AM251, a cannabinoid receptor 1 antagonist, prevents human fibroblasts differentiation and collagen deposition induced by TGF-ß - An in vitro study.

Previous studies showed that cannabinoid 1 receptor (CB1) is linked with skin fibrosis and scar tissue formation in mice. Therefore, the topical use of cannabinoids may have a role in the prevention or treatment of local fibrotic and wound healing diseases as hypertrophic scars or keloids. In this study, we asked whether CB1 activation or inactivation would change fibroblast differentiation into myofibroblast and collagen deposition in skin human fibroblast. Primary cultures of adult human fibroblasts were obtained from abdominal human skin. Cells were stimulated with transforming growth factor-beta (TGF-ß, 10ng/ml) and treated with a CB1 selective agonist (arachidonyl-2-chloroethylamide, ACEA 1 µM) and an antagonist (AM251 1, 5 and 10 µM). Alpha-smooth muscle actin (α-SMA) was quantified using Immunocytochemistry and Western Blot. Collagen was quantified with Sirius Red staining assay. Significance was assessed by One-way ANOVA. P < 0.05 was considered significant. TGF-ß significantly increases α-SMA expression. ACEA 1 µM significantly increases collagen deposition but does not change α-SMA expression. AM251 10 µM added in the absence and the presence of ACEA reduces α-SMA expression and collagen content in TGF-ß treated cells. AM251 shows a concentration-dependent effect over collagen deposition with a pIC50 of 5.5 (4.6-6.4). TGF-ß significantly increases CB1 receptor expression. CB1 inactivation with AM251 prevents fibroblasts differentiation and collagen deposition, induced by TGF-ß in human fibroblasts. The outcome supports that CB1 is a molecular target for wound healing disorders and in vivo and pre-clinical studies should be implemented to clarify this premise.

A new role for anandamide: defective link between the systemic and skin endocannabinoid systems in hypertrophic human wound healing.

The use of cannabinoids to treat fibrotic skin diseases is an emergent issue. Therefore, we aimed to evaluate systemic and skin endocannabinoid responses in the wound-healing process in humans. A prospective study was performed in 50 patients who underwent body-contouring surgery. Anandamide (N-arachidonoylethanolamine, AEA), 2-arachidonoylglycerol (2-AG), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) were quantified using LC-MS/MS. Ten (20%) patients developed hypertrophic (HT) scars. No significant changes were observed between the normal (N) scar and HT scar groups in terms of plasma and skin endocannabinoids. Nevertheless, a positive correlation between plasma and skin AEA concentrations was found in the N group (r = 0.38, p = 0.015), which was absent in the HT group. Moreover, the AEA concentration was significantly lower in HT scar tissue than in normal scar tissue (0.77 ± 0.12 ng/g vs 1.15 ± 0.15 ng/g, p < 0.001). Interestingly, in all patients, the surgical intervention produced a time-dependent effect with a U shape for AEA, PEA and OEA plasma concentrations. In contrast, 2-AG plasma concentrations increased 5 days after surgery and were reduced and stabilized 3 months later. These results suggest crosstalk between systemic and local skin endocannabinoid systems during human wound healing. AEA appears to be the most likely candidate for this link, which is deficient in patients with HT scars.