Receptor-binding domain of SARS-CoV-2 is a functional αv-integrin agonist.

Among the novel mutations distinguishing SARS-CoV-2 from similar coronaviruses is a K403R substitution in the receptor-binding domain (RBD) of the viral spike (S) protein within its S1 region. This amino acid substitution occurs near the angiotensin-converting enzyme 2-binding interface and gives rise to a canonical RGD adhesion motif that is often found in native extracellular matrix proteins, including fibronectin. Here, the ability of recombinant S1-RBD to bind to cell surface integrins and trigger downstream signaling pathways was assessed and compared with RGD-containing, integrin-binding fragments of fibronectin. We determined that S1-RBD supported adhesion of fibronectin-null mouse embryonic fibroblasts as well as primary human small airway epithelial cells, while RBD-coated microparticles attached to epithelial monolayers in a cation-dependent manner. Cell adhesion to S1-RBD was RGD dependent and inhibited by blocking antibodies against αv and ß3 but not α5 or ß1 integrins. Similarly, we observed direct binding of S1-RBD to recombinant human αvß3 and αvß6 integrins, but not α5ß1 integrins, using surface plasmon resonance. S1-RBD adhesion initiated cell spreading, focal adhesion formation, and actin stress fiber organization to a similar extent as fibronectin. Moreover, S1-RBD stimulated tyrosine phosphorylation of the adhesion mediators FAK, Src, and paxillin; triggered Akt activation; and supported cell proliferation. Thus, the RGD sequence of S1-RBD can function as an αv-selective integrin agonist. This study provides evidence that cell surface αv-containing integrins can respond functionally to spike protein and raises the possibility that S1-mediated dysregulation of extracellular matrix dynamics may contribute to the pathogenesis and/or post-acute sequelae of SARS-CoV-2 infection.

Fibronectin as a Marker of Disease Severity in Critically Ill COVID-19 Patients.

The SARS-CoV-2 virus alters the expression of genes for extracellular matrix proteins, including fibronectin. The aim of the study was to establish the relationship between different forms of fibronectin, such as plasma (pFN), cellular (EDA-FN), and proteolytic FN-fragments, and disease severity and mortality of critically ill patients treated in the intensive care unit. The levels of pFN, EDA-FN, and FN-fragments were measured in patients with a viral (N = 43, COVID-19) or bacterial (N = 41, sepsis) infection, using immunoblotting and ELISA. The level of EDA-FN, but not pFN, was related to the treatment outcome and was significantly higher in COVID-19 Non-survivors than in Survivors. Furthermore, EDA-FN levels correlated with APACHE II and SOFA scores. FN-fragments were detected in 95% of COVID-19 samples and the amount was significantly higher in Non-survivors than in Survivors. Interestingly, FN-fragments were present in only 56% of samples from patients with bacterial sepsis, with no significant differences between Non-survivors and Survivors. The new knowledge gained from our research will help to understand the differences in immune response depending on the etiology of the infection. Fibronectin is a potential biomarker that can be used in clinical settings to monitor the condition of COVID-19 patients and predict treatment outcomes.

SARS-CoV-2 deregulates the vascular and immune functions of brain pericytes via Spike protein.

Coronavirus disease 19 (COVID-19) is a respiratory illness caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). COVID-19 pathogenesis causes vascular-mediated neurological disorders via elusive mechanisms. SARS-CoV-2 infects host cells via the binding of viral Spike (S) protein to transmembrane receptor, angiotensin-converting enzyme 2 (ACE2). Although brain pericytes were recently shown to abundantly express ACE2 at the neurovascular interface, their response to SARS-CoV-2 S protein is still to be elucidated. Using cell-based assays, we found that ACE2 expression in human brain vascular pericytes was increased upon S protein exposure. Pericytes exposed to S protein underwent profound phenotypic changes associated with an elongated and contracted morphology accompanied with an enhanced expression of contractile and myofibrogenic proteins, such as α-smooth muscle actin (α-SMA), fibronectin, collagen I, and neurogenic locus notch homolog protein-3 (NOTCH3). On the functional level, S protein exposure promoted the acquisition of calcium (Ca2+) signature of contractile ensheathing pericytes characterized by highly regular oscillatory Ca2+ fluctuations. Furthermore, S protein induced lipid peroxidation, oxidative and nitrosative stress in pericytes as well as triggered an immune reaction translated by activation of nuclear factor-kappa-B (NF-κB) signaling pathway, which was potentiated by hypoxia, a condition associated with vascular comorbidities that exacerbate COVID-19 pathogenesis. S protein exposure combined to hypoxia enhanced the production of pro-inflammatory cytokines involved in immune cell activation and trafficking, namely macrophage migration inhibitory factor (MIF). Using transgenic mice expressing the human ACE2 that recognizes S protein, we observed that the intranasal infection with SARS-CoV-2 rapidly induced hypoxic/ischemic-like pericyte reactivity in the brain of transgenic mice, accompanied with an increased vascular expression of ACE2. Moreover, we found that SARS-CoV-2 S protein accumulated in the intranasal cavity reached the brain of mice in which the nasal mucosa is deregulated. Collectively, these findings suggest that SARS-CoV-2 S protein impairs the vascular and immune regulatory functions of brain pericytes, which may account for vascular-mediated brain damage. Our study provides a better understanding for the mechanisms underlying cerebrovascular disorders in COVID-19, paving the way to develop new therapeutic interventions.

Effect of sleeve gastrectomy on the expression of meteorin-like (METRNL) and Irisin (FNDC5) in muscle and brown adipose tissue and its impact on uncoupling proteins in diet-induced obesity rats.

BACKGROUND: Bariatric surgery is well established as a treatment for obesity and associated complications. This procedure improves metabolic homeostasis through changes in energy expenditure. We hypothesized that sleeve gastrectomy (SG) improves metabolic homeostasis by modulating energy expenditure and enhancing thermogenesis through increasing the expression level of meteorin-like protein (METRNL) and fibronectin type III domain-containing protein 5 (FNDC5/Irisin) through uncoupling proteins 1/2/3 (UCP1, UCP2, and UCP3). OBJECTIVES: To study the effect of SG on the levels of proteins involved in thermogenesis process. SETTING: Laboratory rats at Kuwait University. METHODS: Male Sprague-Dawley rats, aged 4 to 5 weeks, were divided into 2 groups, control (n = 11) and diet-induced obesity (DIO) (n = 22). The control group was fed regular rat chow ad libitum, whereas the DIO group was fed cafeteria diet "high-fat/carbohydrate diet" ad libitum. At 21 weeks, rats in the DIO group that weighed 20% more than the control group animals underwent surgery. These rats were randomly subdivided into Sham and SG operation groups. Gene expression was evaluated, and enzyme-linked immunosorbent assays were employed to assess the changes in gene and protein levels in tissue and circulation. RESULTS: The protein expression data revealed an increase in METRNL levels in the muscles and white adipose tissue of SG animals. METRNL level in circulation in SG animals was reduced compared with control and Sham rats. The level of Irisin increased in the muscle of SG animals compared with the control and Sham group animals; however, a decrease in Irisin level was observed in the white adipose tissue and brown adipose tissue of SG animals compared with controls. Gene expression analysis revealed decreased METRNL levels in muscle tissues in the SG group compared with the control group animals. Increased expression of FNDC5 (Irisin), UCP2, and UCP3 in the muscle tissue of SG animals was also observed. Furthermore, the levels of UCP1, UCP2, UCP3, and METRNL in the brown adipose tissue of SG animals were upregulated. No significant alteration in the gene expression of Irisin was observed in brown adipose tissue. CONCLUSIONS: Sleeve gastrectomy induces weight loss through complex mechanisms that may include browning of fat.

Irisin modulates genes associated with severe coronavirus disease (COVID-19) outcome in human subcutaneous adipocytes cell culture.

Obesity patients are more susceptible to develop COVID-19 severe outcome due to the role of angiotensin-converting enzyme 2 (ACE2) in the viral infection. ACE2 is regulated in the human cells by different genes associated with increased (TLR3, HAT1, HDAC2, KDM5B, SIRT1, RAB1A, FURIN and ADAM10) or decreased (TRIB3) virus replication. RNA-seq data revealed 14857 genes expressed in human subcutaneous adipocytes, including genes mentioned above. Irisin treatment increased by 3-fold the levels of TRIB3 transcript and decreased the levels of other genes. The decrease in FURIN and ADAM10 expression enriched diverse biological processes, including extracellular structure organization. Our results, in human subcutaneous adipocytes cell culture, indicate a positive effect of irisin on the expression of multiple genes related to viral infection by SARS-CoV-2; furthermore, translatable for other tissues and organs targeted by the novel coronavirus and present, thus, promising approaches for the treatment of COVID-19 infection as therapeutic strategy to decrease ACE2 regulatory genes.