ABSTRACT
Introduction: Autopsies of COVID-19 patients demonstrate the presence of SARS-CoV-2 in the brain endothelium, cerebrospinal fluid, glial cells, and neuronal tissue;while emerging clinical data suggest that ~40% of COVID-19 patients develop neurological symptoms. We examined the effects of SARS-COV-2 RBD spike protein on blood brain barrier (BB) integrity, and on the expression of tight junction proteins (TJP) that maintain BBB permeability and function. Materials / Methods: Primary human BMVEC were treated with recombinant SARS-COV-2 Spike protein (BEI Resources Inc) for 24-48 hrs, followed by immunofluorescent staining to quantify ACE2 receptor expression. Pro-inflammatory cytokines were quantified in culture supernatants using BioLegend's LEGENDLplexTM bead-based immunoassay. Additionally, a well validated 2D in-vitro BBB model was used to examine the effects of SARS-COV-2 on BBB integrity as measured by transendothelial electrical resistance (TEER) across the membrane, and TJ protein gene expression levels were measured using real time quantitative PCR. Result(s): Data demonstrates that primary human BMVEC express the ACE2 receptor and treatment with SARS-COV-2 spike protein significant increases in ACE2 receptor expression. We observed a significant increase in the levels of the pro-inflammatory cytokines TNF-alpha (p<0.01), IL-6 (p<0.0001), IL-10 (p<0.05), IL-23 (p<0.05) and IL-33 (p<0.01) in BMVEC treated with SARS-COV-2 spike protein compared to untreated controls. A 30% (p<0.05) decrease in TEER occurred in the BBB treated with SARS-COV-2 spike protein as compared to untreated controls, and SARS-COV-2 decreased TJP expression. Data demonstrates that SARS-COV-2 treatment decreased gene expression for the TJPs- ZO-1 (52%;p<0.05), ZO-2 (92%;p<0.001), Claudin-5 (97%;p<0.001) and JAM-2 (45%;p<0.05) as compared to untreated controls. Discussion(s): SARS COV-2 mediates its effects via the ACE2 receptor and therefore an increase in ACE2 expression on BMVEC suggests that neuroinvasion by SARS- COV2 is mediated via endothelial inflammation. Further, SARS-COV-2 induced levels of pro-inflammatory cytokines IL-6, TNF-alpha, IL-8, and IL-10 corroborates the induction of a neuroinflammatory response, confirming hypercytokinemia, which may underlie neuroinflammation in COVID-19 associated encephalopathy. Our data suggest that the significant decrease in TJP gene expression levels directly affect BBB integrity and function thus enabling neuro-invasion and potential subsequent COVID-19 associated neuropathology. Conclusion(s): BMVEC have a paracrine-autocrine role in maintaining CNS homeostasis and the SARS-COV2 associated endothelial cell dysfunction preludes the neuropathology observed in COVID-19 infected patients. Potentially, anti-cytokine based therapeutics may be effective in treating patients with COVID- 19 associated neurological disease. Acknowledgements: Authors gratefully acknowledge funding from SUNY Research Seed Grant Program 2019-20 -RFP #20-03-COVID that was crucial to obtain data for this pilot project. Learning Objectives: Examine the basic neuromodulatory mechanisms that underlie SARS-COV-2 mediated neuropathology. Keywords: SARS-COV2;transendothelial electrical resistance;Blood Brain Barrier;permeability, Tight junction Copyright © 2022
ABSTRACT
Disorders in pulmonary vascular integrity are a prominent feature in many lung diseases, including acute respiratory distress syndrome (ARDS), capillary leak syndrome, and COVID19. Paracrine signals are enriched in the lung and are critically important in regulating the homeostasis of the functional pulmonary microvasculature. Here, we employed single-cell RNA-sequencing (scRNAseq) to study ligand and receptor interactions in the native human lung microvascular niche, and identified soluble factors that are critical in endothelial integrity. The scRNAseq data reveals a total of 47 cell populations consisting of five vascular endothelial subtypes in human lungs, including general capillary EC, aerocyte capillary EC (EC aCap), arterial EC, pulmonary venous EC, and systemic venous EC. Using EC aCap as a signal receiving core (Receptors) and the putative adjacent cell types (alveolar fibroblast, ATI, ATII, pericyte, plasma cell, etc.) in the EC aCap niche as senders (Ligands), we identified that SLIT2-ROBO4, ANGPT1-TIE1, ADM-RAMP2, VEGFD-KDR, and BMP5-BMPR2 are the top specific and abundant pairs in the niche. Immunostaining and ELISA assays confirmed their spatial information and secretion level. Furthermore, upon treatment with these ligands, real-time resistance recorded using an electric cell-substrate impedance sensing (ECIS) system revealed that VEGFD, ANGPT1 (angiopoietin 1), and ADM (adrenomedullin) could markedly increase the electrical resistance of human lung microvascular, arterial, and venous endothelial cells, suggesting their strong impact on the endothelial barrier function. Deciphering the cell-cell soluble signals that improve endothelial integrity in human lungs lays the foundation for uncovering the pathogenesis of pulmonary vascular disorders and the development of ex vivo functional lung vasculature.
ABSTRACT
Rationale: An increase in endothelial permeability resulting from the disruption of endothelial barrier and aggravated inflammatory responses are two major pathological hallmarks of various lung disorders including the current global pandemic COVID-19. Drugs that enable the preservation and restoration of endothelial function represent attractive therapeutic targets to treat endothelial dysfunction-derived cardiopulmonary diseases. A role of G protein-coupled receptors (GPCRs), especially a sub-family of proton-sensing GPCRs including GPR4 and GPR68, has been suggested in modulation of endothelial function. In this study, we analyzed the barrier protective and anti-inflammatory effects of two recently developed novel class of GPR68 inhibitors: ogremorphins OGM8345 and OGM-1.Methods: Transendothelial electrical resistance (TER) was monitored in human pulmonary arterial endothelial cells (HPAECs) to evaluate endothelial barrier function. Quantitative real time PCR and western blot analyses were performed to determine mRNA and protein expression of endothelial inflammation markers, respectively. Acidic pH (6.5) medium was used to induce acidosis, and luciferase-based Tango assay was employed to evaluate GPR68 activation. C57BL/6 mice were exposed to lipopolysaccharide (LPS from Escherichia coli) or heatkilled Staphylococcus aureus (HKSA), and vascular leak/inflammation was assessed by determining the extravasation of intravenously injected Evans blue tracer into lungs and total cells/protein count in bronchoalveolar lavage samples. Results: A robust dose-dependent increase in basal EC barrier function was observed with OGM8345 (1-5 μM) and OGM-1 (0.3-1.5 μM) evident by an 150-200% increase in TER values. Both inhibitors also effectively rescued LPS- and HKSA-induced EC hyperpermeability. RT-PCR analysis demonstrated that LPS or HKSA-induced upregulation of inflammatory cytokines/chemokines genes TNF-α, ICAM-1, VCAM-1, IL-6, IL-8, IL- 1β, and CXCL5 was significantly attenuated by OGMs. Consistently, both OGMs suppressed LPSand HKSA-induced protein expression of VCAM-1 and ICAM-1. In contrast, pharmacologic inhibition of GPR4 by NE 52-QQ57 failed to alleviate LPS or HKSA-induced EC barrier dysfunction and inflammation. Importantly, LPS, HKSA or acidosis stimulation resulted in increased GPR68 mRNA expression and GPR68 activity that was inhibited by OGMs. Intratracheal injection of LPS or HKSA in C57BL/6 mice caused vascular leak and lung inflammation that was attenuated by both OGMs as illustrated by reduced Evans blue accumulation in the lungs and significant inhibition of accumulation of inflammatory cells and protein content in bronchoalveolar lavage samples. Conclusion: These results establish a critical role of GPR68 in endothelial dysfunction and strongly suggest a therapeutic potential of GPR68-selective inhibitors in improving endothelial dysfunction caused by bacterial infections and acidosis associated with acute and chronic lung injury.