Uncontrolled production or insufficient degradation of lung tissue matrix may alter compliance and gas transfer, causing clinical disorders of ventilation and oxygenation. Certain fibrotic lung diseases demonstrate a high density of mast cells, implicating them in the complex mechanisms of tissue remodeling, an area of investigation focusing on a growing family of matrix-degrading metalloproteinases. The long term objective of this proposal is to determine how resident lung mast cells participate in injury and repair processes during parenchymal inflammation. The preliminary data demonstrate that dog mastocytoma cells secrete and activate their own matrix metalloproteinases (MMPs), supporting the hypothesis that mast cells participate in the regulation of extracellular matrix turnover. Characterization of the proteolytic activity of cell culture supernatants and amino acid analysis of the purified enzyme and its cDNA-predicted peptide sequence suggest secretion of the dog homologue of the human 92-kDa gelatinase. Degranulation studies reveal activation of the gelatinase proenzyme which can be blocked by phenylmethylsulfonyl fluoride (PMSF), further implicating the granule-associated serine proteases, tryptase and chymase, in MMP activation mechanisms. Understanding how mast cells package matrix metalloproteinases may provide insights into interactions with other preformed mediators and in vivo mechanisms of MMP activation.
The specific aims i nclude identification of dog homologues of human MMPs by characterization of substrate specificities and determination of amino acid sequence homology, followed by enzyme purification using sequential chromatographic steps. Activation studies will assess the ability of purified dog mast cell serine proteases to cleave and activate mastocytoma MMPs, and then identify the enzymes responsible for MMP activation in culture. Antibodies will be raised against purified MMPs for use in immunohistochemical studies to determine the cellular localization and tissue distribution of mast cell matrix metalloproteinases. The candidate has completed clinical training in internal medicine, and pulmonary and critical care medicine at UCSF, leading to a strong commitment to academic medicine and an interest in the pathophysiology of lung injury. Research pursued in the laboratory of the sponsor, George Caughey M.D., has focused on mast cell proteases, with the goal of integrating clinical interests in interstitial lung diseases and basic science interests in protein biochemistry and molecular biology. The research training plan includes: an intensive laboratory experience, didactic coursework, weekly seminars and journal clubs, ongoing patient care and clinical teaching, and an advisory committee including the sponsor and experts on matrix metalloproteinases and immunohistopathology, convened to provide scientific guidance and to oversee the applicant's development into an independent investigator in pulmonary research.
