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.

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BACKGROUND AND PURPOSE Melanocortin MC1 and MC3 receptors, mediate the anti‐inflammatory effects of melanocortin peptides. Targeting these receptors could therefore lead to development of novel anti‐inflammatory therapeutic agents. We investigated the expression of MC1 and MC3 receptors on chondrocytes and the role of α‐melanocyte‐stimulating hormone (α‐MSH) and the selective MC3 receptor agonist, [DTRP8]‐γ‐MSH, in modulating production of inflammatory cytokines, tissue‐destructive proteins and induction of apoptotic pathway(s) in the human chondrocytic C‐20/A4 cells. EXPERIMENTAL APPROACH Effects of α‐MSH, [DTRP8]‐γ‐MSH alone or in the presence of the MC3/4 receptor antagonist, SHU9119, on TNF‐α induced release of pro‐inflammatory cytokines, MMPs, apoptotic pathway(s) and cell death in C‐20/A4 chondrocytes were investigated, along with their effect on the release of the anti‐inflammatory cytokine IL‐10. KEY RESULTS C‐20/A4 chondrocytes expressed functionally active MC1,3 receptors. α‐MSH and [DTRP8]‐γ‐MSH treatment, for 30 min before TNF‐α stimulation, provided a time‐and‐bell‐shaped concentration‐dependent decrease in pro‐inflammatory cytokines (IL‐1β, IL‐6 and IL‐8) release and increased release of the chondroprotective and anti‐inflammatory cytokine, IL‐10, whilst decreasing expression of MMP1, MMP3, MMP13 genes.α‐MSH and [DTRP8]‐γ‐MSH treatment also inhibited TNF‐α‐induced caspase‐3/7 activation and chondrocyte death. The effects of [DTRP8]‐γ‐MSH, but not α‐MSH, were abolished by the MC3/4 receptor antagonist, SHU9119. CONCLUSION AND IMPLICATIONS Activation of MC1/MC3 receptors in C‐20/A4 chondrocytes down‐regulated production of pro‐inflammatory cytokines and cartilage‐destroying proteinases, inhibited initiation of apoptotic pathways and promoted release of chondroprotective and anti‐inflammatory cytokines. Developing small molecule agonists to MC1/MC3 receptors could be a viable approach for developing chondroprotective and anti‐inflammatory therapies in rheumatoid and osteoarthritis.

Cartilage loss leads to osteoarthritis, the most common cause of disability for which there is no cure. Cartilage regeneration, therefore, is a priority in medicine. We report that agrin is a potent chondrogenic factor and that a single intraarticular administration of agrin induced long-lasting regeneration of critical-size osteochondral defects in mice, with restoration of tissue architecture and bone-cartilage interface. Agrin attracted joint resident progenitor cells to the site of injury and, through simultaneous activation of CREB and suppression of canonical WNT signaling downstream of β-catenin, induced expression of the chondrogenic stem cell marker GDF5 and differentiation into stable articular chondrocytes, forming stable articular cartilage. In sheep, an agrin-containing collagen gel resulted in long-lasting regeneration of bone and cartilage, which promoted increased ambulatory activity. Our findings support the therapeutic use of agrin for joint surface regeneration.

Arthritic pathologies are a major cause of morbidity within the western world, with rheumatoid arthritis affecting approximately 1% of adults. This review highlights the therapeutic potential of naturally occurring hormones and their peptides, in both arthritic models of disease and patients. The arthritides represent a group of closely related pathologies in which cytokines, joint destruction, and leukocytes play a causal role. Here we discuss the role of naturally occurring pro-opiomelanocortin (POMC)-derived melanocortin peptides (e.g., alpha melanocyte stimulating hormone [a-MSH]) and synthetic derivatives in these diseases. Melanocortins exhibit their biological efficacy by modulating proinflammatory cytokines and subsequent leukocyte extravasation. Their biological effects are mediated via seven transmembrane G-protein-coupled receptors, of which five have been cloned, identified, and termed MC1 to MC5. Adrenocorticotrophic hormone represents the parent molecule of the melanocortins; the first 13 amino acids of which (termed a-MSH) have been shown to be the most pharmacologically active region of the parent hormone. The melanocortin peptides have been shown to display potent anti-inflammatory effects in both animal models of disease and patients. The potential anti-inflammatory role for endogenous peptides in arthritic pathologies is in its infancy. The ability to inhibit leukocyte migration, release of cytokines, and induction of anti-inflammatory proteins appears to play an important role in affording protection in arthritic injury, and thus may lead to potential therapeutic targets.

We hypothesized that exudates collected at the beginning of the resolution phase of inflammation might be enriched for tissue protective molecules; thus an integrated cellular and molecular approach was applied to identify novel chondroprotective bioactions. Exudates were collected 6 h (inflammatory) and 24 h (resolving) following carrageenan-induced pleurisy in rats. The resolving exudate was subjected to gel filtration chromatography followed by proteomics, identifying 61 proteins. Fractions were added to C28/I2 chondrocytes, grown in micromasses, ions with or without IL-1β or osteoarthritic synovial fluids for 48 h. Three proteins were selected from the proteomic analysis, α1-antitrypsin (AAT), hemopexin (HX), and gelsolin (GSN), and tested against catabolic stimulation for their effects on glycosaminoglycan deposition as assessed by Alcian blue staining, and gene expression of key anabolic proteins by real-time PCR. In an in vivo model of inflammatory arthritis, cartilage integrity was determined histologically 48 h after intra-articular injection of AAT or GSN. The resolving exudate displayed protective activities on chondrocytes, using multiple readouts: these effects were retained in low m.w. fractions of the exudate (46.7% increase in glycosaminoglycan deposition; ∼20% upregulation of COL2A1 and aggrecan mRNA expression), which reversed the effect of IL-1β. Exogenous administration of HX, GSN, or AAT abrogated the effects of IL-1β and osteoarthritic synovial fluids on anabolic gene expression and increased glycosaminoglycan deposition. Intra-articular injection of AAT or GSN protected cartilage integrity in mice with inflammatory arthritis. In summary, the strategy for identification of novel chondroprotective activities in resolving exudates identified HX, GSN and AAT as potential leads for new drug discovery programs.

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