Bile acids promote endothelial cell homeostasis via the regulation of cell metabolism and quiescence

Abstract

Abstract Background Endothelial cell (EC) homeostasis is characterized by a quiescent and low-metabolic state. Obesity leads to EC dysfunction and CVD. We demonstrated that bariatric surgery improved obesity-induced EC dysfunction and increased fasting and post-prandial plasma levels of bile acids (BAs)(in particular, cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA)). BAs are emerging as metabolic signaling molecules in several tissues, but their effect on ECs has not been clearly established yet. Purpose We investigate the effect of BA on the maintenance of EC quiescence. Methods Human Aortic Endothelial Cells (HAECs) were treated for 24h with vehicle (0.1% DMSO), CA, CDCA and DCA (50uM each) in 2%FBS EGM-2 media. Percentage of apoptotic cells was assessed with AnnexinV/PI. Seahorse Mitostress test, Mitoplate and glucose uptake with 0.5µCi/ml of 14C-2-deoxy-D-glucose were used to evaluate changes of HAEC metabolism. Immunostaining for Ki67, Crystal Violet staining and scratch assay were performed to assess HAEC proliferation and migration. NO production and endothelial NO synthase (eNOS) activity were measured via DAF-2 probe and Western Blot, respectively. By qPCR genes involved in Notch pathway were evaluated. Mesoscale vascular injury panel, scratch assay, Ki67 immunostaining and glucose uptake were also performed in HAECs pre-treated with 50uM of BAs and stimulated with 0.5ng/mL TNF-α for 24h. Results HAECs treated with BAs increased NO production compared to vehicle and a reduction in both basal and maximal mitochondrial respiration and extracellular acidification rate was shown by Seahorse Mitostress test. Mitoplate assay displayed an overall reduction in the metabolic rate of HAECs treated with CA and DCA and a peculiar metabolic pattern in cells treated with CDCA. Reduction in glucose uptake was also observed after BA treatment, suggesting that BAs are able to decrease EC metabolism. Immunostaining for Ki67 and Crystal Violet highlighted that BAs reduced cell proliferation. The scratch assay also displayed a reduction in EC migration after BA treatment. BAs also reduced EC apoptosis, as significant differences in the percentage of AnnexinV/PI positive cells was observed in BA-treated cells compared to vehicle. qPCR showed increased expression of genes involved in Notch pathway promoting quiescence after BA treatment. Glucose uptake, scratch assay and Ki67 immunostaining performed in TNF-α stimulated HAECs suggested a BA-dependent endothelial protection in conditions of EC pro-inflammatory activation. We did not observed a reduction in pro-inflammatory biomarkers released in the supernatant of HAECs pre-treated with BA and stimulated with TNF-α. Conclusions Our results suggest that BAs may be involved in the maintenance of EC quiescence by regulating EC metabolism and gene expression profile. These findings highlight a potential role of BAs as endothelial-protective molecules.