The activated mesangial cell is critically involved in both the acute inflammatory and chronic fibrosing processes that culminate in glomerulosclerosis Mesangial cells (MC) in culture incompletely model an activated phenotype, and poorly reflect a normal in situ phenotype. We propose that analysis of chronic renal failure-prone animals and culture of MC on extracellular matrices (ECM), which better mimics in vivo conditions, will identify the regulatory processes that control MC activation, a process characterized by myofibroblast conversion. Preliminary data demonstrate that: (1) mesangial cell myofibroblast differentiation in vivo represents an adaptive response to the ECM environment; (2)injury-induced changes in ECM regulate the functional response of MC to pro-inflammatory signals; (3) on ECM basement membrane gels, MC migrate into multi-cellular networks and develop intercellular junctional complexes, similar to MC in normal glomeruli; (4) on Type I collagen gels, MC lack these intercellular junctions, similar to MC in diseased glomeruli; (5) assembly of MC in clusters is regulated by tyrosine kinase and phosphatase systems; and (6) this spatial patterning correlates with ligation and activation of Eph family tyrosine kinase receptors both in vitro and in normal ES+ but not the CRF-prone Es+mice. We hypothesize that, in the normal mesangium, an ECM-regulated dominance of cell-cell contacts, which is mediated in part by EPH family of receptor tyrosine kinases, maintains a normal (quiescent) mesangial cell phenotype. Within the injured glomerulus as ECM abundance and composition change, loss of cell-cell contacts and Eph receptor kinase activity occurs, permitting unopposed mesangial cell-matrix interaction and mesangial myofibroblast differentiation (activation).
Three specific aims are proposed: (1) Does MC myofibroblast differentiation represent an adaptive response to injury induced changes in ECM environment in animal models of progressive CRF?; (2) How does ECM direct the assembly of MC into networks linked by junctional complexes? What are the molecular components of the MC junctions in vitro and in vivo?"""""""" and (3) Does a MC EPH family tyrosine kinase receptor for the B61 ligand concentrate at site of cell-cell contracts and maintain a quiescent MC phenotype? The immediate goal of this proposal is to identify candidate molecules that regulate the transition from a normal to an activate MC phenotype. Understanding these changes should allow development of rational therapies to inhibit or ameliorate ongoing glomerular inflammation and scarring.
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