Exposure of human cerebral microvascular endothelial cells hCMEC/D3 to laminar shear stress induces vascular protective responses

Abstract

AbstractEndothelial cells (ECs) are constantly submitted in vivo to hemodynamical forces derived from the blood circulation, including shear stress (SS). ECs are able to detect SS and consequently adapt their phenotype, thus affecting many endothelial functions. If a plethora of shear stress-regulated molecular networks have been described in peripheral ECs, less is known about the molecular responses of microvascular brain ECs which constitute the blood–brain barrier (BBB). In this work, we investigated the response of human cerebral microvascular ECs to laminar physiological shear stress using the well characterized hCMEC/D3 cell line. Interestingly, we showed that hCMEC/D3 cells responded to shear stress by aligning perpendicularly to the flow direction, contrary to peripheral endothelial cells which aligned in the flow direction. Whole proteomic profiles were compared between hCMEC/D3 cells cultured either in static condition or under 5 or 10 dyn.cm−2 SS for 3 days. 3592 proteins were identified and expression levels were significantly affected for 3% of them upon both SS conditions. Pathway analyses were performed which revealed that most proteins overexpressed by SS refer to the antioxidant defense, probably mediated by activation of the NRF2 transcriptional factor. Regarding down-regulated proteins, most of them participate to the pro-inflammatory response, cell motility and proliferation. These findings confirm the induction of EC quiescence by laminar physiological SS and reveal a strong protective effect of SS on hCMEC/D3 cells, suggesting a similar effect on the BBB. Our results also showed that SS did not significantly increase expression levels nor did it affect the localization of junctional proteins and did not afect either the functional activity of several ABC transporters (P-glycoprotein and MRPs). This work provides new insights on the response of microvascular brain ECs to SS and on the importance of SS for optimizing in vitro BBB models.