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The Molecular Chaperone Heat Shock Protein 90 (Hsp90): A Novel Target For Regulating Smooth Muscle Cell (SMC) Growth In Vascular Injury

Andrew W Hoel¹, Peng Yu¹, Xin Xin Sui¹, Janet Plescia², Dario C. Altieri², Michael S. Conte.^1
1Brigham and Women's Hospital, Boston, MA; ²University of Massachusetts, Boston, MA.

OBJECTIVES: Hsp90 is a molecular chaperone with client proteins, such as survivin (SVV) and Akt, that regulate cellular survival. This function has recently been exploited in the development of new anti-neoplastic agents. Little is known about Hsp90 function in SMC, or its potential relevance to intimal hyperplasia.

METHODS: Hsp90 expression in vascular injury, and its co-localization with SVV, was examined by immunostaining of balloon-injured iliac arteries of New Zealand White Rabbits (N=4). We further examined Hsp90 and SVV associations in primary cultured human saphenous vein SMC by immunoprecipitation (IP) and Western blot. We tested the acute effects of shepherdin, a novel cell-permeable peptide designed to competitively antagonize the Hsp90-SVV interaction, on SMC viability (MTT assay) and apoptosis (DNA content flow cytometry and nuclear morphology by DAPI staining).

RESULTS: Hsp90 expression in normal, uninjured arteries is localized to the luminal endothelium, but is minimal in medial SMC. At 3 days after injury, there is prominent upregulation of Hsp90 throughout the vessel wall and SVV expression co-localizes exclusively to Hsp90 (+) cells. Co-IP experiments confirmed the molecular association between Hsp90 and SVV in SMC in vitro. Exposure of SMC to shepherdin revealed dose-dependent intracellular accumulation of the biotinylated peptide at 30 minutes. At 18 hours after shepherdin treatment, SVV protein levels are decreased by Western blot and a dose-dependent loss of SMC viability is evident (Figure 1). A corresponding induction of SMC apoptosis is demonstrated by DNA content analysis (Figure 2), and confirmed by characteristic alterations in nuclear morphology.

CONCLUSIONS: Analogous to its function in transformed cells, the molecular chaperone Hsp90 is critical for SMC survival. These studies identify the Hsp90-SVV interaction as a potential new therapeutic target for regulation of the vascular injury response.