Introduction: Cell lysis secondary to tissue injury triggers the tissue repair process. Identification of active components within cellular lysate could lead to a discovery of a new class of wound modifiers for treating recalcitrant wounds. In cancer patients, radiation therapy causes a significant impairment in the ability of the irradiated normal tissues to heal - negatively impacting any further surgery that may be necessary as part of the patients' treatment. Agents that ameliorate or overcome the deleterious effects of irradiation on normal tissues could play a pivotal role in reducing this morbidity. Aims & Objectives: Defining cellular signals that trigger healing and identify modifiers that impact the rate of healing recalcitrant wounds. By testing various components of fractionated cellular lysate we determined that a class of intracellular proteins called Heat Shock Proteins (HSPs) are overexpressed and bioactive in injured and healing tissues. This study examines the range of these observations, characterizes cellular mechanisms by which HSPs impact the elements of tissue repair. It explores the use of cytoprotective agents in wounds in irradiated tissue or in systemic diseases that pose a challenge to normal tissue repair. Materials & Methods: In vivo studies were performed using different strains of mice (C57, BALB/c). Full thickness wounds were created dorsally and HSPs were injected locally or systemically and rate of wound healing was measured. Lipopolysaccharide, and a panel of similar proteins were used as controls. Freshly harvested peritoneal macrophages were tested for phagocytosis after in vivo intraperitoneal administration of HSPs, 48 hours prior to the assay. In vitro assessment of HSP-mediated activation was performed using a panel of three HSPs, HSP70 (cytosolic), HSP90 (membrane-bound) and gp96 (endoplasmic reticulum-bound). Macrophage-mediated phagocytosis assay was performed using RAW 264.7 (ATCC) cell line by quantifying the ingestion of latex beads. Assessment was done, both by microscopic cell count and by FACS analysis using fluorescent markers. HSP-mediated activation of both macrophage and fibroblasts (SVT2 cell lines, ATCC) in terms of growth factor release was assessed using QuantkineR ELISA for PDGF and bFGF. Lipopolysaccharide, Beta-galactosidase, mouse serum albumin and buffer were used as controls. A panel of blocking agents was tested to explore possible receptor-mediation. Results: In vivo, HSPs accelerate wound healing in earlier phases and amplify the overall rate of wound contraction. In vitro, HSPs enhance phagocytic activity of cultured macrophages to 1.2 - 2 times the basal rate. When treated with HSPs both cultured macrophages and fibroblasts exhibit increased release of bFGF and PDGF. This activity is specific, dose-titrated and partially receptor mediated via the CD91 ligand. Conclusions: HSPs are abundantly released during tissue injury and provide important cellular signals for the tissue repair process. HSPs stimulate macrophages and fibroblasts in a protein-specific, titratable and a receptor-mediated fashion. In vivo, this benefit translates into faster wound healing specifically in early phases and in the overall rate of contraction. In vitro HSP-mediated cellular activation, its in vivo correlation and the mechanisms delineated by this study suggest a positive role for these cytoprotective molecules in tissue repair.
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