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

Abstract

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.

Molecular and Functional Study of Transient Receptor Potential Vanilloid 1-4 at the Rat and Human Blood–Brain Barrier Reveals Interspecies Differences

“…The hCMEC/D3 cell line kindly given by Cochin Institute (Paris, France). were used from passages 27 to 33 and cultured with the EndoGRO complete medium (Merck), supplemented with 5% FBS,1% streptomycin-penicillin (Gibco, Carlsbad, CA, United States), and 1 ng/mL b-FGF (Sigma) under 5% CO 2 and 37 • C as previously described (Luo et al, 2019). Culture flasks and plates were pre-coated with 150 µg/mL rat tail collagen type I.…”

“…Reverse transcription of total RNA was achieved in a thermocycler (PTC-100 programmable thermal controller, MJ research INC, United States) using the following conditions: 25 • C for 10 min, then at 42 • C for 30 min and at 99 • C for 5 min, as previously reported (Dauchy et al, 2009). cDNAs were stored at −80 • C. Gene expression was determined by SYBR Green fluorescence detection using an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA, United States) as previously reported (Luo et al, 2019). Samples were run in duplicate.…”

“…Thus, the comparative expression levels of TRPV1-4 in the BBB of rodent pharmacological models and human are meaningful in clarifying the possible involvement of each isoform in physiological and pathological BBB. A whole transcriptomic analysis revealed a relative low Trpv2 expression in murine primary BMEC (Zhang et al, 2014) and rat Trpv2 was not immunolocalized in the brain vasculature (Nedungadi et al, 2012), whereas TRPV2 was shown highly expressed in the human cerebral microvessel endothelial cells D3 cells (hCMEC/D3) human cerebral microvessel endothelial cell line (Luo et al, 2019), suggesting interspecies differences in the expression of TRPV between rat and human BMEC.…”

Cannabinoids, Blood–Brain Barrier, and Brain Disposition

“…CBD protection was kept up to two hours after reperfusion and was suppressed by 100 nM of GW9662 (a PPARγ antagonist compound) and to a certain extent diminished by 300 nM of the compound WAY100135 (5-HT1A receptor antagonist). CBD protection was not changed by the addition of CB1R or CB2R antagonists or vanilloid or adenosine A2A receptors [50]. These data suggest that CBD, by triggering 5-HT1A and PPARγ receptors, could have a protective effect against BBB malfunction caused by ischemic events.…”

ABC Transporters at the Blood–Brain Interfaces, Their Study Models, and Drug Delivery Implications in Gliomas

“…Diverse strategies have been devised for the ex vivo study of the brain barriers separately. Isolated microvessels can be employed for the molecular characterization of the BBB and NVU [21,22,95,168,222], used immediately for functionality studies [223][224][225][226][227], or cultured with specific media for a limited time [149]. Whole brains or specific sections (e.g., cortex) are homogenized either mechanically, employing a Potter-Evenheilm homogenizer [115,161,228] by enzymatic dissociation with collagenases that partly degrade the basement membrane [146] or using both [229]; then, the microvessels (diameter <10 µm) are separated from other cells using a density gradient and isolated from bigger vessels by sequential filtration on nylon meshes.…”