(I-341) Effects of THC on Endothelial Cell Barrier Function

Cannabis is a commonly used drug and usage will become more frequent as legalization increases.1 The psychoactive effects of cannabis derive from Δ9-tetrahydrocannabinol (THC) which is a ligand that binds to cannabinoid receptor-1 (CB₁) expressed on endothelial cells.2 CB₁ activation with THC has been shown to increase cytokine gene expression.3 This suggests THC activation of CB₁ contributes to inflammatory processes within endothelial cells. Endothelial cells mediate inflammatory processes through the release of cytokines, the expression of surface adhesion molecules, and tight cell junctions. Thus, I hypothesized that THC would induce an inflammatory phenotype of endothelial cell by increasing surface adhesion molecules, specifically VCAM-1 and degrading cell junctions by decreasing VE-cadherin. To test my hypothesis, we quantified endothelial cell viability, adhesion molecule expression, and permeability in response to THC in a dose dependent manner.

Endothelial cell viability.  Cell culture was performed with human umbilical vein endothelial cells (HUVECs) and seeded in a 96 well plate (3000 cells/well). HUVECs were treated with THC concentrations of 0, 0.1, 0.5, 1, 5, 10, 20µM in quadruplicate for 48 hours before MTS assay (n=3).

Adhesion molecule expression. HUVECs were seeded in an 8 chamber slide (9000 cells/well). HUVECs were treated with THC concentrations of 0, 5, 10, 20 µM in duplicates for 48 hours before fixation (n=3). THC treated HUVECs were then stained with antibodies for VCAM-1, VE-cadherin, and DAPI for immunofluorescent (IF) imaging.

Permeability. HUVECs were seeded in a transwell plate (30,000 cells/well) for 24 hours. HUVECs were treated with THC concentrations of 0 and 5 µM in duplicates for 48 hours before incubating with Evans Blue with bovine serum albumin (BSA) for 30 minutes (n=3). Controls of no treatment, vehicle, and TNF-alpha were used. The absorbance in the lower wells was measured at 620nm in a plate reader.

There was a dose dependent decrease in cell viability due to increasing doses of THC. IF imaging of VCAM-1 and VE-cadherin at endothelial cell junctions shows no changes to these adhesion molecules. THC did not induce permeability in a transwell assay. While our studies did not show adhesion molecule expression changes from THC, there is evidence that cytokine release promotes E- and P-selectin expression prior to VCAM-1 and VE-cadherin. It is known that THC induced CB₁ activation increases cytokine gene expression, therefore, further directions will be assessing the effects of THC induced CB₁ activation on E- and P- selectin expression.  This research will contribute to further our understanding on THC contributions to endothelial cell inflammation processes.