Mapping the Endothelial Cell
S
-Sulfhydrome Highlights the Crucial Role of Integrin Sulfhydration in Vascular Function
Authors: Bibli Sofia-Iris , Hu Jiong , Looso Mario , Weigert Andreas , Ratiu Corina , Wittig Janina , Drekolia Maria Kyriaki , Tombor Lukas , Randriamboavonjy Voahanginirina , Leisegang Matthias S. , Goymann Philipp , Delgado Lagos Fredy , Fisslthaler Beate , Zukunft Sven , Kyselova Anastasia , Justo Alberto Fernando Oliveira , Heidler Juliana , Tsilimigras Diamantis , Brandes Ralf P. , Dimmeler Stefanie , Papapetropoulos Andreas , Knapp Stefan , Offermanns Stefan , Wittig Ilka , Nishimura Stephen L. , Sigala Fragiska , Fleming Ingrid 
Published: December 2020

Background:

In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide–related sulfane sulfur compounds (H
2
S
n
), 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 H
2
S
n
) in human endothelial cells.

Methods:

Liquid chromatography with tandem mass spectrometry was used to identify
S
-sulfhydrated cysteines in endothelial cell proteins and β3 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 H
2
S
n
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 β3 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 β leg. β3 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 β3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 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 H
2
S
n
generation, impaired flow-induced dilatation, and failure to detect β3 integrin
S
-sulfhydration, all of which were rescued after the administration of an H
2
S
n
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 H
2
S
n
supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.