Histamine Potentiates SARS-CoV-2 Spike Protein Entry Into Endothelial Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes coronavirus disease (COVID-19) is one of the most serious global health crises in recent history. COVID-19 patient symptoms range from life-threatening to mild and asymptomatic, which presents unique problems in identifying, quarantining, and treating the affected individuals. The emergence of unusual symptoms among survivors, now referred to as "Long COVID", is concerning, especially since much about the condition and the treatment of it is still relatively unknown. Evidence so far also suggests that some of these symptoms can be attributed to vascular inflammation. Although famotidine, the commonly used histamine H2 receptor (H2R) blocker, was shown to have no antiviral activity, recent reports indicate that it could prevent adverse outcomes in COVID-19 patients. Histamine is a classic proinflammatory mediator, the levels of which increase along with other cytokines during COVID-19 infection. Histamine activates H2R signaling, while famotidine specifically blocks H2R activation. Investigating the effects of recombinant SARS-CoV-2 spike protein S1 Receptor-Binding Domain (Spike) on ACE2 expression in cultured human coronary artery endothelial cells, we found that the presence of histamine potentiated spike-mediated ACE2 internalization into endothelial cells. This effect was blocked by famotidine, protein kinase A inhibition, or by H2 receptor protein knockdown. Together, these results indicate that histamine and histamine receptor signaling is likely essential for spike protein to induce ACE2 internalization in endothelial cells and cause endothelial dysfunction and that this effect can be blocked by the H2R blocker, famotidine.

Thioredoxin Prevents Loss of UCP2 in Hyperoxia via MKK4-p38 MAPK-PGC1α Signaling and Limits Oxygen Toxicity.

Administration of high concentrations of oxygen (hyperoxia) is one of few available options to treat acute hypoxemia-related respiratory failure, as seen in the current coronavirus disease (COVID-19) pandemic. Although hyperoxia can cause acute lung injury through increased production of superoxide anion (O2•-), the choice of high-concentration oxygen administration has become a necessity in critical care. The objective of this study was to test the hypothesis that UCP2 (uncoupling protein 2) has a major function of reducing O2•- generation in the lung in ambient air or in hyperoxia. Lung epithelial cells and wild-type; UCP2-/-; or transgenic, hTrx overexpression-bearing mice (Trx-Tg) were exposed to hyperoxia and O2•- generation was measured by using electron paramagnetic resonance, and lung injury was measured by using histopathologic analysis. UCP2 expression was analyzed by using RT-PCR analysis, Western blotting analysis, and RNA interference. The signal transduction pathways leading to loss of UCP2 expression were analyzed by using IP, phosphoprotein analysis, and specific inhibitors. UCP2 mRNA and protein expression were acutely decreased in hyperoxia, and these decreases were associated with a significant increase in O2•- production in the lung. Treatment of cells with rhTrx (recombinant human thioredoxin) or exposure of Trx-Tg mice prevented the loss of UCP2 protein and decreased O2•- generation in the lung. Trx is also required to maintain UCP2 expression in normoxia. Loss of UCP2 in UCP2-/- mice accentuated lung injury in hyperoxia. Trx activates the MKK4-p38MAPK (p38 mitogen-activated protein kinase)-PGC1α (PPARγ [peroxisome proliferator-activated receptor γ] coactivator 1α) pathway, leading to rescue of UCP2 and decreased O2•- generation in hyperoxia. Loss of UCP2 in hyperoxia is a major mechanism of O2•- production in the lung in hyperoxia. rhTrx can protect against lung injury in hyperoxia due to rescue of the loss of UCP2.

SARS-CoV-2 Spike Protein Induces Degradation of Junctional Proteins That Maintain Endothelial Barrier Integrity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the Angiotensin converting enzyme 2 (ACE2) receptor present on the cell surface to enter cells. Angiotensin converting enzyme 2 is present in many cell types including endothelial cells, where it functions to protect against oxidative damage. There is growing evidence to suggest that coronavirus disease (COVID-19) patients exhibit a wide range of post-recovery symptoms and shows signs related to cardiovascular and specifically, endothelial damage. We hypothesized that these vascular symptoms might be associated with disrupted endothelial barrier integrity. This was investigated in vitro using endothelial cell culture and recombinant SARS-CoV-2 spike protein S1 Receptor-Binding Domain (Spike). Mouse brain microvascular endothelial cells from normal (C57BL/6 mice) and diabetic (db/db) mice were used. An endothelial transwell permeability assay revealed increased permeability in diabetic cells as well as after Spike treatment. The expression of VE-Cadherin, an endothelial adherens junction protein, JAM-A, a tight junctional protein, Connexin-43, a gap junctional protein, and PECAM-1, were all decreased significantly after Spike treatment in control and to a greater extent, in diabetic cells. In control cells, Spike treatment increased association of endothelial junctional proteins with Rab5a, a mediator of the endocytic trafficking compartment. In cerebral arteries isolated from control and diabetic animals, Spike protein had a greater effect in downregulating expression of endothelial junctional proteins in arteries from diabetic animals than from control animals. In conclusion, these experiments reveal that Spike-induced degradation of endothelial junctional proteins affects endothelial barrier function and is the likely cause of vascular damage observed in COVID-19 affected individuals.

Cardiovascular Impacts on COVID-19 Infected Patients.

The SARS-CoV-2 virus has taken more than 2 million lives on a global scale. Over 10 million people were confirmed with COVID-19 infection. The well-known spot of primary infection includes the lungs and the respiratory system. Recently it has been reported that the cardiovascular system and coagulation mechanisms were the second major targets of biological system affected due to the viral replication. The replication mechanism of SARS-CoV-2 involves the angiotensin-converting enzyme 2- (ACE2) surface receptors of endothelial cells belonging to various organs which act as the binding site for the viral spike (S) protein of SARS-CoV-2. The COVID-19 virus has been recently listed as a primary risk factor for the following cardiovascular conditions such as pericarditis, myocarditis, arrhythmias, myocardial injury, cardiac arrest, heart failure and coagulation abnormalities in the patients confirmed with COVID-19 viral infection. Direct and indirect type of tissue damage were the two major categories detected with cardiovascular abnormalities. Direct myocardial cell injury and indirect damage to the myocardial cell due to inflammation were clinically reported. Few drugs were clinically administered to regulate the vital biological mechanism along with symptomatic treatment and supportive therapy.

SARS-CoV-2 spike protein induces degradation of junctional proteins that maintain endothelial barrier integrity

Background SARS-CoV-2 enters cells through the Angiotensin-converting enzyme 2 (ACE-2) receptor present on the cell surface. ACE-2 is present in many cell types including endothelial cells, where it functions to protect against oxidative damage. Reports suggest that COVID-19 patients also showed symptoms related to endothelial damage. We hypothesized that these vascular symptoms might be associated with disrupted endothelial barrier integrity. We investigated this in vitro using endothelial cell culture and recombinant SARS-CoV-2 spike protein S1 Receptor-Binding Domain (S1RBD). Materials and Methods Mouse brain microvascular endothelial cells from normal (C57BL/6 mice) and diabetic (db/db) mice were used. The expression of VE-Cadherin, PECAM-1, JAM-A and Connexin 43, was probed by Western blot in normal and diabetic cells, before and after treatment with S1RBD. Results The expression of VE-Cadherin, an endothelial adherens junction protein, was not affected by diabetes or by S1RBD treatment. PECAM-1 expression showed a small but significant (~15%) decrease after S1RD treatment in control cells. Junctional Adhesion Molecule (JAM-A), a tight junction protein, and the gap junction protein, Connexin-43, decreased by ~50 and 80% respectively in the presence of S1RBD when compared to untreated controls. Expression of PECAM-1, JAM-A and connexin-43 were significantly lower in untreated diabetic endothelial cells, compared to untreated control cells. This expression level further decreased significantly in the presence of S1RBD, with maximal effects observed with JAM-A and Connexin-43 expression (~80 and 90% decrease, respectively). These results indicate that healthy and diabetic endothelial cells respond differently when challenged with SARS-CoV-2 S1RBD. Conclusion S1RBD-induces degradation of endothelial junctional proteins that likely affects endothelial barrier function and causes vascular damage.