Synthetic cannabinoid ajulemic acid exerts potent antifibrotic effects in experimental models of systemic sclerosis.

BACKGROUND: Cannabinoids modulate fibrogenesis in scleroderma. Ajulemic acid (AjA) is a non-psychoactive synthetic analogue of tetrahydrocannabinol that can bind the peroxisome proliferator-activated receptor-γ (PPAR-γ). Recent evidence suggests a key role for PPAR-γ in fibrogenesis. OBJECTIVE: To determine whether AjA can modulate fibrogenesis in murine models of scleroderma. MATERIAL AND METHODS: Bleomycin-induced experimental fibrosis was used to assess the antifibrotic effects of AjA in vivo. In addition, the efficacy of AjA in pre-established fibrosis was analysed in a modified model of bleomycin-induced dermal fibrosis and in mice overexpressing a constitutively active transforming growth factor ß (TGFß) receptor I. Skin fibrosis was evaluated by quantification of skin thickness and hydroxyproline content. As a marker of fibroblast activation, α-smooth muscle actin was examined. To study the direct effect of AjA in collagen neosynthesis, skin fibroblasts from patients with scleroderma were treated with increasing concentrations of AjA. Protein expression of PPAR-γ, and its endogenous ligand 15d-PGJ2, and TGFß were assessed before and after AjA treatment. RESULTS: AjA significantly prevented experimental bleomycin-induced dermal fibrosis and modestly reduced its progression when started 3 weeks into the disease. AjA strongly reduced collagen neosynthesis by scleroderma fibroblasts in vitro, an action which was reversed completely by co-treatment with a selective PPAR-γ antagonist. CONCLUSIONS: AjA prevents progression of fibrosis in vivo and inhibits fibrogenesis in vitro by stimulating PPAR-γ signalling. Since therapeutic doses of AjA are well tolerated in humans, it is suggested that AjA as an interesting molecule targeting fibrosis in patients with scleroderma.

The cannabinoid WIN55, 212-2 abrogates dermal fibrosis in scleroderma bleomycin model.

OBJECTIVES: There is increasing evidence that the endocannabinoid system may be involved in pathological fibrosis, and that its modulation might limit fibrotic responses. The aim of this study was to examine the capacity of a synthetic cannabinoid receptor agonist to modify skin fibrosis in the bleomycin mouse model of scleroderma. METHODS: Skin fibrosis was induced by local injections of bleomycin in two groups of DBA/2J mice. One group was cotreated with the synthetic cannabinoid WIN55,212-2 at 1 mg/kg/day. Skin fibrosis was evaluated by histology and skin thickness and hydroxyproline content were quantified. Markers of fibroblast activation, including α smooth muscle actin and the profibrotic cytokines transforming growth factor (TGF)ß, connective tissue growth factor (CTGF) and platelet-derived growth factor (PDGF)-BB, were examined. Levels of PSMAD2/3, which are crucial in extracellular matrix overproduction, were analysed. RESULTS: Bleomycin treatment induced typical skin fibrosis. Upon WIN55,212-2 treatment dermal fibrosis was completely prevented. Subcutaneous inflammatory cell infiltration, dermal thickness and collagen content resulted similar to those of the control group. The synthetic cannabinoid prevented fibroblasts activation induced by bleomycin, paralleled by a strong inhibition of TGFß, CTGF and PDGF-BB expression. Phosphorylation of SMAD2/3 was significantly downregulated after WIN55,212-2 exposure. CONCLUSIONS: Taken together, the results indicate that the synthetic cannabinoid WIN55,212-2 is capable of preventing skin fibrosis in a mouse model of scleroderma.

Cannabinoids inhibit fibrogenesis in diffuse systemic sclerosis fibroblasts.

OBJECTIVE: It has been demonstrated that the endocannabinoid system is up-regulated in pathologic fibrosis and that modulation of the cannabinoid receptors might limit the progression of uncontrolled fibrogenesis. The aim of this study was to investigate whether the synthetic cannabinoid receptor agonist WIN55,212-2 could modulate fibrogenesis in an in vitro model of dcSSc. METHODS: The expression of cannabinoid receptors CB1 and CB2 was assessed in dcSSc fibroblasts and healthy control fibroblasts. To investigate the effect of WIN55,212-2 on dcSSc fibrogenesis, we studied type I collagen, profibrotic cytokines, fibroblast transdifferentiation into myofibroblasts, apoptotic processes and activation of the extracellular signal-related kinase 1/2 pathway prior to and after the treatment with the synthetic cannabinoid at increasing concentrations. RESULTS: Both CB1 and CB2 receptors were over-expressed in dcSSc fibroblasts compared with healthy controls. WIN55,212-2 caused a reduction in extracellular matrix deposition and counteracted several behavioural abnormalities of scleroderma fibroblasts including transdifferentiation into myofibroblasts and resistance to apoptosis. The anti-fibrogenic effect of WIN55,212-2 was not reverted by selective cannabinoid antagonists. CONCLUSIONS: Our preliminary findings suggest that cannabinoids are provided with an anti-fibrotic activity, thereby possibly representing a new class of agents targeting fibrosis diseases.

Cloning of a rat-specific long PCP4/PEP19 isoform.

We report the identification of a cDNA that encodes a putative protein of 94 amino acids and expected molecular weight of 10.7 kDa, the C-terminal half of which is identical to that of PEP19, a small, brain-specific protein involved in Ca++/calmodulin signaling. The novel rat-specific protein, tentatively named long PEP19 isoform (LPI), is the product of alternative splicing of the rat PCP4 gene encoding PEP19. We found that antibodies raised against the first 13 N-terminal amino acids of LPI, not present in PEP19, recognize a protein enriched in the developing rat brain.

Adenosine A2A receptor activation stimulates collagen production in sclerodermic dermal fibroblasts either directly and through a cross-talk with the cannabinoid system.

Systemic sclerosis (SSc) is a connective tissue disease characterised by exaggerated collagen deposition in the skin and visceral organs. Adenosine A2A receptor stimulation (A2Ar) promotes dermal fibrosis, while the cannabinoid system modulates fibrogenesis in vitro and in animal models of SSc. Moreover, evidence in central nervous system suggests that A2A and cannabinoid (CB1) receptors may physically and functionally interact. On this basis, we investigated A2Ar expression and function in modulating collagen biosynthesis from SSc dermal fibroblasts and analysed the cross-talk with cannabinoid receptors. In sclerodermic cells, A2Ar expression (RT-PCR, Western blotting) was evaluated together with the effects of A2A agonists and/or antagonists on collagen biosynthesis (EIA, Western blotting). Putative physical and functional interactions between the A2A and cannabinoid receptors were respectively assessed by co-immuno-precipitation and co-incubating the cells with the unselective cannabinoid agonist WIN55,212-2, and the selective A2A antagonist ZM-241385. In SSc fibroblasts, (1) the A2Ar is overexpressed and its occupancy with the selective agonist CGS-21680 increases collagen production, myofibroblast trans-differentiation, and ERK-1/2 phosphorylation; (2) the A2Ar forms an heteromer with the cannabinoid CB1 receptor; and (3) unselective cannabinoid receptor stimulation with a per se ineffective dose of WIN55,212-2, results in a marked anti-fibrotic effect after A2Ar blockage. In conclusion, A2Ar stimulation induces a pro-fibrotic phenotype in SSc dermal fibroblasts, either directly, and indirectly, by activating the CB1 cannabinoid receptor. These findings increase our knowledge of the pathophysiology of sclerodermic fibrosis also further suggesting a new therapeutic approach to the disease.