PURPOSE: WIN55,212-2, a potent synthetic agonist of cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2), reduces atherosclerosis in apolipoprotein E (ApoE) null mice. Although pharmacologic evidence suggests the anti-atherosclerotic effects of WIN55,212-2 are mediated CB2, this remains to be confirmed by genetic studies. Therefore, in this study, we investigated the effects of WIN55,212-2 on development of atherosclerosis in low-density lipoprotein receptor (Ldlr) null mice with and without homozygous deletion of the CB2 gene. METHODS: After 6 weeks on an atherogenic diet, groups of CB2 and CB2 Ldlr-null mice received a daily intraperitoneal injection of WIN55,212-2 or vehicle. After two weeks, plasma lipid levels and atherosclerosis in the aortic root were quantified. RESULTS: Plasma cholesterol and triglyceride levels did not differ between CB2 and CB2 mice and WIN55,212-2 had no effect on total cholesterol levels in either genotype. However, triglyceride levels in both CB2 and CB2 mice were significantly lowered by WIN55,212-2. The size of aortic root lesions did not differ significantly between CB2 and CB2 mice with or without WIN55,212-2 treatment. However, WIN55,212-2 treatment significantly lowered lesional macrophage accumulation in CB2 mice, and lesional smooth muscle content in both CB2 and CB2 mice. Lesional apoptosis was also greater in CB2 mice compared to CB2mice, and only reduced by WIN55,212-2 in CB2 mice. Collagen content and elastin fiber fragmentation were unaffected by genotype or WIN55,212-2. CONCLUSIONS: WIN55,212-2 treatment does not alter lesion size in Ldlr null-mice, but does modify lesion cellularity CB2-dependent and CB2-independent mechanisms.

Studies have been conducted to highlight the anti-inflammatory and immunosuppressive properties of cannabinoids and also their potentials for cartilage repair and regeneration. Various wound healing techniques can be used to investigate the mechanisms of chondrocyte repair in monolayers or three dimensional tissue constructs. The effect of different concentrations of the synthetic cannabinoids WIN55, 212-2 (WIN-2), URB602 and HU-308 with and without their antagonists on the wound healing of chondrocyte monolayers was investigated using a simple scratch assay model. The three cannabinoids were found to increase wound healing of chondrocyte monolayers, but at different rates. WIN55, 212-2 at a concentration of 1μM had the highest effect of increasing both migration and proliferation of chondrocytes cultured in a chondrogenic media, which increased the rate of wound closure. It was also found that treating the cells with 2μM of any of the cannabinoids lead to a decrease in cell proliferation and the rate of wound closure. These findings were further investigated, by studying the effect of WIN-2 on nitric oxide (NO) and matrix metalloproteinase-2 (MMP-2) expressed by wounded chondrocyte monolayers. Moreover, expression of collagen type-I, collagen type-II, fibronectin and S100 proteins were detected using immunofluorescence and verified quantitatively using ELISA based techniques, following treatment with 1μM and 2μM of WIN-2, for both 2D monolayers and 3D sheets. Treating chondrocytes with 1μM of WIN-2 significantly increased collagen type-II, fibronectin and S100, and significantly reduced collagen type-I compared to control groups in monolayers and chondrocyte cell sheets. On the other hand, both concentrations of WIN-2 significantly reduced the expression of the inflammation markers NO, and MMP-2, in a dose dependent manner. These findings highlight the potential use of the synthetic cannabinoid for improving the rate of wound closure as well as acting as an antiinflammatory agent, which could be used to enhance tissue engineering protocols aimed at cartilage repair.

Studies have been conducted to highlight the anti-inflammatory and immunosuppressive properties of cannabinoids and also their potentials for cartilage repair and regeneration. Various wound healing techniques can be used to investigate the mechanisms of chondrocyte repair in monolayers or three dimensional tissue constructs. The effect of different concentrations of the synthetic cannabinoids WIN55, 212-2 (WIN-2), URB602 and HU-308 with and without their antagonists on the wound healing of chondrocyte monolayers was investigated using a simple scratch assay model. The three cannabinoids were found to increase wound healing of chondrocyte monolayers, but at different rates. WIN55, 212-2 at a concentration of 1μM had the highest effect of increasing both migration and proliferation of chondrocytes cultured in a chondrogenic media, which increased the rate of wound closure. It was also found that treating the cells with 2μM of any of the cannabinoids lead to a decrease in cell proliferation and the rate of wound closure. These findings were further investigated, by studying the effect of WIN-2 on nitric oxide (NO) and matrix metalloproteinase-2 (MMP-2) expressed by wounded chondrocyte monolayers. Moreover, expression of collagen type-I, collagen type-II, fibronectin and S100 proteins were detected using immunofluorescence and verified quantitatively using ELISA based techniques, following treatment with 1μM and 2μM of WIN-2, for both 2D monolayers and 3D sheets. Treating chondrocytes with 1μM of WIN-2 significantly increased collagen type-II, fibronectin and S100, and significantly reduced collagen type-I compared to control groups in monolayers and chondrocyte cell sheets. On the other hand, both concentrations of WIN-2 significantly reduced the expression of the inflammation markers NO, and MMP-2, in a dose dependent manner. These findings highlight the potential use of the synthetic cannabinoid for improving the rate of wound closure as well as acting as an antiinflammatory agent, which could be used to enhance tissue engineering protocols aimed at cartilage repair. / Egyptian Government

Yes / Studies have been conducted to highlight the anti-inflammatory and immunosuppressive properties of synthetic cannabinoids as well as their potential for cartilage repair. Various wound healing techniques can be used to investigate the mechanisms of chondrocyte repair in monolayers or three dimensional tissues constructs. In this work the effect of WIN55, 212-2 (WIN-2) on nitric oxide (NO) and matrix metalloproteinase-2 (MMP-2) expressed by wounded chondrocyte monolayers was investigated. Moreover, expression of collagen type-I and type-II, fibronectin and S100 proteins were detected using immunofluorescence and quantitatively verified using ELISA based techniques following treatment with 1 μM and 2 μM of WIN-2. Treating chondrocytes with 1 μM of WIN-2 significantly increased expression of collagen type-II, fibronectin and S100, and significantly reduced collagen type-I expressions as compared to the control groups. On the other hand, both concentrations of WIN-2 significantly reduced the expression of the inflammation markers NO and MMP-2 in a dose dependent manner. These findings highlight the potential use of the synthetic cannabinoids for improving cartilage healing properties as well as acting as an anti-inflammatory agent which could be used to enhance tissue engineering protocols aimed at cartilage repair.

The potential antitumor activity of mixed CB1/CB2 cannabinoid receptor agonists, such as the aminoalkylindole WIN55,212-2 (WIN2), has been extensively studied, but little information is available as to their potential interaction with conventional cancer therapies, such as ionizing radiation (IR). In the present work, we investigated the effects of WIN2 on the antiproliferative effects of radiation in human (MCF-7 and MDA-MB-231) and murine (4T1) breast cancer cells, as well as an immortalized human breast epithelial cell line (MCF-10A). WIN2 or radiation alone inhibited breast tumor growth, while the combination of WIN2 and radiation was more effective than either agent alone in breast cancer cells. WIN2 showed lower potency in MCF-10A cells than MCF-7 cells, but was still able to augment the effects of radiation at higher doses. The stereoisomer of WIN2, WIN55,212-3 (WIN3) failed to inhibit growth or potentiate the growth-inhibitory effects of radiation, indicating stereospecificity in all cell lines tested. The combination of WIN2 and IR was examined in vivo but the results were inconclusive. Interestingly, while other aminoalkylindoles, pravadoline and JWH-015, enhanced the antiproliferative effects of radiation, this was not the case for other synthetic cannabinoids (i.e., nabilone, CP55,940 and methanandamide) or phytocannabinoids (i.e., ∆9-tetrahydrocannabinol and cannabidiol). The antiproliferative actions of WIN2 were not ameliorated by CB1, CB2, TRPV1, or PPAR receptor antagonists, suggesting the possibility of a novel site of action. Studies utilizing sphingosine-1-phosphate (S1P) agonists and estradiol suggest that WIN2 interferes with S1P signaling in cell proliferation, but agonist stimulated [³⁵S]GTPγS binding assays show that this antagonism is not occurring at the level of S1P receptors. In addition, WIN2 did not alter radiation-induced DNA damage or the rate of DNA repair based on γH2AX staining. Treatment with WIN2 and radiation promoted both autophagy and senescence, but not apoptosis or necrosis. Time course studies combined with senescence and cell death data suggest that radiation-induced senescence, while WIN2 induced classical growth arrest and the WIN2/IR combination produced parallel mechanisms of both senescent growth arrest and classical growth arrest. Taken together, these findings raise the possibility that aminoalkylindole compounds targeting a novel site of action represents a potential strategy to augment the effectiveness of radiation treatment in breast cancer.