Hyperhomocystinemia impairs endothelial function and eNOS activity via PKC activation

Abstract

Objective: A risk factor for cardiovascular disease, hyperhomocystinemia (HHcy), is associated with endothelial dysfunction. In this study, we examined the mechanistic role of HHcy in endothelial dysfunction.

Methods and results: Through the use of 2 functional models, aortic rings and intravital video microscopy of the cremaster, we found that arterial relaxation in response to the endothelium-dependent vessel relaxant, acetylcholine or the nitric oxide synthase (NOS) activator (A23187), was significantly impaired in cystathionine beta-synthase null (CBS(-/-)) mice. However, the vascular smooth muscle cell (VSMC) response to the nitric oxide (NO) donor (SNAP) was preserved in CBS(-/-) mice. In addition, superoxide dismutase and catalase failed to restore endothelium-dependent vasodilatation. Endothelial nitric oxide synthase (eNOS) activity was significantly reduced in mouse aortic endothelial cells (MAECs) of CBS(-/-) mice, as well as in Hcy-treated mouse and human aortic endothelial cells (HAECs). Hcy-mediated eNOS inhibition--which was not rescued by adenoviral transduction of superoxide dismutase and glutathione peroxidase, or by tetrahydrobiopterin, sepiapterin, and arginine supplementations in MAEC--was associated with decreased protein expression and increased threonine 495 phosphorylation of eNOS in HAECs. Ultimately, a protein kinase C (PKC) inhibitor, GF109203X (GFX), reversed Hcy-mediated eNOS inactivation and threonine 495 phosphorylation in HAECs.

Conclusions: These data suggest that HHcy impairs endothelial function and eNOS activity, primarily through PKC activation.

Figure 1

Plasma Hcy/ADMA levels, body weight, and vasorelaxation to acetylcholine in CBS mice. A. Plasma levels of Hcy and ADMA. Hcy concentrations were measured by liquid chromatography-electrospray tandem mass spectrometry. ADMA levels were measured by high-performance liquid chromatography (n = 12). B. Bodyweight of CBS mice at 10 weeks of age. C. Aortic vasomotor response. Mouse thoracic aortic rings were precontracted with KCl and contracted with phenylephrine. Dose-response relaxation was measured for cumulative increments of acetylcholine at 1 minute intervals (n=12). D. Cremaster microvascular vasomotor response. Mouse cremaster microvasculature was superfused with acetylcholine at 3 minutes intervals. The inner lumen diameter of the arteriole was measured before and after superfusion (n=10). Values are mean±SEM; *P<0.01 vs CBS^+/+ mice; #P<0.01 vs CBS^−/+ mice.

Figure 2

eNOS mediates HHcy impaired endothelium-dependent vasorelaxation. A and B. Vasorelaxation with A23187 and SNAP. Aortic rings were precontracted with KCl and contracted with phenylephrine. Vasorelaxation was measured in response to the addition of 10⁻⁵ M A23187 or 10⁻⁶ M SNAP, at the indicated times (n=9). C and D. Vasorelaxation to acetylcholine following L-NAME and OD+CAT. Aortic rings were precontracted with KCl, treated with 3×10⁻⁵ mol/L L-NAME for 20 minutes, or 200 U/mL SOD plus 140 U/mL CAT for 2 minutes, and contracted with phenylephrine. Dose-response relaxation was measured for cumulative increments of acetylcholine at 1 minute intervals (n=9). *P≤0.01 vs CBS^−/+ or CBS^+/+ mice, with identical treatment.

Figure 3

eNOS activity in CBS MAEC and aortas. A and D. Citrulline conversion in MAEC. B and E. Nitrite production in MAEC. Confluent CBS MAEC or control MAEC were incubated with DL-Hcy for 24 hour. eNOS activity was determined by measuring citrulline conversion and by nitrite production (n=9). C. cGMP levels in CBS aortas. Aortic cGMP accumulation with acetylcholine for 3 minutes in 8 week old CBS^+/+ (n=8), CBS^−/+ (n=8), and CBS^−/− (n=4) mice. *P≤0.01 vs CBS^+/+ mice; #P≤0.01 vs CBS^−/+ mice; †P≤0.05 vs control MAEC without Hcy treatment; ‡P≤0.01 vs MAEC without Hcy treatment.

Figure 4

Effect of SOD or GPX-1 overexpression on eNOS activity in MAEC. Control MAEC at 90% confluence were infected with adenovirus vector (Adv-vector) or adenoviruses expressing ecSOD (Adv-ecSOD) or GPX-1 (Adv-GPX-1) at indicated MOI for 24 hour, and then treated with Hcy for 24 hour. eNOS activity was determined by measuring citrulline conversion. Ectopic gene expression was confirmed by Western blotting with antibodies against SOD or GPX-1. Enzymatic activities of SOD and GPX-1 were examined using commercial kits (n=9).

Figure 5

A. Arg transport activity. Confluent MAEC were incubated with DL-Hcy for 24 hour, and then with 2 μCi/mL ³H-L-Arg for 5 minutes. Arg transport activity is expressed as pmol intracellular ³H-L-Arg per mg protein per min (n=9). B. Effect of Arg, H₄, and sepiapterin on eNOS activity. Confluent MAEC were incubated with DL-Hcy, and with Arg, BH₄, or sepiapterin. eNOS activity was determined by measuring citrulline conversion (n=9). †P≤0.01 vs MAEC control; ‡P≤0.05 vs Hcy-treated MAEC (n=9).

Figure 6

eNOS activity, expression, phosphorylation, and PKC activation in HAEC and MAEC. Confluent HAEC or MAEC were incubated with DL-Hcy for 24 hour. PMA were added for the last 10 minutes. GFX were added for the last 30 minutes. eNOS activity was determined by measuring citrulline conversion (A, E, and F) and by nitrite production (B) (n=9). †P≤0.01 vs HAEC or MAEC control. eNOS protein expression and phosphorylation were examined by Western blotting with antibodies against eNOS, eNOS-pT1177, or eNOS-pS495, and reblotted with β-actin antibody (C and D).