain that activates the complement pathway/coagulation cascade resulting in a systemic procoagulant state as well as the expression of cytokines that produce the cytokine storm. The data predicts a favorable response to therapies based on either removal of circulating viral proteins and/or blunting of the endothelial-induced response. The severe acute respiratory distress syndrome-associated coronavirus-2 (SARS-CoV-2), etiologic agent of Coronavirus disease 2019 (COVID- 19) , was initially identified in Wuhan, Hubei, China in December 2010 (1, 2) and, by mid August there were over 22.5 million confirmed cases and over 800,000 deaths (3) . Respiratory failure, the so-called "cytokine storm", cardiovascular collapse, and a coagulopathy/procoagulant state are associated with fatal disease. Well-documented risk factors include increased age, preexisting medical conditions including diabetes and cardiovascular disease (4, 5) ; obesity is an independent risk factor for severe COVID-19 disease (6, 7) . While the majority of SARS-CoV-2 infections are self-limited, 20% of patients are symptomatic, often requiring hospitalization, and about 3% of all documented cases are fatal. In New York City, where this group is centered, the number of deaths per 1000 person-years greatly increased from a nearly flat monthly death rate, average of 7.83, in 2017 to 9.44 in April and May of 2020 (8) , a 21% increase in all cause mortality. Serious manifestations typically begin within 1-2 weeks after the onset of symptoms, and are heralded by profound difficulty in breathing with further complications related to a hypercoagulable state (9) (10) (11) . Patients who develop severe COVID-19 have extensive microangiopathy and an increased risk of larger vessel thrombosis whereby it has been hypothesized that the complement and coagulation system work synergistically to produce potentially catastrophic sequelae (9. 12-16) . It was recently Of the 26 patients, all but two had severe COVID-19 including 23 with respiratory failure; 12 people died of the disease and had complete post-mortems. The patients ranged in age from 28 to 95 years and were represented by 17 males and 9 females. Of the 24 patients with severe or fatal COVID-19, clinical risk factors included obesity (n=14), arterial hypertension (n=10), diabetes mellitus (n=8), cardiopulmonary disease (n=7), hyperlipidemia (n=4), sickle cell anemia (n=2) and deep vein thrombosis (n=1). Neurologic signs/symptoms were nonspecific and included lethargy and confusion. The two patients with moderate COVID-19 each survived but needed below the knee amputations for deep vein thrombosis; one person was obese and the other had diabetes mellitus. The primary gross abnormality at autopsy was the lungs that were heavy and diffusely congested. The other gross abnormality was macrothrombi that was evident in three autopsies at times associated with tissue infarction. The brains were grossly normal. Histologically, in each of the lung samples the most consistent pattern of vascular injury was one characterized by septal capillary walls exhibiting a disrupted and frayed dyshesive The one pre-mortem lung sample showed an end stage pattern of lung injury characterized by pauci-cellular fibrosis with obliteration of the terminal parenchymal architecture. Microvascular thrombi were evident in many organs including the kidney, brain, heart, skin and liver and were seen in capillaries, venules, and arterioles (figure 2). The pattern of vascular thrombosis varied. One was characterized by bland luminal thrombi or incipient platelet thrombi without endothelial injury (figure 2). This pattern was also seen in much larger vessels in the two below the knee amputations specimens where arterial thrombosis involving small and medium sized blood vessels (figure 2). The second pattern of thrombosis was one accompanied by significant endothelial injury and resultant basement membrane zone disruption and reduplication seen most prominently in the brain and biopsied skin lesions of thrombotic retiform purpura. Interestingly, this same pattern of microvascular damage was seen in grossly normal skin from people who died of COVID-19. Extravascular inflammation apart from inflammation related to ischemic tissue necrosis was not observed. Viral inclusions were not evident. Histologic sections of brain showed rare luminal thrombi comprising large platelets admixed with fibrin in capillaries and venules. However the most striking abnormality was in the context of mummified capillaries devoid of endothelium with associated basement membrane zone reduplication and perivascular edema (figure 2). Red blood cell extravasation and perivascular hemosiderin deposition was noted around capillaries and venules, which is very rare in the perivascular space around microvessels were: normal brain 98.7 microns (9.8), COVID-19 brain, overall 84.1 microns (8.9), COVID-19 brain, microvessels with spike protein 164.7 microns (12.1). The latter was significantly increased versus the normal brain and overall COVID-19 brain using the Tukey-Kramer Multiple Comparisons Test (p < 0.001). Complement studies were conducted on tissue from the lung (n=6), heart (n=4), liver (n=4), kidney (n=4), brain(n=3) and skin (n=20). There was significant endothelial and subendothelial microvascular deposition of C3d, C4d and/or C5b-9 in all cases tested and in none of the controls ( Figure 3 ). The complement expression was temporal and heterogeneous for a particular case. Hence while there could be extensive C4d likely reflective of mannan binding lectin activation, C5b-9 or C3d could be absent. The key was to document substantial deposition of one component of complement activation such as C3d, C4d, and/or C5b-9, the latter in a punctate granular pattern highlighting endothelium and vessel walls. The C5b-9 was deposited in capillary walls, endothelia and intravascular macrophages; the pattern of immunoreactivity included an intracellular one within the endothelium and a surface punctate granular one that outlined the abluminal surface of the vessels. Intravascular platelets also contained C4d and at times other components of complement. In lung samples showing advanced injury with end stage fibrosis there was no vascular complement deposition. Both lesional and nonlesional clinically normal skin exhibited complement deposition where it was most apparent in the deeper dermis and subcutaneous fat. In larger occluded arteries of the lower extremity the deposition was largely confined to the vessel wall but not the endothelia. The extent of complement deposition was greatest in the lung, skin, brain, kidney, and heart. The distribution of SARS-CoV-2 RNA was determined by in situ hybridization. Abundant viral RNA was evident in the lung tissues where it localized to the alveolar macrophages and adjacent septal capillary's endothelia ( Figure 4 ) as evidenced by co-expression with the markers CD68, CD11b, and CD206 as well as the endothelial marker CD31, respectively. Rare signal was evident in alveolar pneumocytes although these cells were often absent from areas with high viral load. Using signal intensity and known standards (16) , viral copy number was estimated at > 500,000 viral genomes/ mm 2 in many fields. Viral RNA and capsid proteins (spike, envelope, membrane) strongly co-expressed and co-localized with C3d, C4d, and C5b-9 as well as to the ACE2 receptor ( Figure 4 ). Interrogation of the spleen, lymph nodes ( Figure 5 ), brain and skin with attached subcutaneous fat did not reveal any cells positive for SARS-CoV-2 RNA. Viral RNA was evident in the liver, heart, and kidney but in rare cells that co-expressed the macrophage marker CD68 ( Figure 5 ); the highest viral RNA load was evident in the liver but this was many fold lower than in the lung (0 -250 genomes/mm 2 ). Immunohistochemistry for the viral spike protein in the COVID-19 cases and negative controls was analyzed in a blinded fashion. In the skin serial section analysis for evidence of the spike glycoprotein receptor ACE2 showed an equivalent distribution to the viral protein. Among the endothelial cells in the various organs by far the strongest expression/unit area for the ACE2 receptor was in the microvessels of the skin ( Figure 6 ) and the lung where >50% of the microvascular endothelia expressed the viral receptor. There was strong expression of the ACE2 receptor in the microvasculature of the brain (i.e. capillaries and venules), albeit focal. Overall ACE-2 expression in the microvasculature's endothelia, as represented by both number of positive staining vessels and the intensity of staining, from highest to lowest follows: skin, lung, J o u r n a l P r e -p r o o f Journal Pre-proof brain, liver, placenta, kidney and heart with no signal evident in the spleen, lymph node, prostate, ovary, bone marrow and esophagus. There was significant viral protein localization in the microvasculature of the skin especially the deeper dermis and subcutaneous fat procured from both thrombosed lesional skin and clinically normal deltoid skin. Multiple foci of viral protein localization within the microvasculature of the brain was also identified although less dense than that noted in the skin. Co-localization experiments documented in both the skin and brain that the endothelial cells with viral spike protein also strongly expressed ACE-2 and that the spike protein co-localized with both the envelope and membrane proteins, suggesting that the capsid proteins circulated as a unit. The viral spike protein was not identified in the spleen ( Figure 5 ) or in any of the negative controls. Rare viral proteins were evident in the endothelia as determined by CD31 coexpression in the heart and kidney from fatal COVID-19 cases but these organs were predominantly negative and reflected the minimal ACE-2 receptor expression. The spike protein was evident in small groups of cells in the liver that were either macrophages or endothelial cells as demonstrated by co-expression with CD68 or CD31, respectively (data not shown). In the heart tissue, the ACE2 receptor and spike protein were more prevalent in the microvessels of the fat surrounding the heart than in the heart muscle proper (data not shown). The primary findings of this study are threefold. 1) Histologic. Besides the microangiopathy in the septal capillaries of the lung as previously described (9), there is systemic vascular disease marked either by thrombi in large vessels or endothelial cell damage/necrosis, basement membrane duplication, perivascular edema, and microthrombi in microvessels; the latter are most prominent in the skin, brain, and liver. 2) Viral. SARS-CoV-2 RNA/protein (infectious virus) in high copy number is evident only in the septal capillaries/macrophages in the lung whereas pseudovirions (spike, envelope, membrane proteins without viral RNA) are evident systemically where they are endocytosed by ACE2+ endothelia, which dominate in the skin, brain, and liver. 3) Host response. Complement activation is evident with both the infectious virus and pseudovirions. However, cytokine up regulation including IL6, IL8, IL1 , TNF a, and p38 as well as caspase 3 expression is evident only with the pseudovirions where they strongly co-express with the viral capsid proteins. In the brain, the microvasculitis is mainly confined to the endothelial cells with mminimal evidence of neuronal or other cell death. A graphic summary of these mechanisms is provided in Figure 8 . In an earlier study we showed that a critical aspect of the pathophysiology of severe COVID-19 is one of complement mediated vascular injury in the lung (9) . In the 5 cases presented in the original study, the lung and skin of all patients had evidence of vascular injury characterized by endothelial necrosis and thrombosis associated with microvascular deposition of complement including C4d, MASP2, and C5b-9 indicative of mannan binding lectin (MBL) pathway activation (9) . By demonstrating that endothelial based SARS CoV-2 viral capsid proteins co-expressed with these complement proteins, it was assumed that infectious virus was inducing the MBL pathway. This prior study did not specifically address whether or not the viral Given the millions of cases worldwide, the clinical correlates of severe COVID-19 are well documented. These include an independent association with obesity, strong correlation with preexisting conditions including diabetes, a hypercoagulable state, so-called cytokine storm and neurologic signs/symptoms that often become manifest 1-2 weeks after pneumonia (4) (5) (6) (7) (8) . The current study by correlating the light microscopic, viral, and host response, including cytokine expression and complement analysis of significantly ill COVID-19 patients, has provided data that may help to understand the foundation for these clinical correlates. The extremely high copy viral load in the lung induces a microangiopathy that destroys the infected endothelial cell and, thus, could release large numbers of pseudovirions into the circulation. The spike protein in these pseudovirions will dock on ACE2+ endothelia. The largest reservoir for the latter is the subcutaneous fat, which will be much increased in the obese. The endocytosed viral proteins induce a strong complement cascade activation and marked cytokine response including IL6, IL8, IL1  , p38, and TNF . These cellular responses would cause many systemic medical problems, especially in those with pre-existing medical conditions. The observation that the microvascular bed in the liver and brain also contains many ACE2+ endothelial cells that, as demonstrated here, shows much evidence of viral-induced damage, could clearly be related to dermal and subcutaneous vasculature were observed in the autopsy cases in grossly normal skin. This reinforces the potential use of deltoid skin biopsies in symptomatic COVID-19 as a means to identify patients in whom substantial viral capsid protein docking has occurred and, thus, may benefit from blood thinning or anti-cytokine therapy. Prior studies utilizing RT-PCR based methodologies require tissue destruction, which prevents cellular microanatomic localization of the virus. These studies have found active replicating virus in the blood, brain, brain, spleen, placenta, liver, large and small intestine, skeletal muscle, and lymph nodes (20) (21) (22) . To our knowledge, replicating virus has not been detected in skin samples (23-25). What has been reported as viral inclusions have been found in the kidney endothelium, and apoptotic blebbing with microvesicles has been seen in the endothelium of other organ systems as well; active replication was not proven (26, 27). Viruslike particles have also been seen in the endothelium in the brain with concomitant detection of viral RNA from minced FFPE tissue, though localization to the endothelium could not documented (20) . Given the widespread expression of ACE-2 receptors across many tissue types, it is possible that the aforementioned studies detected viral replication in non-vascular tissue (28); this study did document rare macrophages in the heart/liver/kidney that contained infectious virus that could produce a positive result with RTPCR. It is postulated that the corporeally. In the setting of MERS there was reduced infectivity and reduced concentrations of circulating viral particles over time with apheresis. Both MERS-CoV (pseudovirus) and MARV soluble glycoproteins were effectively eliminated by lectin affinity plasmapheresis (60) . With the extensive complement activation and widespread endothelial destruction, inhibition of complement pathways may also represent a favorable therapeutic target (61, 62) . Compassionate use of the C3 inhibitor AMY-101, as well as the anti-CD5 monoclonal antibody eculizumab have shown anecdotal success (63) (64) (65) . Mannan binding lectin has also been proposed as a therapeutic target given its gatekeeping role in complement activity and endothelial damage (66) . A number of trials are now underway with inhibitors to complement and mannan binding lectin as novel therapeutic targets (67) . and spleen (not shown) in cases where a very high viral load was present in the lung (panels D and E). 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