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Endothelial hyperperme ability leading to vascular leak is an important consequence of sepsis and sepsis-induced lung injury. We previously reported that heat shock protein (hsp) 90 inhibitor pretreatment improved pulmonary barrier dysfunction in a murine model of sepsis-induced lung injury. We now examine the effects of hsp90 inhibitors on LPS-mediated endothelial hyperpermeability, as reflected in changes in transendothelial electrical resistance (TER) of bovine pulmonary arterial endothelial cells (BPAEC). Vehicle-pretreated cells exposed to endotoxin exhibited a concentration-dependent decrease in TER, activation of pp60(Src), phosphorylation of the focal adhesion protein paxillin, and reduced expression of the adherens junction proteins, vascular endothelial (VE)-cadherin and beta-catenin. Pretreatment with the hsp90 inhibitor, radicicol, prevented the decrease in TER, maintained VE-cadherin and beta-catenin expression, and inhibited activation of pp60(Src) and phosphorylation of paxillin. Similarly, when BPAEC hyperpermeability was induced by endotoxin-activated neutrophils, pretreatment of neutrophils and/or endothelial cells with radicicol protected against the activated neutrophil-induced decrease in TER. Increased paxillin phosphorylation and decreased expression of beta-catenin and VE-cadherin were also observed in mouse lungs 12 h after intraperitoneal endotoxin and attenuated in mice pretreated with radicicol. These results suggest that hsp90 plays an important role in sepsis-associated endothelial barrier dysfunction.

Vascular soluble guanylate cyclase (sGC) exists in multimeric complexes with endothelial nitric oxide (NO) synthase (eNOS) and heat shock protein 90 (hsp90). Whereas disruption of hsp90-eNOS complexes clearly attenuates eNOS-dependent vascular relaxation, the contribution of sGC-hsp90 complexes to eNOS- or NO donor-dependent relaxations remains unclear. Isolated rat thoracic aortic rings were preincubated with structurally diverse hsp90 binding inhibitors, radicicol (RA) or geldanamycin (GA), or vehicle for 0.5, 1, or 15 h. Preconstricted vessels were exposed to ACh, 8-bromo-cGMP (8-BrcGMP), forskolin, or one of three NO donors: nitroglycerin (NTG), sodium nitroprusside, or spermine NONOate (SNN). Both RA and GA inhibited endothelium-dependent relaxations dose dependently. Indomethacin or the antioxidant tiron did not affect the inhibition of ACh-induced relaxations by GA. Long-term (15 h) exposure to RA inhibited all NO donor-induced relaxations; however, GA inhibited SNN-induced relaxation only. The effects of GA and RA appeared to be selective because 15-h treatment with either agent did not affect forskolin-induced relaxations and only slightly decreased 8-BrcGMP-induced relaxations. Similarly to their effects on NO-donor-induced relaxation, 15-h exposure to RA, but not to GA, decreased hsp90-bound sGC protein expression and NTG-stimulated cGMP formation in aortic rings, whereas RA more than GA reduced SNN-stimulated cGMP formation. We conclude that RA, much more so than GA, selectively inhibits sGC-dependent relaxations of aortic rings by reducing sGC expression, disrupting sGC-hsp90 complex formation and decreasing cGMP formation. These studies suggest that hsp90 regulates both eNOS- and sGC-dependent relaxations.