The synthetic cannabinoid 5F-MDMB-PICA enhances the metabolic activity and angiogenesis in human brain microvascular endothelial cells by upregulation of VEGF, ANG-1, and ANG-2

Abstract

Brain angiogenesis, the formation of new blood vessels from existing brain vasculature, has been previously associated with neural plasticity and addictive behaviors related to substances. Synthetic cannabinoids (SCs) have become increasingly popular due to their ability to mimic the effects of cannabis, offering high potency and easy accessibility. In the current study, we reveal that the SC 5F-MDMB-PICA, the most common SC in the United States in 2019, increases cell metabolic activity and promotes angiogenesis in human brain microvascular endothelial cells (HBMECs). First, we performed an MTT assay to evaluate the effects of 5F-MDMB-PICA treatment at various concentrations (0.0001 μM, 0.001 μM, 0.01 μM, 0.1 μM, and 1 μM) on HBMECs metabolic activity. The results demonstrated higher concentrations of the SC improved cell metabolic activity. Furthermore, 5F-MDMB-PICA treatment enhanced tube formation and migration of HBMECs in a dosage-dependent manner. Additionally, the mRNA, secreted protein, and intracellular protein levels of vascular endothelial growth factor, angiopoietin-1, and angiopoietin-2, which are involved in the regulation of angiogenesis, as well as the protein levels of cannabinoid receptor type-1, were all increased following treatment with 5F-MDMB-PICA. Notably, the phosphorylation levels at Serine 9 residue of glycogen synthase kinase-3β were also increased in the 5F-MDMB-PICA treated HBMECs. Collectively, our findings demonstrate that 5F-MDMB-PICA can enhance angiogenesis in HBMECs, suggesting the significant role of angiogenesis in the response to SCs. Manipulating this interaction may pave the way for innovative treatments targeting SC addiction and angiogenesis-related conditions.

Keywords: 5F-MDMB-PICA; ANG-1 and -2; VEGF; angiogenesis; cannabinoid receptors; synthetic cannabinoids.

Fig. 1

The chemical structure of 5F-MDMB-PICA.

Fig. 2

The expression of CB1R has been observed to rise during angiogenesis stimulation. HBMECs were cultured in proangiogenic media supplemented with bFGF (10 ng/ml) for 24, 48, and 72 hours. Subsequently, (a) western blot images were acquired, and CB1R bands were analyzed using ImageJ software. The findings are presented in (b) with a quantitative analysis of the CB1R expression rates. Data were quantified and presented as mean ± SEM (n = 3). (^**) means P > 0.01, (^***) means P > 0.0001, and (^****) means P > 0.0001.

Fig. 3

5F-MDMB-PICA treatment improves HBMECs metabolic activity of HBMECs. Cell metabolic activity rate was analyzed using an MTT assay after treatment with 5F-MDMB-PICA. First, 5 × 10³ HBMECs were seeded in a 96-well plate for 24 hours. Five different concentrations of 5F-MDMB-PICA (0.0001 μM–1 μM) were incubated for 24 hours. After that, the media containing 5F-MDMB-PICA was discarded and replaced with MTT (5 mg/ml) and further incubated for 4 hours at 37 °C and 5% CO₂. Finally, the formazan crystals were dissolved using DMSO and the absorbance was measured at 570 nm in an ELISA reader. The treatment significantly enhanced cell viability in the concentrations 0.001 μM–1 μM when compared to the control. Data were quantified and presented as mean ± SEM (n = 3). (^**) means P > 0.01 and (^****) means P > 0.0001.

Fig. 4

5F-MDMB-PICA treatment enhances cell migration in HBMECs. HBMECs were grown in a 12-well plate for 24 hours. Next, the cell monolayer was scratched using a 1,000 μl pipette tip after the cells reached the appropriate confluency. (a) Microscopic images of HBMEC migration at baseline (zero time) and at 24 hours after treatment with different doses of 5F-MDMB-PICA. (b) Quantitative analysis of the migration data. 5F-MDMB-PICA doses (0.001 μM–1 μM demonstrated significantly higher migration rates compared to the control. Data were quantified from 3 experiments, where each was done in duplicates and presented as mean ± SEM (n = 3). (^**) means P > 0.01 and (^****) means P > 0.0001.

Fig. 5

The tubulogenic activity of HBMECs is increased by 5F-MDMB-PICA treatment. A tube-formation assay was performed to measure the effects of 5F-MDMB-PICA on the angiogenic capacity. BME-coated plates were seeded with 2 × 104 HBMECs suspended in SFM treated with 5F-MDMB-PICA (0.0001 μM, 0.01 μM, and 1 μM). (a) Microscopic images of tubular structures formed by HBMECs treated with 5F-MDMB-PICA after 24 hours of treatment. Significant increases have been observed in all angiogenic measurements including (b) the number of tube-like structures, (c) loops, (d) branch points, and (e) total tube lengths. Data presented as mean ± SEM (n = 3). (^***) means P > 0.001 and (^****) means P > 0.0001.

Fig. 6

5F-MDMB-PICA upregulates mRNA levels of proangiogenic factors VEGF, ANG-1, and ANG-2. RT-qPCR was performed on 5F-MDMB-PICA treated cells (0.0001 μM–1 μM) to quantify the expression level of (a) VEGF, (b) ANG-1, and (c) ANG-2 on the mRNA level. Data presented as mean ± SEM (n = 3). (^*) means P < 0.05, (^**) means P < 0.01, (^***) means P < 0.001, and (^****) means P < 0.0001.

Fig. 7

5F-MDMB-PICA treatment increases proangiogenic factors VEGF, ANG-1, ANG-2, and p-Ser9-GSK-3β protein levels in HBMECs. (a) Western blot bands images of GSK-3β, p-GSK-3β, VEGF, ANG-1, ANG-2, and β-actin isolated from HBMECs treated with 5F-MDMB-PICA. Western blot was performed on 5F-MDMB-PICA treated HBMECs (0.0001 μM, 0.01 μM, and 1 μM) to measure protein levels of VEGF, ANG-1, ANG-2, GSK-3β, and p-GSK-3β in the cells, while the reference protein used for the measurements was β-actin. Briefly, proteins were extracted using RIPA lysis buffer combined with phosphate-protease inhibitors followed by measuring the protein concentration of each sample. Then, SDS-PAGE was used to load 20 μg of proteins from each sample followed by blotting onto a polyvinylidene fluoride membrane. Blocking with 2% bovine serum albumin was performed before the addition of primary antibodies against the target proteins and overnight incubation. Finally, HRP-conjugate secondary antibodies were incubated with the membrane, and the signals were detected using enhanced chemiluminescence. Quantification of the expression rates of (b) VEGF, (c) ANG-1, (d) ANG-2, and (e) phospho-Ser9-GSK-3β. Data presented as mean ± SEM (n = 2). (^**) means P < 0.01 and (^****) means P < 0.0001.

Fig. 8

5F-MDMB-PICA treatment increases serum levels of VEGF, ANG-1, and ANG-2. ELISA was performed on 5F-MDMB-PICA treated HBMECs (0.0001 μM–1 μM) to measure secretion levels of proangiogenic factors VEGF, ANG-1, and ANG-2 in the media. Conditioned media from wells treated with concentrations had significantly higher secreted angiogenic factor levels compared to the control (a-c). Data presented as mean ± SEM (n = 3). (^****) means P < 0.0001.