key: cord-1024061-qw0crnqo authors: Syed, F.; Li, W.; Relich, R. F.; Russell, P. M.; Zhang, S.; Zimmerman, M. K.; Yu, Q. title: Excessive matrix metalloproteinase-1 and hyperactivation of endothelial cells occurred in COVID-19 patients and were associated with the severity of COVID-19 date: 2021-01-20 journal: medRxiv : the preprint server for health sciences DOI: 10.1101/2021.01.19.21250115 sha: 5d35a438dd10aabd0c2bbee54bab7238196bd73c doc_id: 1024061 cord_uid: qw0crnqo COVID-19 starts as a respiratory disease that can progress to pneumonia, severe acute respiratory syndrome (SARS), and multi-organ failure. Growing evidence suggests that COVID-19 is a systemic illness that primarily injures the vascular endothelium, yet the underlying mechanisms remain unknown. SARS-CoV-2 infection is believed to trigger a cytokine storm that plays a critical role in the pathogenesis of endothelialitis and vascular injury, eventually leading to respiratory and multi-organ failure in COVID-19 patients. We used a multiplex immunoassay to systematically profile and compare 65 inflammatory cytokines/chemokines/growth factors in plasma samples from 24 hospitalized (severe/critical) COVID-19 patients, 14 mild/moderate cases, and 13 healthy controls (HCs). Patients with severe/critical and mild/moderate COVID-19 had significantly higher plasma levels of 20 analytes than HCs. Surprisingly, only one cytokine (MIF) was among these altered analytes, while the rest were chemokines and growth factors. In addition, only MMP-1 and VEGF-A were significantly elevated in hospitalized COVID-19 patients when compared to mild/moderate cases. Given that excessive MMP-1 plays a central role in tissue destruction in a wide variety of vascular diseases and that elevated VEGF-A, an EC activation marker, increases vascular permeability, we further studied MMP-1 enzymatic activity and other EC activation markers such as soluble forms of CD146, ICAM-1, and VCAM-1. We found that plasma MMP-1 enzymatic activity and plasma levels of MMP-1 and EC activation markers were highly dysregulated in COVID-19 patients. Some dysregulations were associated with patient age or gender, but not with race. Our results demonstrate that COVID-19 patients have distinct inflammatory profiles that are distinguished from the cytokine storms in other human diseases. Excessive MMP-1 and hyperactivation of ECs occur in COVID-19 patients and are associated with the severity of COVID-19. Coronavirus disease 2019 (COVID- 19) , an infectious disease caused by a novel coronavirus (severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2), has created an unprecedented global health and economic crisis. By the end of 2020, confirmed COVID-19 cases surpassed 84 million globally, resulting in over 1.8 million deaths. COVID-19 patients can experience a range of manifestations, from being asymptomatic, to having mild, moderate, severe symptoms, to having a critical illness. Growing evidence shows that COVID-19 is a vascular illness, not solely a respiratory disease. Histopathological examinations of post-mortem tissues of COVID-19 patients have revealed (1) diffuse alveolar damage with perivascular infiltration of inflammatory cells [1] [2] [3] [4] [5] , (2) extensive damage to the lining of blood vessels throughout the body 6-8 , (3) severe endothelial injury and widespread thrombosis in the lungs, heart, liver, kidney, and small intestine 1,5-8 , (4) viral particles in endothelial cells (ECs) of the glomerular capillary loops 6 , and (5) caspase-3-positive apoptotic ECs in the lung and intestine tissues 6 . In addition, a recent study has shown that COVID-19 patients in intensive care units (ICUs) have higher counts of circulating ECs (CECs) than non-ICU patients 9 . CECs are stressed cells detached from injured blood vessels, thereby indicating severe vascular injury 10 . Collectively, patients with severe COVID-19 exhibit impaired endothelial and microcirculatory functions across vascular beds of different organs, which may be particularly relevant for vulnerable individuals with pre-existing endothelial dysfunctions such as diabetes, obesity, hypertension, and cardiovascular diseases, all of which are associated with adverse outcomes in COVID-19 6, [11] [12] [13] [14] . The pathological mechanisms underlying vascular injury in COVID-19 remain unclear, although cytokine storm syndrome (CSS) and direct SARS-CoV-2 infection are considered contributors. SARS-CoV-2 is a member of the family Coronaviridae, genus Betacoronavirus, and is closely related to SARS-CoV that caused the 2003 SARS pandemic [15] [16] [17] . CSS plays a critical role in the pathogenesis of SARS-CoV infection and represents a major cause of All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 20, 2021. ; https://doi.org/10.1101/2021.01. 19 .21250115 doi: medRxiv preprint morbidity in SARS patients 18, 19 . Elevated circulating concentrations of IL-6, IFN-γ, IL-8, IL-18, TGF-β, IP-10, MCP-1, and MIG have been reported in SARS patients when compared with healthy controls (HCs) 20, 21 . Circulating IL-6 levels are also elevated in COVID-19 patients and are cited as evidence of COVID- 19 CCS 22, 23 . However, the levels of IL-6 and other inflammatory cytokines such as IL-8 are significantly less elevated in patients with critical COVID-19 than the values typically reported in patients with CSS such as septic shock with and without acute respiratory distress syndrome (ARDS) 24 . Indeed, the concentrations of IL-6, IL-8, and TNF-α, three of the most important inflammatory mediators in human diseases with CSS 25 , in COVID- 19 patients are similar to those found in ICU patients with cardiac arrest or trauma, conditions that are not notable for cytokine storms 24 . These findings bring into question whether a cytokine storm occurs in COVID-19, and whether IL-6, IL-8, and TNF-α act as key inflammatory mediators for fatal manifestations in patients with severe or critical COVID-19 25 . Our results echo recent NIH guidelines that note there are insufficient data to recommend IL-6 inhibitors for the treatment of COVID-19 26 . In fact, efforts to combat cytokine storm in patients with severe COVID-19 have proven unsuccessful 27 . SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as a primary receptor for viral binding and entry [28] [29] [30] [31] [32] . Viral entry is also facilitated by transmembrane protease serine 2 (TMPRSS2) and neuropilin-1 (NRP1) 31, 33, 34 . ACE2, TMPRSS2, and NRP1 are highly abundant on the lung alveolar type II epithelial (AT2) cells, rendering AT2 cells highly susceptible to productive SARS-CoV-2 infection 33, [35] [36] [37] . ACE2, TMPRSS2, and NRP1 are also expressed on the surface of ECs 33, 35, [38] [39] [40] , albeit at lower levels than in AT2 cells 33 , suggesting that these molecules may facilitate direct SARS-CoV-2 infection of ECs. Indeed, SARS-CoV-2 particles were observed in ECs in kidney tissues from COVID-19 patients 8 . However, a recent unreferenced preprint reported that ECs derived from human peripheral blood mononuclear cells (PBMCs), human lung microvascular ECs, and human aortic ECs did not have detectable mRNA of ACE2 and TMPRSS2 and were resistant to SARS-CoV-2 infection in vitro 41 . Although this study shows results that are not consistent with both their own report 42 and previous studies 33, 35, [38] [39] [40] , it highlights the need for further research on the pathogenic mechanisms underlying vascular injury in COVID-19. To explore the profile and key players of the cytokine storm in COVID-19, we used a multiplex immunoassay to simultaneously measure 65 inflammatory cytokines/chemokines/growth factors in plasma samples from hospitalized (severe/critical) COVID-19 patients, mild/moderate cases, and HCs. Surprisingly, matrix metalloproteinase-1 (MMP-1) and vascular endothelial growth factor A (VEGF-A), not conventional inflammatory cytokines such as IL-6, were two of the most unambiguously elevated inflammatory factors in hospitalized COVID-19 patients when compared to mild/moderate cases or HCs. Spearman correlation analysis revealed that the plasma levels of MMP-1 and VEGF-A in hospitalized COVID-19 patients were positively correlated, suggesting that there may be an interaction between MMP-1 and VEGF-A in COVID-19. Given that excessive MMP-1 plays a central role in tissue destruction in a wide variety of vascular diseases and that elevated VEGF-A, an EC activation markers, increases vascular permeability 43 , we further studied MMP-1 enzymatic activity and other EC activation markers including soluble forms of CD146, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1) in our cohort of COVID-19 patients and HCs. We performed correlation analysis among plasma levels of MMP-1, EC activation markers, and inflammatory cytokines/chemokines, MMP-1 enzymatic activity, and patients' demographics and clinical parameters. We found that excessive MMP-1 and hyperactivation of ECs occurred in COVID-19 patients and were correlated with the severity of COVID-19. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 20, 2021. ; This study was performed with the approval of the Institutional Review Boards at Indiana University School of Medicine. Blood samples were drawn after each participant provided a written informed consent form. Combined nasopharyngeal-oropharyngeal swab specimens collected from study subjects were tested using either the cobas ® SARS-CoV-2 Test ( 44 ) or critical disease with complications such as respiratory failure, disseminated intravascular coagulation (DIC), and/or multi-organ failure 44 . Some of these hospitalized patients were admitted to ICUs. Patients with mild/moderate COVID-19 were characterized with mild respiratory symptoms (nasal congestion, runny nose, and a sore throat) or mild pneumonia 44 . All COVID-19 patients were treated in Indiana University (IU) Health hospitals in Indianapolis, Indiana, during May -December 2020. Plasma samples from HCs were selected from our banked blood samples that were collected before the COVID-19 pandemic as described in our previous reports 45, 46 . Demographics of HCs were matched with COVID-19 patients so that there were no significant differences between age or gender of HCs and COVID-19 subjects. The demographic and clinical characteristics of COVID-19 patients and HC demographics were summarized in Table 1 . Peripheral blood was collected in heparin-coated BD Vacutainer Blood Collection tubes Briefly, plasma samples were incubated with beads at room temperature for 2 h with shaking at 500 rpm, and subsequently washed twice using a magnetic plate washer (eBioscience, San Diego, CA). The beads were incubated with the biotinylated antibodies for 1 h at room temperature with shaking at 500 rpm. After washing, beads were incubated with streptavidin-PE for 30 min at room temperature with shaking at 500 rpm. After washing twice, beads were Plasma levels of soluble CD146 (sCD146), soluble ICAM-1 (sICAM-1), soluble VCAM-1 (sVACM-1), and intestinal fatty-acid binding protein (I-FABP) were quantified using the Human All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Enzymatic activity of plasma MMP-1 was determined using the Human Active MMP-1 Fluorokine E kit (F1M00, R&D Systems, Minneapolis, MN) as per manufacturer's instructions. Briefly, diluted plasma samples and MMP-1 standards were added to the wells that were precoated with a monoclonal antibody specific for human MMP-1. After washing to remove unbound substances, amino-phenyl mercuric acetate (APMA), an activation reagent of MMP-1, was added to the standards, but not the plasma samples. After washing, a fluorogenic substrate linked to a quencher molecule was added and any active enzyme present would cleave the peptide linker between the fluorophore and the quencher molecule, generating a fluorescent signal that is proportional to the amount of enzyme activity in an individual sample. Thus, plasma levels of active MMP-1 were quantitatively detected using a Synergy H1 Hybrid Multi-Mode Reader (BioTek, Winooski, VT), where fluorescence emission was recorded in relative fluorescence unit (RFU). Statistical analysis was performed using GraphPad Prism 6.0 (La Jolla, CA). Data were expressed as mean ± standard error of the mean (SEM) or mean ± standard deviation (SD) unless otherwise indicated. All data were tested for suitability for parametric or non-parametric analysis. Differences between two groups were calculated using the Mann Whitney test. Kruskal-Wallis test with Dunn's corrections was used for comparisons among three groups. Chisquare test was used for comparison between groups for categorical variables. The linear All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 20, 2021. ; https://doi.org/10.1101/2021.01.19.21250115 doi: medRxiv preprint relationship between two variables was analyzed using the Spearman correlation test. P <0.05 was considered statistically significant. The study subjects included 24 hospitalized patients with severe/critical COVID-19, 14 patients with mild/moderate COVID-19, and 13 HCs. Severe/critical COVID-19 patients were hospitalized in Indiana University (IU) Health hospitals in Indianapolis, Indiana and mild/moderate COVID-19 patients visited IU Health hospitals between May -December 2020. HCs were recruited before the COVID-19 pandemic for our research on immunopathogenesis of HIV infection and alcoholic hepatitis as described in our previous reports [45] [46] [47] [48] . The demographics and clinical characteristics of these subjects were summarized in Table 1 . There were no differences in age, gender, and race distributions among these three groups. Hospitalized COVID-19 patients had lower absolute lymphocyte count (ALC), higher erythrocyte sedimentation rate (ESR), and higher levels of D-dimer, C-reactive protein (CRP), and ferritin than patients with mild/moderate COVID-19 (Table 1 ). There were no differences in white blood cell (WBC) or neutrophil counts between the hospitalized patients and patients with mild/moderate COVID ( Table 1) . COVID-19 patients present varied clinical features, ranging from asymptomatic, mild/moderate, severe, to critical illness. The pathogenesis of severe or critical COVID-19 is complex and has been suggested to include a cytokine storm that sustains an aberrant systemic immune response 49 . To elucidate the profile and key pathogenic inflammatory mediators of the cytokine storm in COVID-19 patients, we used a multiplex immunoassay to simultaneously All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 20, 2021. ; https://doi.org/10.1101/2021.01.19.21250115 doi: medRxiv preprint detect plasma levels of 65 cytokines/chemokines/growth factors in hospitalized (severe/critical) patients, mild/moderate cases, and HCs. In comparison to HCs, hospitalized and mild/moderate COVID-19 patients had significantly higher plasma levels of 20 of these analytes (Table 2) Table 2) . Surprisingly, MMP-1 and VEGF-A were the only two elevated inflammatory factors that were significantly higher in hospitalized COVID-19 patients when compared to mild/moderate cases (Table 2 and Figure 1A ), while all others did not show differences between hospitalized and mild/moderate COVID-19 patients (Table 2) MCP-1, MCP-2, IL-6, and IL-18 were the 4 analytes that were significantly increased in hospitalized patients, but not in mild/moderate cases, when compared to HCs (Table 2 and Figure 1A ). However, plasma levels of IL-6 and IL-18 were elevated in some, but not all, patients with severe/critical COVID-19. As shown in Figure 1A , IL-6 was detected in 0 HCs ( All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 20, 2021. ; https://doi.org/10.1101/2021.01.19.21250115 doi: medRxiv preprint Nineteen analytes were detected in all three groups of research subjects, but there were no differences between these groups (Table 2) . Twenty-two analytes, primarily inflammatory cytokines such as IL-8 and TNF-α, were below the limit of detection in the majority of hospitalized cases, mild/moderate cases, and HCs (Table 2) . Our data support previous findings that suggested IL-6, IL-8, and TNF-α did not act as key inflammatory mediators for fatal manifestations in patients with severe or critical COVID-19 25 , and also question whether cytokine storm occurs in COVID-19. MMP-1 and VEGF-A were two of the most unambiguously elevated inflammatory factors in hospitalized COVID-19 patients. Their plasma levels were positively correlated (Table 1 and 50 . This study measured plasma MMP-9 levels using an enzyme immunoassay, and showed that hospitalized COVID-19 patients had higher MMP-9 levels than HCs 50 . However, none of the MMPs have been studied for their enzymatic activity in COVID-19 patients. We analyzed enzymatic activity of MMP-1 in plasma samples from HCs and patients with mild/moderate or hospitalized COVID- 19 . We found that the enzymatic activity of MMP-1 was significantly increased in hospitalized COVID-19 patients when compared to HCs and those with mild/moderate COVID-19 ( Figure 2A ). Thus, both the levels and the enzymatic activity of MMP-1 were up-regulated in the peripheral blood of COVID-19 patients, particularly in patients with severe/critical COVID-19. Next, we used ELISA assays to measure the levels of sCD146, sVCAM-1, and sICAM-1, surrogate markers of EC activation, in the plasma samples from hospitalized COVID-19 patients, mild/moderate cases, and HCs. As shown in Figure 2B , hospitalized COVID-19 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 20, 2021. ; https://doi.org/10.1101/2021.01.19.21250115 doi: medRxiv preprint patients had significantly higher plasma levels of sCD146, sICAM-1, and sVCAM-1, than HCs. Mild/moderate COVID-19 patients also had higher levels of sCD146, but neither sICAM-1 nor sVCAM-1, than HCs ( Figure 2B ). There were no differences in the plasma levels of sCD146, sICAM-1, or sVCAM-1 between hospitalized and mild/moderate COVID-19 patients ( Figure 2B ). Age is a well-known factor that influences the severity and fatality of COVID-19 53 . The risk for severe illness with COVID-19 increases with age, with older adults at higher risk 53 . Other demographic factors such as sex and race have been also linked with risk for severity of COVID-19 illness. We analyzed the relationship between demographic factors and circulating MMP-1 and EC activation markers in hospitalized COVID-19 patients. Twenty-four hospitalized All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Figure 3A , plasma VEGF-A levels were significantly higher in hospitalized COVID-19 patients >55 years old when compared to cases ≤ 50 years old. Plasma levels of MMP-1, sICAM-1, sVCAM-1, active MMP-1 showed an increased trend in the older group ( Figure 3A ). We also analyzed the associations of sex and race with MMP-1 and EC activation markers. As shown in Figure 3B and 3C, plasma sVCAM-1 levels were significantly higher in males (n=10) when compared to females (n=14) ( Figure 3B ). There were no differences in the plasma levels of MMP-1, active MMP-1, and EC activation markers in African-Americans (n=10) when compared to Caucasian Americans (n=13) ( Figure 3C ). In the present study, we systematically profiled and compared 65 inflammatory cytokines/chemokines/growth factors in the plasma samples from hospitalized (severe/critical) COVID-19 patients, mild/moderate COVID-19 cases, and HCs. As shown in Table 2 , (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 49, 54, 55 , and also question whether a cytokine storm occurs in COVID-19. The term "cytokine storm" was first coined to describe the excessive release of inflammatory cytokines by immune cells in graft-versus-host disease (GVHD) 49, 54, 55 . Since then, CSS has been reported in a wide range of other human diseases including cancer patients undergoing chimeric antigen receptor (CAR) T cell therapy 56, 57 , pancreatitis 58 , multiple organ dysfunction syndrome 59 69 . In HCs, mean levels of circulating IL-6 have been reported to be <5 pg/mL 70 , which can be increased to >10,000 pg/mL in cancer patients on CAR T cell therapy 69 . The substantial elevation of IL-6 and its correlation with disease severity have resulted in IL-6 inhibitors such as sarilumab, siltuximab, and tocilizumab becoming therapeutic agents that can effectively treat CSS-associated diseases 69, 71 . Circulating IL-6 levels are also elevated in COVID-19 patients and is cited as evidence of COVID- 19 CCS 22, 23, 66, [72] [73] [74] [75] [76] [77] [78] . However, as previously reported, elevated IL-6 levels in COVID-19 patients are minuscule compared to those found in individuals on CAR T cell therapy and other CSS-associated diseases 25 , suggesting that IL-6 does not act as a key inflammatory mediator for fatal manifestations in patients with severe or critical COVID-19 25 . In line with this finding, IL-6 inhibitors are not recommended to be used as therapeutic agents for the treatment of COVID-19 as noted by NIH guidelines 26 . Our results provide evidence that explains why efforts to combat cytokine storm have proven unsuccessful in severe COVID-19 patients. Our study implicates a chemokine storm, not a cytokine storm, All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 20, 2021. ; https://doi.org/10.1101/2021.01.19.21250115 doi: medRxiv preprint occurs in COVID-19. This chemokine storm plays an important role in the pathogenesis of COVID-19 via recruitment of inflammatory cells to the lungs and other organs or tissues 1-5 . Thus, chemokines and chemokine receptors as therapeutic targets for COVID-19 treatment need to be studied. Table 2 patients. In addition to VEGF-A, other EC activation markers including sCD146, sICAM-1, and sVACM-1 were highly elevated in COVID-19. In contrast, plasma I-FABP levels were no All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 20, 2021. Age is a well-known factor that influences the severity and fatality of COVID-19 53 . The risk for severe illness with COVID-19 increases with age, with older adults at highest risk 53 . Other demographic factors such as gender and race have been also linked with risk for the severity of COVID-19 illness. We found that excessive MMPs and/or circulating markers of EC activation increased with age or were higher in hospitalized male COVID-19 patients (Figure 3 ), but not affected by race ( Figure 3 ). However, we realize that our sample size of hospitalized COVID-19 patients, after splitting into 2 demographic groups, is too small to make statistical inferences. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 20, 2021. ; (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 20, 2021. ; (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. patients. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 20, 2021. ; 7.9 (7.9 -7.9) 7.9 (7.9 -7.9) 41.9 (7.9 -76.1)* IL-18 9.7 (4.2 -9.7) 9.7 (9.1 -20.5) 21.8 (9.7 -56.5)*** CD40L (CD154) 4 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 20, 2021. ; https://doi.org/10.1101/2021.01.19.21250115 doi: medRxiv preprint Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19 Gross and histopathological pulmonary findings in a COVID-19 associated death during self-isolation Pathological findings of COVID-19 associated with acute respiratory distress syndrome Pathological study of the 2019 novel coronavirus disease (COVID-19) through postmortem core biopsies Targeting potential drivers of COVID-19: Neutrophil extracellular traps Endothelial cell infection and endotheliitis in COVID-19 Postmortem examination of COVID-19 patients reveals diffuse alveolar damage with severe capillary congestion and variegated findings in lungs and other organs suggesting vascular dysfunction Autopsy Findings and Venous Thromboembolism in Patients With COVID-19: A Prospective Cohort Study Circulating Endothelial Cells as a Marker of Endothelial Injury in Severe COVID -19 Circulating endothelial cells. Biomarker of vascular disease Could a family history of type 2 diabetes be a risk factor to the endothelial damage in the patient with COVID-19? Med Hypotheses Clinical Characteristics of Coronavirus Disease 2019 in China Diabetes mellitus is associated with increased mortality and severity of disease in COVID-19 pneumonia -A systematic review, metaanalysis, and meta-regression Prevention and management of COVID-19 among patients with diabetes: an appraisal of the literature Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus Cytokine release syndrome in severe COVID-19 Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology An interferon-gamma-related cytokine storm in SARS patients Cytokine Levels in Critically Ill Patients With COVID-19 and Other Conditions Rethinking interleukin-6 blockade for treatment of COVID-19 Guideline] NIH. Interleukin-6 Inhibitors. COVID-19 Treatment Guidelines Distinct inflammatory profiles distinguish COVID-19 from influenza with limited contributions from cytokine storm Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus Interaction of SARS-CoV-2 and Other Coronavirus With ACE (Angiotensin-Converting Enzyme)-2 as Their Main Receptor: Therapeutic Implications SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity TMPRSS2 and COVID-19: Serendipity or Opportunity for Intervention? Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes Influenza and SARS-coronavirus activating proteases TMPRSS2 and HAT are expressed at multiple sites in human respiratory and gastrointestinal tracts Potential Biomarker for COVID-19 Outcomes SARS-CoV-2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells Devil and angel in the renin-angiotensin system: ACEangiotensin II-AT1 receptor axis vs. ACE2-angiotensin-(1-7)-Mas receptor axis Resistance of endothelial cells to SARS-CoV-2 infection in vitro Cardiorenal Tissues Express SARS-CoV-2 Entry Genes and Basigin (BSG/CD147) Increases With Age in Endothelial Cells Vascular permeability factor/vascular endothelial cell growth factor-mediated permeability occurs through disorganization of endothelial junctional proteins Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study Alcohol abstinence ameliorates the dysregulated immune profiles in patients with alcoholic hepatitis: A prospective observational study Alcohol Abstinence Does Not Fully Reverse Abnormalities of Mucosal-Associated Invariant T Cells in the Blood of Patients With Alcoholic Hepatitis Immune Checkpoint Axes Are Dysregulated in Patients With Alcoholic Hepatitis MMPs/TIMPs imbalances in the peripheral blood and cerebrospinal fluid are associated with the pathogenesis of HIV-1-associated neurocognitive disorders Into the eye of the cytokine storm Distinct and early increase in circulating MMP-9 in COVID-19 patients with respiratory failure The role of I-FABP as a biomarker of intestinal barrier dysfunction driven by gut microbiota changes in obesity The intestinal fatty acidbinding protein as a marker for intestinal damage in gastroschisis The coronavirus is most deadly if you are older and male -new data reveal the risks Cytokine dysregulation as a mechanism of graft versus host disease Interleukin-1 is a critical effector molecule during cytokine dysregulation in graft versus host disease to minor histocompatibility antigens Cytokine Release Syndrome: Current Perspectives Toxicity management for patients receiving novel T-cell engaging therapies Cytokine storm in acute pancreatitis The cytokine storm and factors determining the sequence and severity of organ dysfunction in multiple organ dysfunction syndrome The cytokine storm in multiple sclerosis Role of cytokines as a double-edged sword in sepsis New fronts emerge in the influenza cytokine storm Innate immune responses to influenza A H5N1: friend or foe? Temporal changes in cytokine/chemokine profiles and pulmonary involvement in severe acute respiratory syndrome Persistence of lung inflammation and lung cytokines with highresolution CT abnormalities during recovery from SARS SARS-CoV-2: a storm is raging MERS-CoV infection in humans is associated with a pro-inflammatory Th1 and Th17 cytokine profile Clinical Progression and Cytokine Profiles of Middle East Respiratory Syndrome Coronavirus Infection Current status of chimeric antigen receptor therapy for haematological malignancies Serum cytokine profiles in healthy young and elderly population assessed using multiplexed bead-based immunoassays Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan Clinical features of patients infected with 2019 novel coronavirus in Wuhan Potential role of fibroblast growth factor 21 in the deterioration of bone quality in impaired glucose tolerance Reduction and Functional Exhaustion of T Cells in Patients With Coronavirus Disease 2019 (COVID-19) Risk Factors Associated With Acute Respiratory Distress Syndrome and Death in Patients With Coronavirus Disease Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease Matrix metalloproteases and PAR1 activation Integration of endothelial protease-activated receptor-1 inflammatory signaling by ubiquitin Protective signaling pathways of activated protein C in endothelial cells Protease-activated receptor 1 is the primary mediator of thrombinstimulated platelet procoagulant activity Proteaseactivated receptors 1 and 4 mediate activation of human platelets by thrombin Role of protease-activated receptors in inflammatory responses, innate and adaptive immunity Protease activated receptor-1 regulates macrophage-mediated cellular senescence: a risk for idiopathic pulmonary fibrosis International Union of Pharmacology. XXVIII. Proteinase-activated receptors Thrombin and protease-activated receptors (PARs) in atherothrombosis Dynamics and plasticity of Weibel-Palade bodies in endothelial cells Matrix metalloproteinase-1-mediated up-regulation of vascular endothelial growth factor-2 in endothelial cells VEGF receptor signallingin control of vascular function