EXOGENOUS HSP70 BECOMES CELL-ASSOCIATED, BUT NOT INTERNALIZED, BY STRESSED ARTERIAL SMOOTH-MUSCLE CELLS

Cell death within atherosclerotic plaques leads to necrosis and ruptur e, resulting in vascular occlusion. We have previously demonstrated th at addition of exogenous 70 kDa heat shock protein (HSP70) to arterial smooth muscle cells (aSMCs) in vitro can protect against toxins that may initiate necrosis. To determine whether exogenous HSP70 enters aSM Cs or acts from outside cells to preserve viability, cultured rabbit a SMCs were stressed by serum deprivation and treated with fluorescently labeled (7-aminomethyl-4-coumarin-3-acetate) or I-125-radiolabeled HS P70. Cell-associated HSP70 was analyzed using Western blotting, fluore scence spectroscopy, and gamma counting/autoradiography. Surface bindi ng of HSP70 to aSMCs was differentiated from uptake by using trypsin t reatment to degrade non-internalized HSP70. Specificity of HSP70 bindi ng was tested by inhibiting uptake of I-125-HSP70 with excess unlabele d HSP70 or bovine serum albumin (BSA). The effect of unlabeled exogeno us HSP70 on endogenous HSP synthesis was also tested. Exogenous HSP70 increased total cell-associated HSP70 2.9- to 3.6-fold over levels pre sent in unstressed aSMCs. However, <5% of the exogenous HSP70 was tryp sin-insensitive, indicating that bound HSP70 was not internalized. Bin ding of I-125-HSP70 was inhibited by both unlabeled HSP70 and BSA, imp lying a non-specific interaction with the plasmalemma. Exogenous HSP70 significantly lowered overall protein synthesis by serum-deprived aSM Cs, but it did not specifically inhibit synthesis of endogenous HSPs a fter heat shock. The results indicate that exogenous HSP70 protects vi ability of stressed aSMCs through interactions with the cell surface r ather than via internalization.