Mapping the Endothelial Cell S-Sulfhydrome Highlights the Crucial Role of Integrin Sulfhydration in Vascular Function

Background: In vascular endothelial cells, cysteine metabolism by the cystathionine gamma lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the S-sulfhydrome (ie, the spectrum of proteins targeted by H2Sn) in human endothelial cells. Methods: Liquid chromatography with tandem mass spectrometry was used to identify S-sulfhydrated cysteines in endothelial cell proteins and beta 3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction. Results: Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell S-sulfhydrome consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on beta 3 integrin in detail we found that S-sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the beta leg. beta 3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between beta 3 integrin and G alpha 13 (guanine nucleotide-binding protein subunit alpha 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation, and failure to detect beta 3 integrin S-sulfhydration, all of which were rescued after the administration of an H2Sn supplement. Conclusions: Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.

Automated summary

This study investigated the S-sulfhydrome in human endothelial cells and found that S-sulfhydration plays a crucial role in regulating endothelial cell functions, especially in integrins and mechanotransduction. Impaired S-sulfhydration was associated with vascular dysfunction, while H2Sn supplementation improved vascular reactivity, suggesting a potential therapeutic target for vascular diseases.