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" i.e. the spectrum of proteins targeted by H 2 S n in human endothelial cells. Methods: LC-MS/MS was used to identify S-sulfhydrated cysteines in endothelial cell proteins and β3 integrin intra-protein 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 knock out mice and 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 intra-protein 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, resulting in the constitutive activation of RhoA 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 a failure to detect β3 integrin S -sulfhydration, all of which were rescued following the administration of an H 2 S 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.

Conventional Ly6Chi monocytes have developmental plasticity for a spectrum of differentiated phagocytes. Here we show, using conditional deletion strategies in a mouse model of Toll-like receptor (TLR) 7-induced inflammation, that the spectrum of developmental cell fates of Ly6Chi monocytes, and the resultant inflammation, is coordinately regulated by TLR and Notch signaling. Cell-intrinsic Notch2 and TLR7-Myd88 pathways independently and synergistically promote Ly6Clo patrolling monocyte development from Ly6Chi monocytes under inflammatory conditions, while impairment in either signaling axis impairs Ly6Clo monocyte development. At the same time, TLR7 stimulation in the absence of functional Notch2 signaling promotes resident tissue macrophage gene expression signatures in monocytes in the blood and ectopic differentiation of Ly6Chi monocytes into macrophages and dendritic cells, which infiltrate the spleen and major blood vessels and are accompanied by aberrant systemic inflammation. Thus, Notch2 is a master regulator of Ly6Chi monocyte cell fate and inflammation in response to TLR signaling.

Secreted modular calcium binding protein 1 (SMOC1) is an osteonectin/SPARC related matricellular protein, whose expression is regulated by miR-223. Given that platelets are rich in miR-223, this study investigated the expression of SMOC1 and its contribution to platelet function. Both human and murine platelets expressed SMOC1, whereas platelets from SMOC1+/- mice presented no detectable mature SMOC1 protein. Platelets from SMOC1+/- mice demonstrated attenuated responsiveness to thrombin (platelet neutrophil aggregate formation, aggregation, clot formation, Ca2+ increase and β3 integrin phosphorylation), while responses to other platelet agonists were unaffected. SMOC1 has been implicated in TGFb singling but no link to this pathway was detected in platelets. Rather, the SMOC1 kazal domain directly bound thrombin to potentiate its activity in vitro as well as its actions on isolated platelets. The latter effects were prevented by monoclonal antibodies against SMOC1. Platelets from miR-223-deficient mice expressed high levels of SMOC1 and exhibited hyper-reactivity to thrombin that was also reversed by pre-incubation with monoclonal antibodies against SMOC1. Similarly, SMOC1 levels were markedly upregulated in platelets from individuals with type 2 diabetes and the SMOC1 antibody abrogated platelet hyper-responsiveness to thrombin. Taken together, we have identified SMOC1 as a novel thrombin-activating protein that makes a significant contribution to the pathophysiological changes in platelet function associated with type 2 diabetes. Thus, strategies that target SMOC1 or its interaction with thrombin may be attractive therapeutic approaches to normalize platelet function in diabetes.