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.

Aims Receptor-type vascular endothelial protein tyrosine phosphatase (VE-PTP) dephosphorylates Tie-2 as well as CD31, VE-cadherin and VEGFR2. The latter form a signal transduction complex that mediates the endothelial cell response to shear stress, including the activation of the endothelial nitric oxide (NO) synthase (eNOS). As VE-PTP expression is increased in diabetes, we investigated the consequences of VE-PTP inhibition (using AKB-9778) on blood pressure in diabetic patients and the role of VE-PTP in the regulation of eNOS activity and vascular reactivity. Methods and Results In diabetic patients AKB-9778 significantly lowered systolic and diastolic blood pressure. This could be linked to elevated NO production, as AKB increased NO generation by cultured endothelial cells and elicited the NOS inhibitor-sensitive relaxation of endothelium–intact rings of mouse aorta. At the molecular level, VE-PTP inhibition increased the phosphorylation of eNOS on Tyr81 and Ser1177 (human sequence). The PIEZO1 activator Yoda1, which was used to mimic the response to shear stress, also increased eNOS Tyr81 phosphorylation, an effect that was enhanced by VE-PTP inhibition. Two kinases i.e. abelson-tyrosine protein kinase (ABL)1 and Src were identified as eNOS Tyr81 kinases as their inhibition and downregulation significantly reduced the basal and Yoda1-induced tyrosine phosphorylation and activity of eNOS. VE-PTP, on the other hand, formed a complex with eNOS in endothelial cells and directly dephosphorylated eNOS Tyr81 in vitro. Finally, phosphorylation of eNOS on Tyr80 (murine sequence) was found to be reduced in diabetic mice and diabetes-induced endothelial dysfunction (isolated aortic rings) was blunted by VE-PTP inhibition. Conclusions VE-PTP inhibition enhances eNOS activity to improve endothelial function and decrease blood pressure indirectly, through the activation of Tie-2 and the CD31/VE-cadherin/VEGFR2 complex, and directly by dephosphorylating eNOS Tyr81. VE-PTP inhibition, therefore, represents an attractive novel therapeutic option for diabetes-induced endothelial dysfunction and hypertension. Translational perspective Diabetes and hypertension are associated with endothelial dysfunction, therefore, strategies that increase NO bioavailability are likely to be beneficial. In this study, VE-PTP inhibition using AKB-9778 lowered systolic and diastolic blood pressure in diabetic patients. Mechanistically, VE-PTP inhibition improved endothelial function by activating different signaling pathways that converged to increase eNOS activity. VE-PTP expression was increased in diabetic mice and the VE-PTP inhibitor abrogated diabetes-induced endothelial dysfunction. Thus, this study highlights the clinical feasibility of VE-PTP inhibition to improve endothelial dysfunction and treat hypertension.

Campomanesia adamantium is a medicinal plant of the Brazilian Cerrado. Different parts of its fruits are used in popular medicine to treat gastrointestinal disorders, rheumatism, urinary tract infections and inflammations. Despite its widespread use by the local population, the mechanisms involving platelet aggregation and the inhibition of cyclooxygenase by C. adamantium are unknown. This study evaluated the chemical composition, antioxidant activities and potential benefits of the C. adamantium peel extract (CAPE) and its components in the platelet aggregation induced by arachidonic acid in platelet-rich plasma. Aspects of the pharmacological mechanism were investigated as follows: platelet viability, calcium mobilization, levels of the cyclic nucleotides cAMP and cGMP, thromboxane B2 levels, and the inhibitory effects on COX-1 and COX-2 were studied in vitro and using molecular docking in the catalytic domain of these proteins. The major CAPE constituents standing out from the chemical analysis are the flavonoids, namely those of the flavones and chalcones class. The results showed that CAPE, quercetin and myricetin significantly decreased arachidonic acid-induced platelet aggregation; the assays showed that CAPE and quercetin decreased the mobilization of calcium and thromboxane B2 levels in platelets and increased cAMP and cGMP levels. Moreover, CAPE inhibited the activity of COX-1 and COX-2, highlighting that quercetin could potentially prevent the access of arachidonic acid more to the catalytic site of COX-1 than COX-2. These results highlight CAPE’s potential as a promising therapeutic candidate for the prevention and treatment of diseases associated with platelet aggregation.

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