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

Endothelial 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.

Cannabidiol Increases Proliferation, Migration, Tubulogenesis, and Integrity of Human Brain Endothelial Cells through TRPV2 Activation.

The effect of cannabidiol (CBD), a high-affinity agonist of the transient receptor potential vanilloid-2 (TRPV2) channel, has been poorly investigated in human brain microvessel endothelial cells (BMEC) forming the blood-brain barrier (BBB). TRPV2 expression and its role on Ca2+ cellular dynamics, trans-endothelial electrical resistance (TEER), cell viability and growth, migration, and tubulogenesis were evaluated in human primary cultures of BMEC (hPBMEC) or in the human cerebral microvessel endothelial hCMEC/D3 cell line. Abundant TRPV2 expression was measured in hCMEC/D3 and hPBMEC by qRT-PCR, Western blotting, nontargeted proteomics, and cellular immunofluorescence studies. Intracellular Ca2+ levels were increased by heat and CBD and blocked by the nonspecific TRP antagonist ruthenium red (RR) and the selective TRPV2 inhibitor tranilast (TNL) or by silencing cells with TRPV2 siRNA. CBD dose-dependently induced the hCMEC/D3 cell number (EC50 0.3 ± 0.1 µM), and this effect was fully abolished by TNL or TRPV2 siRNA. A wound healing assay showed that CBD induced cell migration, which was also inhibited by TNL or TRPV2 siRNA. Tubulogenesis of hCMEC/D3 cells in 3D matrigel cultures was significantly increased by 41 and 73% after a 7 or 24 h CBD treatment, respectively, and abolished by TNL. CBD also increased the TEER of hPBMEC monolayers cultured in transwell, and this was blocked by TNL. Our results show that CBD, at extracellular concentrations close to those observed in plasma of patients treated by CBD, induces proliferation, migration, tubulogenesis, and TEER increase in human brain endothelial cells, suggesting CBD might be a potent target for modulating the human BBB.

RCBTB1 Deletion Is Associated with Metastatic Outcome and Contributes to Docetaxel Resistance in Nontranslocation-Related Pleomorphic Sarcomas.

Half of soft-tissue sarcomas are tumors with complex genomics, which display no specific genetic alterations and respond poorly to treatment. It is therefore necessary to find new therapeutic targets for these sarcomas. Despite genetic heterogeneity across samples, oncogenesis may be driven by common pathway alterations. Therefore, genomic and transcriptomic profiles of 106 sarcomas with complex genomics were analyzed to identify common pathways with altered genes. This brought out a gene belonging to the "cell cycle" biological pathway, RCBTB1 (RCC1 And BTB Domain Containing Protein 1), which is lost and downregulated in 62.5% of metastatic tumors against 34% of non-metastatic tumors. A retrospective study of three sarcoma cohorts revealed that low RCBTB1 expression is prognostic for metastatic progression, specifically in patients that received chemotherapy. In vitro and in vivo, RCBTB1 overexpression in leiomyosarcoma cells specifically sensitized to docetaxel-induced apoptosis. This was associated with increased mitotic rate in vitro and higher growth rate of xenografts. By contrast, RCBTB1 inhibition decreased cell proliferation and protected sarcoma cells from apoptosis induced by docetaxel. Collectively, these data evidenced that RCBTB1 is frequently deleted in sarcomas with complex genomics and that its downregulation is associated with a higher risk of developing metastasis for patients receiving chemotherapy, likely due to their higher resistance to docetaxel.