Hyperhomocysteinemia potentiates diabetes-impaired EDHF-induced vascular relaxation: Role of insufficient hydrogen sulfide

Abstract

Insufficient hydrogen sulfide (H₂S) has been implicated in Type 2 diabetic mellitus (T2DM) and hyperhomocysteinemia (HHcy)-related cardiovascular complications. We investigated the role of H₂S in T2DM and HHcy-induced endothelial dysfunction in small mesenteric artery (SMA) of db/db mice fed a high methionine (HM) diet. HM diet (8 weeks) induced HHcy in both T2DM db/db mice and non-diabetic db/+ mice (total plasma Hcy: 48.4 and 31.3 µM, respectively), and aggravated the impaired endothelium-derived hyperpolarization factor (EDHF)-induced endothelium-dependent relaxation to acetylcholine (ACh), determined by the presence of eNOS inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME) and prostacyclin (PGI₂) inhibitor indomethacin (INDO), in SMA from db/db mice but not that from db/+ mice. A non-selective Ca²⁺-active potassium channel (K_Ca) opener NS309 rescued T2DM/HHcy-impaired EDHF-mediated vascular relaxation to ACh. EDHF-induced relaxation to ACh was inhibited by a non-selective K_Ca blocker TEA and intermediate-conductance K_Ca blocker (IK_Ca) Tram-34, but not by small-conductance K_Ca (SK_Ca) blocker Apamin. HHcy potentiated the reduction of free sulfide, H₂S and cystathionine γ-lyase protein, which converts L-cysteine to H₂S, in SMA of db/db mice. Importantly, a stable H₂S donor DATS diminished the enhanced O₂^- production in SMAs and lung endothelial cells of T2DM/HHcy mice. Antioxidant PEG-SOD and DATS improved T2DM/HHcy impaired relaxation to ACh. Moreover, HHcy increased hyperglycemia-induced IK_Ca tyrosine nitration in human micro-vascular endothelial cells. EDHF-induced vascular relaxation to L-cysteine was not altered, whereas such relaxation to NaHS was potentiated by HHcy in SMA of db/db mice which was abolished by ATP-sensitive potassium channel blocker Glycolamide but not by K_Ca blockers.

Conclusions: Intermediate HHcy potentiated H₂S reduction via CSE-downregulation in microvasculature of T2DM mice. H₂S is justified as an EDHF. Insufficient H₂S impaired EDHF-induced vascular relaxation via oxidative stress and IK_Ca inactivation in T2DM/HHcy mice. H₂S therapy may be beneficial for prevention and treatment of micro-vascular complications in patients with T2DM and HHcy.

Keywords: Calcium-activated potassium channel (K(Ca)); Endothelial dysfunction; Hydrogen sulfide; Micro-vasculature; T2DM.

Fig. 1

HHcy aggravated glucose intolerance and reduced body weight of db/db mice. A. Body weight. B. Total plasma homocysteine (tHcy) levels. C. Blood glucose levels. D. Oral glucose tolerance test (OGTT). E. 24 h water intake. F. 24 h urine excretion. At the age of 8-week, db/+ and db/db mice were fed with a high methionine diet (HM, methionine: 2% w/w) for 8 weeks. db/+ and db/db mice fed with a control diet (CT, methionine: 0.37% w/w) served as controls. n = 5–10, * p < 0.05 vs db/+ mice on CT diet (db/+_CT); †p < 0.05 vs db/+ mice on HM diet; ‡p < 0.05 vs db/db mice on CT diet (db/db_CT). HHcy, hyperhomocysteinemia.

Fig. 2

HHcy aggravated T2DM-impaired endothelium-dependent vascular relaxation to ACh in SMA of db/db mice. A and B. Vascular contractile response to potassium chloride (KCl, A) and phenylephrine (PE, B). C. Endothelium-dependent vascular relaxation to acetylcholine (ACh). D. Endothelium-independent vascular relaxation to sodium nitroprusside (SNP). Small mesenteric arterial rings were pre-contracted with phenylephrine (1 µM) and examined for relaxation response to cumulative additions of ACh or SNP. n = 5–10, *p < 0.05 vs db/+ mice on CT diet (db/+ _CT); ‡p < 0.05 vs db/db mice on CT diet (db/db_CT). SMAs, small mesenteric arteries.

Fig. 3

HHcy aggravated impaired EDHF-induced relaxation to ACh in SMA of db/db mice via IK_Cainhibition. EDHF-induced relaxation was determined by pretreated the SMA with NOS inhibitor L-NAME (100 µM) and COX inhibitor (INDO, 10 µM) and indicated inhibitors, precontracted with phenylephrine (1 µM), and examined for relaxation to acetylcholine (ACh). A. EDHF-induced relaxation to ACh. B. EDHF-induced relaxation in the presence of SK_Ca/Ik_Ca blocker TEA (1 mM). C. EDHF-induced relaxation in the presence of SK_Ca blocker Apamin (1 μM) and IK_c blocker Tram-34 (1 μM). D. EDHF-induced relaxation in the presence of Ik_Ca blocker Tram-34. E. EDHF-induced relaxation in the presence of SK_ca blocker Apamin. F. EDHF-induced relaxation in the presence of SK_Ca/IK_Ca activator NS309 (10 μM). n = 5–10. *p < 0.05 vs db/+ mice on CT diet (db/+_CT); ‡p < 0.05 vs db/db mice on CT diet (db/db_CT). COX, cyclooxygenase; HHcy, hyperhomocysteinemia; INDO, indomethacin; L-NAME, N^G-Nitro-L-arginine methyl ester; NOS, NO synthase.

Fig. 4

HHcy potentiated H₂S deficiency and CSE downregulation in SMA of db/db mice. A. Reprehensive images of intracellular H₂S production in SMAs stained with fluorescent probe sulfidefluor 7AM (SF-7AM, 25 µM, for 30 min). B. Quantification of Intracellular H₂S production in SMAs. C. Free sulfide levels in SMAs measured by reversed phase HPLC. D. CSE protein levels in SMAs. n = 3–5 *p < 0.05 vs db/+ mice on CT diet (db/+_CT); ‡p < 0.05 vs db/db mice on CT diet (db/db_CT). CSE, cystathionine γ-lyase.

Fig. 5

H₂S donor DATS rescued HHcy-induced oxidative stress and -impaired EDHF-induced relaxation to ACh in SMA of db/db mice with HHcy. A. Representative images (Left panel) and quantifications (Right panel) of in situ O₂^- production in SMAs stained with DHE. The SMAs from db/db mice fed with HM diet were treated with or without DATS (5 µM) for 30 min before DHE staining. B. Identification of primary mouse lung endothelial cells (MLECs) isolated from lung of db/+ and db/db by Dil-ac-LDL uptake and CD31 staining. C. Representative images (Left panel) and quantifications of O₂^- production (Right panel) in mouse MLECs (DHE staining). The MLECs from db/db mice fed with HM diet were treated with or without DATS (5 µM) for 30 min. D. EDHF-induced relaxation to ACh in SMAs of db/db mice fed with HM diet in the presence of L-NAME+INDO (30 min) with or without PEG-SOD (150 U/ml) for 1 h. E EDHF-induced relaxation to ACh in SMAs of db/db mice fed with HM diet in the presence or L-NAME+INDO with or without DATS (5 µM) for 30 min. Then the SMAs were contracted with phenylephrine (1 µM), and examined for relaxation to ACh. n = 3–5. ^*p < 0.05 vs db/+ mice on CT diet (db/+_CT); ‡p < 0.05 vs db/db mice on CT diet (db/db_CT); $p < 0.05 vs db/db mice on HM diet (db/db_HM). DATS, diallyl trisulfide; DHE, dihydroethidium. PEG-SOD, polyethylene glycol superoxide dismutase.

Fig. 6

HHcy potentiated hyperglycemia-induced tyrosine nitration of IK_Ca. A. IK_Ca levels in HCMVECs after immunoprecipitation with 3-NT antibody. HCMVECs were treated with D-glucose (D-Glu, 25 mM) or D-Glu plus DL-homocysteine (DL-Hcy, 500 µM) for 48 h. *p < 0.05 vs HCMVECs treated with vehicle; ‡p < 0.05 vs HCMVECs treated with D-Glu). B. Representative images of co-staining of 3-nitrotyrosine (3-NT, green) and IK_Ca (red) and DAPI (blue) in SMAs of db/db mice fed with HM diet. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

HHcy sensitized H₂S donor NaHS-induced vascular relaxation in SMA of db/db mice via activation of K_ATP. EDHF-induced relaxation in SMA was determined by pretreated the SMA with NOS inhibitor L-NAME (100 µM) and COX inhibitor (INDO, 10 µM) and indicated inhibitors, precontracted with phenylephrine (1 µM), and examined for relaxation to L-cysteine or NaHS. A. EDHF-induced relaxation by L-cysteine (10 µM). B. EDHF-induced relaxation by NaHS. C. EDHF-induced relaxation to NaHS in the presence of IK_Ca inhibitor Tram-34 (1 μM). D. EDHF-induced relaxation to NaHS in the presence of IK_Ca inhibitor Tram-34 and SK_Ca inhibitor Apamin (1 μM). E. EDHF-induced relaxation to NaHS in the presence of K_Ca inhibitor TEA (1 mM). F. EDHF-induced relaxation to NaHS in the presence of K_ATP inhibitor glibenclamide (GLB, 3 µM). n = 5–8 *p < 0.05 vs db/+ mice on CT diet (db/+_CT); ‡p < 0.05 vs db/db mice on CT diet (db/db_CT).

Fig. 8

Mechanism of HHcy-potentiated diabetes-impaired EDHF-induced vascular relaxation. Working model of T2DM/HHcy-induced endothelial dysfunction in micro-vasculature.