Immune-Mediated Glycocalyx Remodeling in Hospitalized COVID-19 Patients.

PURPOSE: Vascular and immune dysfunction are hallmarks of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections and coronavirus disease 2019 (COVID-19). Although our understanding of the pathogenesis of COVID-19 has rapidly evolved, much of the focus has been on the immune mechanisms underlying COVID-19. In addition to immune dysfunction, vascular injury is also associated with COVID-19 and is a major driver of clinical deterioration in SARS-CoV-2 infections. The glycocalyx (GAC), a sugar-based shell that surrounds all mammalian cells, is an important regulator of vascular and immune responses. In sepsis, vascular dysfunction contributes to acute respiratory distress syndrome (ARDS) by altering vessel integrity, promoting thrombosis, and accelerating inflammation, all of which are also present in COVID-19. Observational studies in sepsis have found an association between levels of circulating GAC degradation products with both organ dysfunction and mortality. Although vascular dysfunction is a hallmark of COVID-19, it remains unclear whether GAC disruption occurs in COVID-19 and if GAC disruption contributes to the clinical progression of COVID-19. METHODS: In this prospective cohort study, we measured the GAC components syndecan-1 (SDC1) and hyaluronan (Hyal) along with inflammatory cytokines in 12 hospitalized COVID-19 patients and 8 healthy controls (HC). RESULTS: In agreement with other studies, we found that inflammatory cytokines are elevated in hospitalized COVID-19 patients compared with HC [median (IQR), all units picograms per milliliter: IL-6 4.65 (3.32-9.16) vs 0.69 (0.55-0.89), p < 0.001; TNFα 4.49 (1.87-8.03) vs 0.04 (0.04-0.84), p < 0.001]. Additionally, we found that the GAC components SDC1 and Hyal are also elevated in COVID-19 patients [median (IQR), all units picograms per milliliter: SDC1: 247.37 (101.43-458.26) vs 84.8 (52.88-123.59), p = 0.036; Hyal: 26.41 (16.4-35.1) vs 3.01 (1.66-4.61), p < 0.001]. CONCLUSION: We propose that GAC markers offer insights into the pathobiology of COVID-19, potentially guide therapeutic approaches, and could aid in early risk stratification that is particularly beneficial in phasic diseases such as COVID-19.

Vascular mechanisms of post-COVID-19 conditions: Rho-kinase is a novel target for therapy.

BACKGROUND: In post-coronavirus disease-19 (post-COVID-19) conditions (long COVID), systemic vascular dysfunction is implicated, but the mechanisms are uncertain, and the treatment is imprecise. METHODS AND RESULTS: Patients convalescing after hospitalization for COVID-19 and risk factor matched controls underwent multisystem phenotyping using blood biomarkers, cardiorenal and pulmonary imaging, and gluteal subcutaneous biopsy (NCT04403607). Small resistance arteries were isolated and examined using wire myography, histopathology, immunohistochemistry, and spatial transcriptomics. Endothelium-independent (sodium nitroprusside) and -dependent (acetylcholine) vasorelaxation and vasoconstriction to the thromboxane A2 receptor agonist, U46619, and endothelin-1 (ET-1) in the presence or absence of a RhoA/Rho-kinase inhibitor (fasudil), were investigated. Thirty-seven patients, including 27 (mean age 57 years, 48% women, 41% cardiovascular disease) 3 months post-COVID-19 and 10 controls (mean age 57 years, 20% women, 30% cardiovascular disease), were included. Compared with control responses, U46619-induced constriction was increased (P = 0.002) and endothelium-independent vasorelaxation was reduced in arteries from COVID-19 patients (P < 0.001). This difference was abolished by fasudil. Histopathology revealed greater collagen abundance in COVID-19 arteries {Masson's trichrome (MT) 69.7% [95% confidence interval (CI): 67.8-71.7]; picrosirius red 68.6% [95% CI: 64.4-72.8]} vs. controls [MT 64.9% (95% CI: 59.4-70.3) (P = 0.028); picrosirius red 60.1% (95% CI: 55.4-64.8), (P = 0.029)]. Greater phosphorylated myosin light chain antibody-positive staining in vascular smooth muscle cells was observed in COVID-19 arteries (40.1%; 95% CI: 30.9-49.3) vs. controls (10.0%; 95% CI: 4.4-15.6) (P < 0.001). In proof-of-concept studies, gene pathways associated with extracellular matrix alteration, proteoglycan synthesis, and viral mRNA replication appeared to be upregulated. CONCLUSION: Patients with post-COVID-19 conditions have enhanced vascular fibrosis and myosin light change phosphorylation. Rho-kinase activation represents a novel therapeutic target for clinical trials.

TNF/iNOS/NO pathway mediates host susceptibility to endothelial-dependent circulatory failure and death induced by betacoronavirus infection.

Poor disease outcomes and lethality are directly related to endothelial dysfunction in betacoronavirus infections. Here, we investigated the mechanisms underlying the vascular dysfunction caused by the betacoronaviruses MHV-3 and SARS-CoV-2. Wild-type C57BL/6 (WT) and knockout mice for inducible nitric oxide synthase (iNOS-/-) or TNF receptor 1 (TNFR1-/-) were infected with MHV-3, and K18-hACE2 transgenic mice expressing human ACE2 were infected with SARS-CoV-2. Isometric tension was used to evaluate vascular function. Protein expression was determined by immunofluorescence. Tail-cuff plethysmography and Doppler were used to assess blood pressure and flow, respectively. Nitric oxide (NO) was quantified with the DAF probe. ELISA was used to assess cytokine production. Survival curves were estimated using Kaplan-Meier. MHV-3 infection reduced aortic and vena cava contractility, arterial blood pressure, and blood flow, resulting in death. Resistance mesenteric arteries showed increased contractility. The contractility of the aorta was normalized by removing the endothelium, inhibiting iNOS, genetically deleting iNOS, or scavenging NO. In the aorta, iNOS and phospho-NF-kB p65 subunit expression was enhanced, along with basal NO production. TNF production was increased in plasma and vascular tissue. Genetic deletion of TNFR1 prevented vascular changes triggered by MHV-3, and death. Basal NO production and iNOS expression were also increased by SARS-CoV-2. In conclusion, betacoronavirus induces an endothelium-dependent decrease in contractility in macro-arteries and veins, leading to circulatory failure and death via TNF/iNOS/NO. These data highlight the key role of the vascular endothelium and TNF in the pathogenesis and lethality of coronaviruses.

The Role of Extracellular Vesicles in COVID-19 Pathology.

Extracellular vesicles (EVs) have become a trending topic in recent years; they constitute a new intercellular communication paradigm. Extracellular vesicles are 30-4000 nanometers in diameter particles that are limited by a phospholipid bilayer and contain functional biomolecules, such as proteins, lipids, and nucleic acids. They are released by virtually all types of eukaryotic cells; through their cargoes, EVs are capable of triggering signaling in recipient cells. In addition to their functions in the homeostatic state, EVs have gained attention because of their roles in pathological contexts, eventually contributing to disease progression. In the Coronavirus disease 2019 (COVID-19) pandemic, aside from the scientific race for the development of preventive and therapeutic interventions, it is critical to understand the pathological mechanisms involved in SARS-CoV-2 infection. In this sense, EVs are key players in the main processes of COVID-19. Thus, in this review, we highlight the role of EVs in the establishment of the viral infection and in the procoagulant state, cytokine storm, and immunoregulation of innate and adaptive immune responses.

Inflammation and vascular dysfunction: The negative synergistic combination of diabetes and COVID-19.

AIMS: Several reports indicate that diabetes determines an increased mortality risk in patients with coronavirus disease 19 (COVID-19) and a good glycaemic control appears to be associated with more favourable outcomes. Evidence also supports that COVID-19 pneumonia only accounts for a part of COVID-19 related deaths. This disease is indeed characterised by abnormal inflammatory response and vascular dysfunction, leading to the involvement and failure of different systems, including severe acute respiratory distress syndrome, coagulopathy, myocardial damage and renal failure. Inflammation and vascular dysfunction are also well-known features of hyperglycemia and diabetes, making up the ground for a detrimental synergistic combination that could explain the increased mortality observed in hyperglycaemic patients. MATERIALS AND METHODS: In this work, we conduct a narrative review on this intriguing connection. Together with this, we also present the clinical characteristics, outcomes, laboratory and histopathological findings related to this topic of a cohort of nearly 1000 subjects with COVID-19 admitted to a third-level Hospital in Milan. RESULTS: We found an increased mortality in subjects with COVID-19 and diabetes, together with an altered inflammatory profile. CONCLUSIONS: This may support the hypothesis that diabetes and COVID-19 meet at the crossroads of inflammation and vascular dysfunction. (ClinicalTrials.gov NCT04463849 and NCT04382794).

Molecular Cross-Talk between Integrins and Cadherins Leads to a Loss of Vascular Barrier Integrity during SARS-CoV-2 Infection.

The vascular barrier is heavily injured following SARS-CoV-2 infection and contributes enormously to life-threatening complications in COVID-19. This endothelial dysfunction is associated with the phlogistic phenomenon of cytokine storms, thrombotic complications, abnormal coagulation, hypoxemia, and multiple organ failure. The mechanisms surrounding COVID-19 associated endotheliitis have been widely attributed to ACE2-mediated pathways. However, integrins are emerging as possible receptor candidates for SARS-CoV-2, and their complex intracellular signaling events are essential for maintaining endothelial homeostasis. Here, we showed that the spike protein of SARS-CoV-2 depends on its RGD motif to drive barrier dysregulation by hijacking integrin αVß3, expressed on human endothelial cells. This triggers the redistribution and internalization of major junction protein VE-Cadherin which leads to the barrier disruption phenotype. Both extracellular and intracellular inhibitors of integrin αVß3 prevented these effects, similarly to the RGD-cyclic peptide compound Cilengitide, which suggests that the spike protein-through its RGD motif-binds to αVß3 and elicits vascular leakage events. These findings support integrins as an additional receptor for SARS-CoV-2, particularly as integrin engagement can elucidate many of the adverse endothelial dysfunction events that stem from COVID-19.

Spontaneous Muscle Hematoma in Japanese Patients with Severe COVID-19 Treated with Unfractionated Heparin: Two Case Reports.

In hospitalized coronavirus disease 2019 (COVID-19) patients, anticoagulation therapy is administered to prevent thrombosis. However, anticoagulation sometimes causes bleeding complications. We herein report two Japanese cases of severe COVID-19 in which spontaneous muscle hematomas (SMH) developed under therapeutic anticoagulation with unfractionated heparin. Although the activated partial prothrombin time was within the optimal range, contrast-enhanced computed tomography (CECT) revealed SMH in the bilateral iliopsoas muscles in both cases, which required emergent transcatheter embolization. Close monitoring of the coagulation system and the early diagnosis of bleeding complications through CECT are needed in severe COVID-19 patients treated with anticoagulants.

Increased Autotaxin Levels in Severe COVID-19, Correlating with IL-6 Levels, Endothelial Dysfunction Biomarkers, and Impaired Functions of Dendritic Cells.

Autotaxin (ATX; ENPP2) is a secreted lysophospholipase D catalyzing the extracellular production of lysophosphatidic acid (LPA), a pleiotropic signaling phospholipid. Genetic and pharmacologic studies have previously established a pathologic role for ATX and LPA signaling in pulmonary injury, inflammation, and fibrosis. Here, increased ENPP2 mRNA levels were detected in immune cells from nasopharyngeal swab samples of COVID-19 patients, and increased ATX serum levels were found in severe COVID-19 patients. ATX serum levels correlated with the corresponding increased serum levels of IL-6 and endothelial damage biomarkers, suggesting an interplay of the ATX/LPA axis with hyperinflammation and the associated vascular dysfunction in COVID-19. Accordingly, dexamethasone (Dex) treatment of mechanically ventilated patients reduced ATX levels, as shown in two independent cohorts, indicating that the therapeutic benefits of Dex include the suppression of ATX. Moreover, large scale analysis of multiple single cell RNA sequencing datasets revealed the expression landscape of ENPP2 in COVID-19 and further suggested a role for ATX in the homeostasis of dendritic cells, which exhibit both numerical and functional deficits in COVID-19. Therefore, ATX has likely a multifunctional role in COVID-19 pathogenesis, suggesting that its pharmacological targeting might represent an additional therapeutic option, both during and after hospitalization.

Blunted peripheral but not cerebral vasodilator function in young otherwise healthy adults with persistent symptoms following COVID-19.

Recent findings suggest that COVID-19 causes vascular dysfunction during the acute phase of the illness in otherwise healthy young adults. To date, to our knowledge, no studies have investigated the longer-term effects of COVID-19 on vascular function. Herein, we hypothesized that young, otherwise healthy adults who are past the acute phase of COVID-19 would exhibit blunted peripheral [brachial artery flow-mediated dilation (FMD) and reactive hyperemia] and cerebral vasodilator function (cerebral vasomotor reactivity to hypercapnia; CVMR) and increased central arterial stiffness. Sixteen young adults who were at least 4 wk past a COVID-19 diagnosis and 12 controls who never had COVID-19 were studied. Eight subjects with COVID-19 were symptomatic (SYM) and eight were asymptomatic (ASYM) at the time of testing. FMD and reactive hyperemia were not different between COVID and control groups. However, FMD was lower in SYM (3.8 ± 0.6%) compared with ASYM (6.8 ± 0.9%; P = 0.007) and control (6.8 ± 0.6%; P = 0.003) with no difference between ASYM and control. Similarly, peak blood velocity following cuff release was lower in SYM (47 ± 8 cm/s) compared with ASYM (64 ± 19 cm/s; P = 0.025) and control (61 ± 14 cm/s; P = 0.036). CVMR and arterial stiffness were not different between any groups. In summary, peripheral macrovascular and microvascular function, but not cerebral vascular function or central arterial stiffness were blunted in young adults symptomatic beyond the acute phase of COVID-19. In contrast, those who were asymptomatic had similar vascular function compared with controls who never had COVID-19.NEW & NOTEWORTHY This study was the first to investigate the persistent effects of COVID-19 on vascular function in otherwise healthy young adults. We demonstrated that peripheral macrovascular and microvascular vasodilation was significantly blunted in young adults still symptomatic from COVID-19 beyond the acute phase (>4 wk from diagnosis), whereas those who become asymptomatic have similar vascular function compared with controls who never had COVID-19. In contrast, cerebral vascular function and central arterial stiffness were unaffected irrespective of COVID-19 symptomology.

Heparin prevents in vitro glycocalyx shedding induced by plasma from COVID-19 patients.

The severe forms and worsened outcomes of COVID-19 (coronavirus disease 19) are closely associated with hypertension and cardiovascular disease. Endothelial cells express Angiotensin-Converting Enzyme 2 (ACE2), which is the entrance door for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The hallmarks of severe illness caused by SARS-CoV-2 infection are increased levels of IL-6, C-reactive protein, D-dimer, ferritin, neutrophilia and lymphopenia, pulmonary intravascular coagulopathy and microthrombi of alveolar capillaries. The endothelial glycocalyx, a proteoglycan- and glycoprotein-rich layer covering the luminal side of endothelial cells, contributes to vascular homeostasis. It regulates vascular tonus and permeability, prevents thrombosis, and modulates leukocyte adhesion and inflammatory response. We hypothesized that cytokine production and reactive oxygen species (ROS) generation associated with COVID-19 leads to glycocalyx degradation. A cohort of 20 hospitalized patients with a confirmed COVID-19 diagnosis and healthy subjects were enrolled in this study. Mechanisms associated with glycocalyx degradation in COVID-19 were investigated. Increased plasma concentrations of IL-6 and IL1-ß, as well as increased lipid peroxidation and glycocalyx components were detected in plasma from COVID-19 patients compared to plasma from healthy subjects. Plasma from COVID-19 patients induced glycocalyx shedding in cultured human umbilical vein endothelial cells (HUVECs) and disrupted redox balance. Treatment of HUVECs with low molecular weight heparin inhibited the glycocalyx perturbation. In conclusion, plasma from COVID-19 patients promotes glycocalyx shedding and redox imbalance in endothelial cells, and heparin treatment potentially inhibits glycocalyx disruption.

Endothelial Cells in Emerging Viral Infections.

There are several reasons to consider the role of endothelial cells in COVID-19 and other emerging viral infections. First, severe cases of COVID-19 show a common breakdown of central vascular functions. Second, SARS-CoV-2 replicates in endothelial cells. Third, prior deterioration of vascular function exacerbates disease, as the most common comorbidities of COVID-19 (obesity, hypertension, and diabetes) are all associated with endothelial dysfunction. Importantly, SARS-CoV-2's ability to infect endothelium is shared by many emerging viruses, including henipaviruses, hantavirus, and highly pathogenic avian influenza virus, all specifically targeting endothelial cells. The ability to infect endothelium appears to support generalised dissemination of infection and facilitate the access to certain tissues. The disturbed vascular function observed in severe COVID-19 is also a prominent feature of many other life-threatening viral diseases, underscoring the need to understand how viruses modulate endothelial function. We here review the role of vascular endothelial cells in emerging viral infections, starting with a summary of endothelial cells as key mediators and regulators of vascular and immune responses in health and infection. Next, we discuss endotheliotropism as a possible virulence factor and detail features that regulate viruses' ability to attach to and enter endothelial cells. We move on to review how endothelial cells detect invading viruses and respond to infection, with particular focus on pathways that may influence vascular function and the host immune system. Finally, we discuss how endothelial cell function can be dysregulated in viral disease, either by viral components or as bystander victims of overshooting or detrimental inflammatory and immune responses. Many aspects of how viruses interact with the endothelium remain poorly understood. Considering the diversity of such mechanisms among different emerging viruses allows us to highlight common features that may be of general validity and point out important challenges.

Mechanisms of Pulmonary Hypertension in Acute Respiratory Distress Syndrome (ARDS).

Background: Acute respiratory distress syndrome (ARDS) is a severe and often fatal disease. The causes that lead to ARDS are multiple and include inhalation of salt water, smoke particles, or as a result of damage caused by respiratory viruses. ARDS can also arise due to systemic complications such as blood transfusions, sepsis, or pancreatitis. Unfortunately, despite a high mortality rate of 40%, there are limited treatment options available for ARDS outside of last resort options such as mechanical ventilation and extracorporeal support strategies. Aim of review: A complication of ARDS is the development of pulmonary hypertension (PH); however, the mechanisms that lead to PH in ARDS are not fully understood. In this review, we summarize the known mechanisms that promote PH in ARDS. Key scientific concepts of review: (1) Provide an overview of acute respiratory distress syndrome; (2) delineate the mechanisms that contribute to the development of PH in ARDS; (3) address the implications of PH in the setting of coronavirus disease 2019 (COVID-19).