C. Di Filippo

Association of CNR1 and FAAH endocannabinoid gene polymorphisms with anorexia nervosa and bulimia nervosa: evidence for synergistic effects

Endocannabinoids modulate eating behavior; hence, endocannabinoid genes may contribute to the biological vulnerability to eating disorders. The rs1049353 (1359 G/A) single nucleotide polymorphism (SNP) of the gene coding the endocannabinoid CB1 receptor (CNR1) and the rs324420 (cDNA 385C to A) SNP of the gene coding fatty acid amide hydrolase (FAAH), the major degrading enzyme of endocannabinoids, have been suggested to have functional effects on mature proteins. Therefore, we explored the possibility that those SNPs were associated to anorexia nervosa and/or bulimia nervosa. The distributions of the CNR1 1359 G/A SNP and of the FAAH cDNA 385C to A SNP were investigated in 134 patients with anorexia nervosa, 180 patients with bulimia nervosa and 148 normal weight healthy controls. Additive effects of the two SNPs in the genetic susceptibility to anorexia nervosa and bulimia nervosa were also tested. As compared to healthy controls, anorexic and bulimic patients showed significantly higher frequencies of the AG genotype and the A allele of the CNR1 1359 G/A SNP. Similarly, the AC genotype and the A allele of the FAAH cDNA 385C to A SNP were significantly more frequent in anorexic and bulimic individuals. A synergistic effect of the two SNPs was evident in anorexia nervosa but not in bulimia nervosa. Present findings show for the first time that the CNR1 1359 G/A SNP and the FAAH cDNA 385C to A SNP are significantly associated to anorexia nervosa and bulimia nervosa, and demonstrate a synergistic effect of the two SNPs in anorexia nervosa.

MC-3 receptor and the inflammatory mechanisms activated in acute myocardial infarct

Investigation of the mechanisms activated by endogenous inhibitory pathways can lead to identification of novel targets for cardiovascular inflammatory pathologies. Here we exploited the potential protective role that melanocortin receptor type 3 (MC3‐R) activation might play in a myocardial ischemia‐reperfusion injury model. In resting conditions, mouse and rat heart extracts expressed MC3‐R mRNA and protein, without changes following ischemia‐reperfusion. At the cellular level heart macrophages, but not fibroblasts or cardiomyocytes, expressed this receptor, as demonstrated by immunogold labeling. In vivo, administration of the melanocortin agonist MTII (10 μg per mouse equivalent to 9.3 nmol) 30 min prior to ischemia (25 min) attenuated mouse heart 2 h reperfusion injury by ∼40%, an effect prevented by the mixed MC3/4‐R antagonist SHU9119 but not by the selective MC4‐R antagonist HS204. Similar results were obtained when the compound was given at the beginning of the reperfusion period. Importantly, delayed myocardial damage as measured 24 h post‐reperfusion was equally protected by administration of 10 μg MTII. The focus on MC3‐R was also substantiated by analysis of the recessive yellow (e/e) mouse, bearing a mutated (inactive) MC1‐R, in which MTII was fully protective. Myocardial protection was associated with reduced markers of systemic and local inflammation, including cytokine contents (interleukin‐1 and KC) and myeloperoxidase activity. In conclusion, this study has highlighted a previously unrecognized protective role for MC3‐R activation on acute and delayed heart reperfusion injury. These data may open new avenues for therapeutic intervention against heart and possibly other organ ischemia‐reperfusion injury.

Protection from Endotoxic Uveitis by Intravitreal Resolvin D1: Involvement of Lymphocytes, miRNAs, Ubiquitin-Proteasome, and M1/M2 Macrophages

This study investigated the protective effects of intravitreal Resolvin D1 (RvD1) against LPS-induced rat endotoxic uveitis (EIU). RvD1 was administered into the right eye at a single injection of 5 μL volume containing 10–100–1000 ng/kg RvD1 1 h post-LPS injection (200 μg, Salmonella minnesota) into thefootpad of Sprague-Dawley rats. 24 h later, the eye was enucleated and examined for clinical, biochemical, and immunohistochemical evaluations. RvD1 significantly and dose-dependently decreased the clinical score attributed to EIU, starting from the dose of 10 ng/kg and further decreased by 100 and 1000 ng/kg. These effects were accompanied by changes in four important determinants of the immune-inflammatory response within the eye: (i) the B and T lymphocytes, (ii) the miRNAs pattern, (iii) the ubiquitin-proteasome system (UPS), and (iv) the M1/M2 macrophage phenotype. LPS+RvD1 treated rats showed reduced presence of B and T lymphocytes and upregulation of miR-200c-3p, miR 203a-3p, miR 29b-3p, and miR 21-5p into the eye compared to the LPS alone. This was paralleled by decreases of the ubiquitin, 20S and 26S proteasome subunits, reduced presence of macrophage M1, and increased presence of macrophage M2 in the ocular tissues. Accordingly, the levels of the cytokine TNF-α, the chemokines MIP1-α and NF-κB were reduced.

Activation of Melanocortin Receptors MC1 and MC5 Attenuates Retinal Damage in Experimental Diabetic Retinopathy

We hypothesize that melanocortin receptors (MC) could activate tissue protective circuit in a model of streptozotocin- (STZ-) induced diabetic retinopathy (DR) in mice. At 12–16 weeks after diabetes induction, fluorescein angiography (FAG) revealed an approximate incidence of 80% microvascular changes, typical of DR, in the animals, without signs of vascular leakage. Occludin progressively decreased in the retina of mice developing retinopathy. qPCR of murine retina revealed expression of two MC receptors, Mc1r and Mc5r. The intravitreal injection (5 μL) of the selective MC1 small molecule agonist BMS-470539 (33 μmol) and the MC5 peptidomimetic agonist PG-901 (7.32 nM) elicited significant protection with regular course and caliber of retinal vessels, as quantified at weeks 12 and 16 after diabetes induction. Mouse retina homogenate settings indicated an augmented release of IL-1α, IL-1β, IL-6, MIP-1α, MIP-2α, MIP-3α, and VEGF from diabetic compared to nondiabetic mice. Application of PG20N or AGRP and MC5 and MC1 antagonist, respectively, augmented the release of cytokines, while the agonists BMS-470539 and PG-901 almost restored normal pattern of these mediators back to nondiabetic values. Similar changes were quantified with respect to Ki-67 staining. Finally, application of MC3-MC4 agonist/antagonists resulted to be inactive with respect to all parameters under assessment.

Interplay between Intravitreal RvD1 and Local Endogenous Sirtuin-1 in the Protection from Endotoxin-Induced Uveitis in Rats

Rat endotoxin-induced uveitis (EIU) is a well-established model of human uveitis. In this model, intravitreal injection of resolvin D1 (RvD1, 10–100–1000 ng/kg) 1 hour after subcutaneous treatment of Sprague-Dawley rats with lipopolysaccharide (LPS, 200 μg/rat) significantly prevented the development of uveitis into the eye. RvD1 dose-dependently increased the expression of sirtuin-1 (SIRT1) within the eye, while it decreased the expression of acetyl-p53 and acetyl-FOXO1. These effects were accompanied by local downregulation of some microRNAs related to the expression and activity of SIRT1. These were miR-195-5p, miR-200a-3p, miR-34a-5p, and miR-145-5p. An increase of manganese superoxide dismutase and decrease of caspase 3 were evident after RvD1 treatment. In another set of experiments, the protective effects of RvD1 (1000 ng/kg) were partly abolished by the pretreatment of the rats with EX527 (10 mg/kg/day, i.p.), a specific inhibitor of SIRT1 activity, for 7 days prior to the induction of EIU in rats. Similarly, the effects of RvD1 (1000 ng/kg) on the SIRT1 protein expression were abolished by Boc2, N-t-butoxycarbonyl-PLPLP, a specific formyl-peptide receptor type 2/lipoxin A receptor antagonist. Therefore, an interplay of the SIRT1 activity on the RvD1 mediated resolution of EIU is argued.

Characterizing the anti-inflammatory and tissue protective actions of a novel Annexin A1 peptide.

Inflammation in now appreciated to be at the centre of may diseases that affect Western civilization. Current therapeutics for managing these conditions may interfere with the host response leading to immune suppression. We recently developed an annexin (Anx) A1-derived peptide, coined CR-AnxA12-50, which displays potent pro-resolving and tissue protective actions. Herein, we designed a novel peptide using CR-AnxA12-50 as a template that was significantly more resistant to neutrophil-mediated degradation. This peptide, termed CR-AnxA12-48, retained high affinity and specificity to the pro-resolving Lipoxin A4 receptor (ALX) with an IC50 of ~20nM. CR-AnxA12-48 dose dependently (100fM-10nM) promoted the efferocytosis of apoptotic neutrophils, an action that was mediated by the murine orthologue of human ALX. The neutrophil-directed actions were also retained with human primary cells were CR-AnxA12-48 reduced human neutrophil recruitment to activated endothelial cells at concentrations as low as 100 pM. This protective action was mediated by human ALX, since incubation of neutrophils with an anti-ALX antibody reversed this anti-inflammatory actions of CR-AnxA12-48. Administration of this peptide to mice during dermal inflammation led to a significant and dose dependent decrease in neutrophil recruitment. This reduction in neutrophil numbers was more pronounced than that displayed by the parent peptide CR-AnxA12-50. CR-AnxA12-48 was also cardioprotecitve reducing infarct size and systemic chemokine (C-C motif) ligand 5 concentration following ischemia reperfusion injury. These findings identify CR-AnxA12-48 as a new ALX agonist that regulates phagocyte responses and displays tissue-protective actions.

Effects of Timolol and of Timolol with Tamarind Seed Polysaccharide on Intraocular Pressure in Rabbits

Tamarind seed polysaccharide (TS-P) has high viscosity and mucoadhesive properties which make it a suitable candidate for addition to ophthalmic solutions of β-adrenergic blockers to increase the residence time on the cornea. The effect of an ophthalmic preparation containing timolol and TS-P on intraocular pressure (IOP) was evaluated in rabbits. Ocular administration of 0.5% timolol or 0.5% timolol with 1 and 2% TS-P, into the conjunctival sac had no effect on normal IOP, but significantly reduced betamethasone-induced ocular hypertension, over 20 days. The effect lasted up to 12 h after treatment with timolol + TS-P, and was still present on day 20 of treatment. Timolol in association with TS-P had a prolonged duration of action and is suitable for ocular administration in cases of elevated IOP.