Extracellular transsulfuration generates hydrogen sulfide from homocysteine and protects endothelium from redox stress

Abstract

Homocysteine, a cardiovascular and neurocognitive disease risk factor, is converted to hydrogen sulfide, a cardiovascular and neuronal protectant, through the transsulfuration pathway. Given the damaging effects of free homocysteine in the blood and the importance of blood homocysteine concentration as a prognosticator of disease, we tested the hypotheses that the blood itself regulates homocysteine-hydrogen sulfide metabolism through transsulfuration and that transsulfuration capacity and hydrogen sulfide availability protect the endothelium from redox stress. Here we show that the transsulfuration enzymes, cystathionine β-synthase and cystathionine γ-lyase, are secreted by microvascular endothelial cells and hepatocytes, circulate as members of the plasma proteome, and actively produce hydrogen sulfide from homocysteine in human blood. We further demonstrate that extracellular transsulfuration regulates cell function when the endothelium is challenged with homocysteine and that hydrogen sulfide protects the endothelium from serum starvation and from hypoxia-reoxygenation injury. These novel findings uncover a unique set of opportunities to explore innovative clinical diagnostics and therapeutic strategies in the approach to homocysteine-related conditions such as atherosclerosis, thrombosis, and dementia.

Fig. 1.

Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL) expression in human plasma and serum. A: Western blots for CBS (showing 2 bands) and CGL in plasma (P) and serum (S) for 6 subjects. B and C: quantification of band densities shown in A (no significant differences were found). D: under nonreducing conditions, both CBS and CGL are found in monomeric and (predominantly) oligomeric form.

Fig. 2.

Transsulfuration activity in human blood. A: hydrogen sulfide (H₂S) production in human blood from substrates homocysteine (Hcy) and cysteine (Cys). Substrates were incubated (2 mmol/l each) in samples from healthy subjects for the indicated time periods at 37°C along with cofactor [pyridoxal-5′-phosphate (PLP)] in MOPS buffer. Control assays were buffer + 2 mmol/l Cys (Ctrl 1), buffer + 2 mmol/l Hcy + 2 mmol/l Cys (Ctrl 2), buffer + serum (Ctrl 3), or buffer + 2 mmol/l methionine (Ctrl 4). At each time point, *P < 0.05 for Hcy + Cys vs. all other conditions (Cys and Ctrl 1, 2, 3) and for Cys vs. all other conditions (Hcy + Cys, Ctrl 1, 2, 3, 4) (n = 10–12 subjects for each condition at each time point). B: parallel experiments to those shown in A with specificity for CBS and CGL enzymes showing reduced H₂S production at 2 h when CBS or CBS and CGL are inhibited (CBS inhibition: hydroxylamine 2 mmol/l; CGL inhibition: propargylglycine 2 mmol/l). C: Cys production (2-h assay) from serine and sodium sulfide (Na₂S) substrates is specific for CBS activity. Inhibiting CGL (propargylglycine, 2 mmol/l) further increased Cys yield because CGL otherwise uses the CBS-produced Cys as a substrate (n = 6/group, *P < 0.05 for all pairwise comparisons). D: Cys degradation during transsulfuration activity assay. Controls were substrate alone in buffer (Ctrl 1) and blood sample alone without added Cys (Ctrl 2) [n = 6–10/group, P < 0.05 vs. all other points (*) and vs. all other points except Ctrl 2 values (#)].

Fig. 3.

Effects of H₂S on endothelial cells following stress. Human umbilical vein endothelial cells (HUVECs) were serum starved or subjected to 18 h hypoxia (Po₂ <4 mmHg) followed by 6 h of reoxygenation (Po₂ ∼130 mmHg). H₂S was applied to the cells as 10% final media volume (using dissolved Na₂S) at the start of serum starvation or at the start of reoxygenation. A: viability was assessed using the MTT assay, and data are expressed as a percent of normal culture controls. B: oxidative damage to DNA was assessed by 8-hydroxydeoxyguanosine (8-OHdG) ELISA and expressed as ng 8-OHdG/ml cell lysate. *P < 0.05 compared with zero (control) treatment (P < 0.05) (n = 6–8/group/condition).

Fig. 4.

CBS and CGL expression in blood. CBS/CGL expression in plasma/serum of female (A) and male (B) subjects detected by Western blot. C: variance of CBS and CGL expression in samples shown in A and B (P < 0.05 CBS vs. CGL). D: correlation between CBS and CGL Western blot band densities within subjects (P < 0.05, n = 22 females, 17 males). Subjects were all Caucasian, nonsmokers, without cardiovascular disease.

Fig. 5.

Effect of CBS concentration in media bathing endothelial cells during Hcy treatment. CBS and CGL enzymes were removed from human serum by immunoprecipitation (IP); serum was then incubated with 200 μM Hcy for 15 h at 37°C and used to spike HUVEC media (10% final volume). As controls, pseudo-IP was performed using an antibody to vascular cell adhesion molecule (VCAM) or cells were left untreated (Control = CTRL). A: iNOS expression is induced by Hcy treatment, and this is exacerbated when transsulfuration capacity is diminished. *P < 0.05 for all pairwise comparisons. B: protein nitrosylation (3-nitrotyrosine; 3-NT) is increased by Hcy treatment but rescued when extracellular transsulfuration activity is intact. *P < 0.05 compared with control and VCAM. C: claudin-5 expression is reduced by Hcy treatment and is further reduced when transsulfuration capacity is diminished. *P < 0.05 for all pairwise comparisons. D: consistent with the barrier properties of claudin-5, monolayer permeability is significantly greater when cells are treated with Hcy-spiked media void of transsulfuration enzymes compared with the VCAM-IP (pseudo-IP) control condition where transsulfuration enzymes were active and able to reduce the Hcy burden. P < 0.05 compared with control (*) and compared with control and VCAM (#). A-D: n = 4/group.

Fig. 6.

Secretion of CBS and CGL enzymes by liver and endothelial cells. A: representative Western blots of conditioned media proteins from mouse hepatocytes or endothelial cells incubated for 1, 20, or 40 h. B: optical density of bands shown in A. P < 0.05 compared with 1 h (*) and for all pairwise comparisons (#).

Fig. 7.

Transsulfuration enzyme expression in plasma and serum of two mouse models of hyperhomocysteinemia (HHcy). A: dietary model (high methionine, low B6/B12 vitamins). B: CBS deficiency (+/−) (*P < 0.05, n = 6/group).

Fig. 8.

Schematic of the ideas and findings of this study. Transsulfuration enzymes CBS and CGL are secreted into the blood stream by the liver and by vascular endothelial cells. This pathway metabolizes Hcy and produces H₂S. When Hcy levels are high, nitric oxide pathways are disturbed and endothelial barrier integrity is compromised. H₂S protects endothelium from stress conditions, serum starvation, and hypoxia-reoxygenation.