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Cardiovascular Engineering

Cardiovascular Engineering - Poster Session E

Poster H14 - The Effects of THC and CBD on Endothelial Glycocalyx and Viability

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  As cannabis use has become more prevalent, the need for cannabis-based research also increases. Cannabis contains numerous cannabinoids, including ∆9-tetrahydrocannabinol (THC) and cannabidiol (CBD), that bind to their corresponding receptors: cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2), respectively, which are naturally expressed in endothelial cells. These receptors are embedded within and protrude out of the glycocalyx. The glycocalyx is a coating of sugars, proteins, and lipids that protect all cells from inflammation. THC has been shown to increase inflammation of endothelial cells, while CBD has anti-inflammatory effects. Additionally, THC has been shown to induce a dose-dependent effect on cell viability. Endothelial dysfunction, including dysregulated glycocalyx, has been linked to conditions such as cardiovascular disease (CVD) and atherosclerosis. However, the direct effect of THC and CBD on the glycocalyx and the effect of CBD on endothelial cell viability have yet to be studied. Using cultured human aortic endothelial cells (HAECs), we measured the effects of THC and CBD on the glycocalyx, specifically the expression of heparan sulfate (HS), and cell viability. We hypothesized that the HAECs treated with THC would have decreased expression of HS and decreased cell viability, while those treated with CBD would have no change in HS and cell viability.

Materials and

Methods:  HAECs were cultured and seeded into 8 chamber slides at 30,000 cells /cm2 in 10% FBS EGM. At 16 hours after the plating, the media was changed to 2% FBS, 0.5% BSA EBM. After 6 hours, HAECs were treated in duplicate with either vehicle (methanol, negative control), 5µM THC, 5µM CBD, or 10ng/mL IL-1β (positive control).
48 hours post treatment, the MTT assay was performed on the HAECs to assess cell viability and the remaining HAECs were fixed in 2% paraformaldehyde for 15 minutes for immunofluorescent (IF) staining of heparan sulfate expression. In each plate, there was one vehicle well that was only stained with the secondary antibody, to test for nonspecific binding. All other wells were stained with a mouse anti-human-heparan sulfate antibody and a rabbit anti-mouse-488 secondary antibody. The stained cells were then imaged with a confocal spinning disk microscope to take z-stack images.
To analyze the confocal microscope images of the IF stained cells, we used ImageJ software. For each well, we used the Z Project feature that overlays all the slices onto one image. The Z project image compressed the maximum intensity of staining for each slice, to quantify total heparan sulfate expression. Then we used ImageJ to measure the integrated density of 1 image per well. Integrated density considers both the area and brightness of positive staining. After measuring the integrated densities for each well, we averaged each separate treatment and normalized the data to the vehicle.

Results, Conclusions, and Discussions: The secondary only control confirmed there was no nonspecific binding in the secondary antibody and was used to set exposure time. The vehicle and Il-1β treated cells had the highest expressions of heparan sulfate. In IL-1β, this was expected because IL-1β is known to cause an increase in HS. The THC treated HAECs had decreased expression of heparan sulfate, as hypothesized. The CBD treated HAECs also had decreased expression of heparan sulfate, contrary to our hypothesis. These results indicate that both THC and CBD negatively impacts heparan sulfate expression in the glycocalyx. Additionally, the MTT assay showed that at 5µM THC and 5µM CBD, both decreased cell viability. Ultimately, these changes to the glycocalyx and cell viability could leave endothelial cells vulnerable to coagulation and inflammation, which may impact cardiovascular disease.

Acknowledgements (Optional):