Hydrogen Sulfide Metabolism Regulates Endothelial Permeability

Hydrogen sulfide (H 2 S) is an endogenous gaseous signaling molecule that plays important roles in the cardiovascular system. However, the impact of H 2 S on endothelial barrier function is not known. In endothelial cells, the predominant source of H 2 S is cystathionine γ‐lyase (CSE). After generation, H 2 S is converted into biological pools of sulfane sulfurs and acid labile sulfurs. Recent studies suggest that these H 2 S metabolites are also biologically active. Thus, we sought to determine how exogenous and endogenous H 2 S and its metabolites regulate endothelial permeability. In vitro endothelial permeability was evaluated by albumin leakage or transendothelial electrical resistance (TEER). Exogenous H 2 S was studied by treating endothelial monolayers with different H 2 S donors including sodium sulfide (Na 2 S), GYY4137 and diallyl trisulfide (DATS). To investigate the effect of endogenous H 2 S, endothelial cells were isolated from aorta (MAECs) of wild type and CSE knockout mice. Alternatively, CSE knockdown was achieved using siRNA targeting CSE mRNA. Cell morphology and junction protein are examined by immunostaining and immunoblotting. Cell survival was examined by the lactate dehydrogenase (LDH) release and cleaved caspase 3. p‐Nitrophenyl Phosphate (pNPP) assay was also used to measure protein tyrosine phosphatase (PTP) activity. In vivo endothelial permeability was evaluated by a modified Miles assay. Biological pools of H 2 S were measured using monobromobimane followed by detection of sulfide‐dibimane with reverse phase HPLC. DATS treatment in vitro induced rapid albumin leakage across endothelial monolayers. Comparatively, Na 2 S and GYY4137 increased permeability only at later time points and to a lesser extent. The increased permeability was accompanied by the disruption of the intercellular junction and an increase in stress fiber formation. The effective dose of DATS increased both free H 2 S and bound sulfane sulfur compared to the vehicle control and other donors. In comparison, both free H 2 S and bound sulfane sulfur were reduced in CSE deficient MAECs, while the lack of CSE enhanced endothelial barrier function compared to wild‐type endothelial cells. CSE knockdown in HUVEC also exhibited better junction structures and increased claudin 5 expression. Interestingly, Na2S and GYY4137 but not DATS decreased PTP activity, while MAECs lacking CSE showed higher PTP activity. However, sulfide donors failed to increase permeability of mouse skin, although CSE knockout mice were less responsive to VEGF stimulated permeability. In summary, both exogenous and endogenous free H 2 S and bound sulfane sulfur increase endothelial permeability with decreased PTP activity possibly associated with endogenous sulfide regulation of permeability but not DATS induced permeability. Support or Funding Information This work is supported by an NIH grant (HL113303) to Dr. CG Kevil and a graduate research fellowship from the Center for Cardiovascular Diseases and Sciences, LSU Health Science Center‐Shreveport to S Yuan.