Metformin improves diabetic endothelial barrier integrity by increasing ACE-2 receptor expression

Background: The COVID-19 virus gains entry into cells through the Angiotensinconverting enzyme 2 (ACE-2) receptor present on the cell surface The ACE-2 receptor is present in many cell types including endothelial cells, where it helps protect against oxidative damage Recent reports suggest that some patients who test positive for COVID-19 also showed symptoms related to endothelial damage We investigated this effect in vitro using endothelial cell culture and commercially procured recombinant COVID-19 spike protein S1 Receptor-Binding Domain (S1RBD) Materials and Methods: Mouse brain microvascular endothelial cells from normal (C57BL/6) and diabetic (db/db) mice were used ACE-2 receptor expression was studied using real time-PCR (RT-PCR) and Western blot A commercial endothelial transwell permeability assay kit was used to assess endothelial barrier integrity in vitro Results: ACE-2 mRNA and total protein expression decreased by ∼50% in diabetic endothelial cells Endothelial permeability increased ∼3-fold in diabetic cells compared to normal cells which indicated impaired barrier integrity Incubation of normal cells for 18h with PD123319, a specific ACE-2 receptor blocker, or recombinant S1RBD, decreased ACE-2 total protein by ∼50% and ∼60% respectively, and also increased endothelial permeability by ∼2 5-fold each, compared to untreated controls Addition of PD123319 and S1RBD to diabetic cells caused a further reduction in ACE-2 total protein to ∼55% and ∼63% respectively, and ∼3-fold increase in endothelial permeability compared to normal untreated controls Metformin alone had no significant effect on ACE-2 receptor expression in normal cells but increased expression almost 3-fold and decreased permeability by ∼40% in diabetic cells compared to untreated diabetic controls Pre-treatment of diabetic cells for 12h with metformin and then co-incubating with the S1RBD for 18h, not only prevented loss of ACE-2 protein but also increased expression ∼2-fold and decreased endothelial permeability ∼2 5-fold compared to S1RBD treatment alone Conclusion: Metformin protects endothelial barrier integrity against recombinant S1RBD challenge by stabilizing ACE-2 receptor expression in diabetic cells