Magdalena Kaneva

A role for MC3R in modulating lung inflammation

In this study we set out to ascertain whether melanocortin peptides could be potential therapeutic agents in allergic and non-allergic models of lung inflammation by identifying the receptor(s) involved using a molecular, genetic and pharmacological approach. Western blot analyses revealed expression of the melanocortin receptor (MCR) type 1 and 3 on alveolar macrophages from wild-type mice. Alveolar macrophage incubation, with the selective MC3R agonist [D-TRP(8)]-gamma-MSH and pan-agonist alpha-MSH but not the selective MC1R agonist MS05, led to an increase in cAMP in wild-type macrophages. This increase occurred also in macrophages taken from recessive yellow (e/e; bearing a mutant and inactive MC1R) mice but not from MC3R-null mice. In an allergic model of inflammation, the pan-agonist alpha-MSH and selective MC3R agonist [D-TRP(8)]-gamma-MSH displayed significant attenuation of both eosinophil and lymphocyte accumulation but not IL-5 levels in wild-type and recessive yellow e/e mice. However in MC3R-null mice, alpha-MSH failed to cause a significant inhibition in these parameters, highlighting a preferential role for MC3R in mediating the anti-inflammatory effects of melanocortins in this model. Utilising a non-allergic model of LPS-induced lung neutrophilia, the pan-agonist alpha-MSH and selective MC3R agonist [D-TRP(8)]-gamma-MSH displayed significant attenuation of neutrophil accumulation and inhibition of TNF-alpha release. Thus, this study highlights that melanocortin peptides inhibit leukocyte accumulation in a model of allergic and non-allergic inflammation and this protective effect is associated with activation of the MC3R. The inhibition of leukocyte accumulation is via inhibition of TNF-alpha in the non-allergic model of inflammation but not IL-5 in the allergic model. These data have highlighted the potential for selective MC3R agonists as novel anti-inflammatory therapeutics in lung inflammation.

Chondroprotective and anti-inflammatory role of melanocortin peptides in TNF-α activated human C-20/A4 chondrocytes

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.

Melanocortin Peptide Therapy for the Treatment of Arthritic Pathologies

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.

Melanocortin peptides protect chondrocytes from mechanically induced cartilage injury

INTRODUCTION Mechanical injury can greatly influence articular cartilage, propagating inflammation, cell injury and death - risk factors for the development of osteoarthritis. Melanocortin peptides and their receptors mediate anti-inflammatory and pro-resolving mechanisms in chondrocytes. This study aimed to investigate the potential chondroprotective properties of α-MSH and [DTRP(8)]-γ-MSH in mechanically injured cartilage explants, their ability to inhibit pro-inflammatory and stimulate anti-inflammatory cytokines in in situ and in freshly isolated articular chondrocytes. METHODS The effect of melanocortins on in situ chondrocyte viability was investigated using confocal laser scanning microscopy of bovine articular cartilage explants, subjected to a single blunt impact (1.14N, 6.47 kPa) delivered by a drop tower. Chondroprotective effects of α-MSH, [DTRP(8)]-γ-MSH and dexamethasone on cytokine release by TNF-α-activated freshly isolated articular chondrocytes/mechanically injured cartilage explants were investigated by ELISA. RESULTS A single impact to cartilage caused discreet areas of chondrocyte death, accompanied by pro-inflammatory cytokine release; both parameters were modulated by α-MSH, [DTRP(8)]-γ-MSH and dexamethasone. Melanocortin pre-treatment of TNF-α-stimulated freshly isolated chondrocytes resulted in a bell-shaped inhibition in IL-1β, IL-6 and IL-8, and elevation of IL-10 production. The MC3/4 antagonist, SHU9119, abrogated the effect of [DTRP(8)]-γ-MSH but not α-MSH on cytokine release. CONCLUSION Melanocortin peptide pre-treatment prevented chondrocyte death following mechanical impact to cartilage and led to a marked reduction of pro-inflammatory cytokines, whilst prompting the production of anti-inflammatory/pro-resolving cytokine IL-10. Development of small molecule agonists towards melanocortin receptors could thus be a viable approach for preventing chondrocyte inflammation and death within cartilage and represent an alternative approach for the treatment of osteoarthritis.

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.

Analyses on the mechanisms that underlie the chondroprotective properties of calcitonin

INTRODUCTION Calcitonin (CT) has recently been shown to display chondroprotective effects. Here, we investigate the putative mechanisms by which CT delivers these actions. METHODS Immortalized C-28/I2 cells or primary adult human articular chondrocytes (AHAC) were cultured in high-density micromasses to investigate: (i) CT anabolic effects using qPCR and immuhistochemistry analysis; (ii) CT anti-apoptotic effects using quantitation of Bax/Bcl gene products ratio, TUNEL assay and caspase-3 expression; (iii) CT effects on CREB, COL2A1 and NFAT transcription factors. RESULTS CT (10(-10)-10(-8)nM) induced significant up-regulation of cartilage phenotypic markers (SOX9, COL2A1 and ACAN), with down-regulation of catabolic (MMP1 and MMP13 and ADAMTS5) gene products both in resting and inflammatory conditions. This was mirrored by an augmented production of type II collagen and accumulation of glycosaminoglycan- and proteoglycan-rich extracellular matrix in vitro. Mechanistic analyses revealed only partial involvement of cyclic AMP formation in these effects of CT. Congruently, using reporter assays for specific transcription factors, there was no indication for CREB activation, whereas the COL2A1 promoter was genuinely and directly activated by cell exposure to CT. Phenotypically, these mechanisms supported the ability of CT, whilst inactive on its own, to counteract the pro-apoptotic effects of IL-1β, demonstrated by TUNEL-positive staining of chondrocytes and ratio of BAX/BCL genes products. CONCLUSION These data may provide a novel lead for the development of CT-based chondroprotective strategies that rely on the engagement of mechanisms that lead to augmented chondrocyte anabolism and inhibited chondrocyte apoptosis.

Identification of Novel Chondroprotective Mediators in Resolving Inflammatory Exudates.

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.

The Phenotypic Characterization of A13/BACii, a Novel Bovine Chondrocytic Cell Line with Differentiation Potential

In cartilage research bovine articular cartilage is used as an alternative to human tissue. However, animal material is subject to availability and primary cultures undergo senescence, limiting their use. Here we report the immortalization of primary bovine chondrocytes, which could be used as a surrogate for freshly isolated chondrocytes. Chondrocytes were isolated from cartilage explants and immortalized using 1.0 µg/ml benzo[alpha]pyrene. For 3-dimensional culture, chondrocytes were resuspended in 0.5% low-melt agarose at high density (HD) and cultured for 24 h prior to determining changes in expression profile and morphology. A13/BACii chondrocytes acquired a ‘flat’ irregular morphology and a foetal-like cell volume (1,509.59 ± 182.04 µm3). The human cell line C-20/A4 showed a statistically similar volume and length to A13/BACii. Two-dimensional-cultured A13/BACii expressed elevated levels of type I collagen (col1), reduced levels of type II collagen (col2) compared to freshly isolated chondrocytes and an overall col2 to col1 expression ratio (col2:col1) of 0.11 ± 0.01. Upon 3-dimensional encapsulation, there was a significant rise in col2 expression in both A13/BACii and C-20/A4, suggesting a capacity for redifferentiation in both cell lines with a return of col2:col1 values of A13/BACii to values previously observed in primary chondrocytes. A13/BACii chondrocytes expressed aggrecan, matrix metalloproteinase (MMP)-3, MMP-9 and MMP-13, further supporting indications of the differentiated phenotype. Here we report the creation of a novel chondrocytic cell line and demonstrate its strong potential for redifferentiation upon HD 3-dimensional encapsulation, providing an alternative to conventional dedifferentiated cell lines and primary culture.