Mast Cell/Proteinase Activated Receptor 2 (PAR2) Mediated Interactions in the Pathogenesis of Discogenic Back Pain.

Mast cells (MCs) are present in the painful degenerate human intervertebral disc (IVD) and are associated with disease pathogenesis. MCs release granules containing enzymatic and inflammatory factors in response to stimulants or allergens. The serine protease, tryptase, is unique to MCs and its activation of the G-protein coupled receptor, Protease Activated Receptor 2 (PAR2), induces inflammation and degradation in osteoarthritic cartilage. Our previously published work has demonstrated increased levels of MC marker tryptase in IVD samples from discogenic back pain patients compared to healthy control IVD samples including expression of chemotactic agents that may facilitate MC migration into the IVD. To further elucidate MCs' role in the IVD and mechanisms underlying its effects, we investigated whether (1) human IVD cells can promote MC migration, (2) MC tryptase can mediate up-regulation of inflammatory/catabolic process in human IVD cells and tissue, and (3) the potential of PAR2 antagonist to function as a therapeutic drug in in vitro human and ex vivo bovine pilot models of disease. MC migration was quantitatively assessed using conditioned media from primary human IVD cells and MC migration examined through Matrigel. Exposure to soluble IVD factors significantly enhanced MC migration, suggesting IVD cells can recruit MCs. We also demonstrated significant upregulation of MC chemokine SCF and angiogenic factor VEGFA gene expression in human IVD cells in vitro in response to recombinant human tryptase, suggesting tryptase can enhance recruitment of MCs and promotion of angiogenesis into the usually avascular IVD. Furthermore, tryptase can degrade proteoglycans in IVD tissue as demonstrated by significant increases in glycosaminoglycans released into surrounding media. This can create a catabolic microenvironment compromising structural integrity and facilitating vascular migration usually inhibited by the anti-angiogenic IVD matrix. Finally, as a "proof of concept" study, we examined the therapeutic potential of PAR2 antagonist (PAR2A) on human IVD cells and bovine organ culture IVD model. While preliminary data shows promise and points toward structural restoration of the bovine IVD including down-regulation of VEGFA, effects of PAR2 antagonist on human IVD cells differ between gender and donors suggesting that further validation is required with larger cohorts of human specimens.

Inhibition of hyperglycemia-induced angiogenesis and breast cancer tumor growth by systemic injection of microRNA-467 antagonist.

Abnormal angiogenesis in multiple tissues is a key characteristic of the vascular complications of diabetes. However, angiogenesis may be increased in one tissue but decreased in another in the same patient at the same time point in the disease. The mechanisms of aberrant angiogenesis in diabetes are not understood. There are no selective therapeutic approaches to target increased neovascularization without affecting physiologic angiogenesis and angiogenesis in ischemic tissues. We recently reported a novel miRNA-dependent pathway that up-regulates angiogenesis in response to hyperglycemia in a cell- and tissue-specific manner. The goal of the work described herein was to test whether systemic administration of an antagonist of miR-467 would prevent hyperglycemia-induced local angiogenesis in a tissue-specific manner. We examined the effect of the antagonist on hyperglycemia-induced tumor growth and angiogenesis and on skin wound healing in mouse models of diabetes. Our data demonstrated that the systemic injection of the antagonist prevented hyperglycemia-induced angiogenesis and growth of mouse and human breast cancer tumors, where the miR-467 pathway was active in hyperglycemia. In tissues where the miR-467-dependent mechanism was not activated by hyperglycemia, there was no effect of the antagonist: the systemic injection did not affect skin wound healing or the growth of prostate tumors. The data show that systemic administration of the miR-467 antagonist could be a breakthrough approach in the treatment and prevention of diabetes-associated breast cancer in a tissue-specific manner without affecting physiologic angiogenesis and angiogenesis in ischemic tissues.

Control of organization and function of muscle and tendon by thrombospondin-4.

Thrombospondins (TSPs) are multifunctional proteins that are deposited in the extracellular matrix where they directly affect the function of vascular and other cell types. TSP-4, one of the 5 TSP family members, is expressed abundantly in tendon and muscle. We have examined the effect of TSP-4 deficiency on tendon collagen and skeletal muscle morphology and function. In Thbs4(-/-) mice, tendon collagen fibrils are significantly larger than in wild-type mice, and there is no compensatory over-expression of TSP-3 and TSP-5, the two TSPs most highly homologous to TSP-4, in the deficient mice. TSP-4 is expressed in skeletal muscle, and higher levels of TSP-4 protein are associated with the microvasculature of red skeletal muscle with high oxidative metabolism. Lack of TSP-4 in medial soleus, red skeletal muscle with predominant oxidative metabolism, is associated with decreased levels of several specific glycosaminoglycan modifications, decreased expression of a TGFß receptor beta-glycan, decreased activity of lipoprotein lipase, which associates with vascular cell surfaces by binding to glycosaminoglycans, and decreased uptake of VLDL. The soleus muscle is smaller and hind- and fore-limb grip strength is reduced in Thbs4(-/-) mice compared to wild-type mice. These observations suggest that TSP-4 regulates the composition of the ECM at major sites of its deposition, tendon and muscle, and the absence of TSP-4 alters the organization, composition and physiological functions of these tissues.