Journal Logo

Original Article

Impairment of Endothelium-Dependent Relaxation of Rat Aortas by Homocysteine Thiolactone and Attenuation by Captopril

Liu, Yu-Hui PhD*; You, Yu MD; Song, Tao PhD*; Wu, Shu-Jing PhM*; Liu, Li-Ying PhD*

Author Information
Journal of Cardiovascular Pharmacology: August 2007 - Volume 50 - Issue 2 - p 155-161
doi: 10.1097/FJC.0b013e31805c9410
  • Free



There is growing evidence that elevated plasma levels of homocysteine (Hcy) are suggested to be a powerful independent risk factor for premature atherosclerosis and other inflammatory vascular diseases.1,2 Recent studies hypothesized that the Hcy-induced vascular damage could be due to homocysteine thiolactone (HTL), an Hcy-reactive product formed in several cell types as a result of editing reactions of some amionacyl-tRNA synthetase (AARS).3,4 In fact, chronic infusions of homocysteine thiolactone in experimental animals, including rabbits and baboons, cause atherosclerosis.5,6 It was proposed that HTL could react with lysine residues of protein and damage protein structure.7 The interaction between HTL and low-density lipoprotein (LDL) causes LDL aggregation and higher uptake of homocysteinylated LDL (Hcy-LDL) by cultured macrophages more rapidly than native LDL.8 And Hcy-LDLs could reduce the production of nitric oxide and increase generation of peroxynitrite, a highly reactive nitrogen species originating from nitric oxide and superoxide anion radicals.9 Moreover, it has been demonstrated in vitro that HTL could induce apoptosis of both cultured vascular endothelial cells and human promyeloid HL-60 cells by elevated hydrogen peroxide, and the latter were diminished by antioxidants such as N-Acetylcysteine, vitamin C, and vitamin E.10,11

Our previous study has shown that the captopril could ameliorate the endothelial dysfunction by high-dose of methionine. The aim of this study is to explore the effect of captopril against endothelial dysfunction of isolated rat aortic rings induced by high HTL and to investigate its mechanisms.


Drugs and Chemicals

Acetylcholine, phenylephrine, D,L-homocysteine thiolactone, captopril (Cap), Enalaprilat (Ena), N-acetylcysteine (NAC), sodium nitroprusside, superoxide dismutase (SOD), p-hydroxymercurybenzoate (PHMB), Nomega-nitro-l-arginine methyl ester (L-NAME), apocynin (Apo), and L-arginine were purchased from Sigma Chemical Co. Losartan (Los) was purchased from Merck. They were dissolved in distilled water and diluted with Krebs buffer before use. Other agents were chemically analyzed for purity. The kits for measuring NO, malondialdehyde (MDA), and protein content were products of the Nanjing Jiancheng Bioengineer Institute (Jiangsu, China).

Organ Chamber Experiment

Male Sprague-Dawley rats (200 to 220 g; supplied by the animal Center of Xiangya Medical College of Central South University) were anesthetized with pentobarbital sodium.12 A segment of the thoracic aorta (6 to 7 cm) was removed and immediately placed in 4 mL of Krebs' buffer, which contained the following composition (in mM): NaCl 118.3; KCl 4.7; CaCl2 2.5; MgSO4 1.2; KH2PO4 1.2; NaHCO3 25.0; glucose 11.0). The perivascular tissue of blood vessel was carefully cleaned. The aortic segment was cut into 3 to 4 mM rings. The rings were suspended horizontally between 2 stirrups in organ chambers filled with 5 mL of Krebs' buffer solution gassed with 95% O2 and 5% CO2 at 37°C. One stirrup was connected to an anchor, and the other was connected to a force transducer. The tension of the aortic ring was monitored by a force transducer and recorded on a polygraph (Model RM6240B/C; Chengdu Instruments, China).

The solution in the chamber was changed every 15 minutes. Rings were allowed to equilibrate for 90 minutes under 2 g of resting tension. The rings were then precontracted with a submaximal concentration (1 μM) of phenylephrine (Phe) and relaxed with cumulative concentrations of acetylcholine (0.03 to 3 μM) at the plateau phase of the phenylephrine contraction to evaluate the integrity of the endothelium. The rings with maximal relaxation ratio (Emax) to ACh of 3 μM more than 75% were considered to have intact endothelium and were used in the study.

Experimental Protocol

The experiments were divided into as fallowing groups, and the drugs were added to the chambers of contained Krebs' buffer, respectively. The Krebs' buffer control group; homocysteine thiolactone-treated group in which 3, 10, and 30 mM HTL were respectively added to Krebs' buffer; the captopril-treated groups in which various concentrations of captopril (0.003 to 0.03 mM) alone or with HTL (30 mM) were added to Krebs' buffer. SOD-treated group in which SOD (200 U/mL) alone or with homocysteine thiolactone (30 mM) were added to Krebs' buffer. The same procedure was used in groups with treatment of L-NAME (0.01 mM), p-hydroxymercurybenzoate (PHMB, 0.05 mM). After incubation of 90 min with the above incubations, all rings were recontracted with 1 μM Phe and concentration response curves induced by ACh (0.03 to 3 μM) and sodium nitroprusside (0.03 to 3 μM) were repeatedly recorded. To determine whether captopril exerts scavenging oxygen free radicals and enhances NO production, we compared the effects of SOD (200 U/mL) with those of captopril. Some rings were co-incubated with SOD alone (200 U/mL) or with 30 mM homocysteine thiolactone for 90 min. The same procedure was used in the NO synthase (NOS) inhibitor L-NAME (0.01 mM) group and the free sulfhydryl group blocking agent PHMB (0.05 mM) group.

Assay of NO Concentration and MDA Content

After 1.5 h of incubation of aortic segments, the aortic segments were blotted dry, weighed, and then made into 5% tissue homogenate in ice-cold 0.9% NaCl solution. A supernatant was obtained from tissue homogenate by centrifugalization (1000 × g, 4°C, 10 min). The MDA concentration (thiobarbituric acid reactive substances, TBARS) in the supernatant was measured. Briefly, 1.0 mL of 20% trichloroacetic acid and 1.0 mL of 1% TBARS reagent were added to 100 μL of supernatant and then mixed and incubated at 100°C for 80 min. After cooling on ice, samples were centrifuged at 1000× g for 20 min and the absorbance of the supernatant was read at 532 nm. TBARS results were expressed as MDA equivalents using tetraethoxypropane as standard.13

NO was assayed by the Griess method. Because NO is a compound with a short half life and is rapidly converted to the stable end products nitrate (NO3-) and nitrite (NO2-), the principle of the assay is the conversion of nitrate into nitrite by cadmium and followed by color development with Griess reagent (sulfanilamide and N-naphthyl ethylenediamine) in acidic medium. The total nitrite was measured by Griess reaction. The absorbance was determined with a spectrophotometer to be 540 nM.

Statistical Analysis

Relaxation response was calculated and expressed as percentage of the contraction elicited by phenylephrine. The half maximum effective concentration (EC50) to acetylcholine was estimated by linear regression from log concentration-effect curves. Results are expressed as mean ± SD. Differences between groups were tested for statistical significance by analysis of variance followed by Newman-Keuls test. P < 0.05 was considered significant.


Effects of HTL on Endothelium-Dependent Relaxation of Isolated Rat Aortic Ring

As shown in Figure 1, concentration response curves for ACh (0.01 to 3 μM) evoked endothelium-dependent relaxation (EDR) of isolated rat aortic rings in control group, the Emax reached 83.9 ± 2.5%, and the half maximum effective concentration (EC50) value was 93.9 ± 7.3 nmol/L. Incubation of aortic rings with 3, 10, and 30 mM HTL for 90 minutes significantly attenuated relaxation to ACh in a concentration-dependent mode compared with control group, the Emax response was reduced from 83.9 ± 2.5% to 66.2 ± 2.7%, 51.3 ± 2.1%, and 40.6 ± 2.2%, respectively (n = 6 in each case; P < 0.01). EC50 was 164.4 ± 8.5, 221.3 ± 9.1, 234.3 ± 9.7 nmol/L, respectively (n = 6 in each case; P < 0.01). However, the maximum of endothelium-independent relaxation to SNP (3 μM) was not affected by HTL (Table 1, P > 0.05).

The maximum endothelium-independent relaxation to SNP (3 μM) in isolated rat aortic rings
Effects of HTL on endothelium-dependent relaxation (EDR) in isolated rat aortic rings. After 90-min incubation of aortic rings with 3 to 30 mM homocysteine thiolactone (HTL), the EDR induced by 0.03 to 3 μM Ach was significantly inhibited. Each values are mean ± SD, n = 6. *P < 0.01 versus control group.

Effects of Drugs on Endothelium-Dependent Relaxation of Isolated Rat Aortic Ring

Treatment with captopril in different concentrations (0.003, 0.1, and 0.03 mM; n = 6, respectively) significantly prevented inhibition of EDR induced by HTL (Figure 2). Emax was 58.4 ± 2.5%, 66.2 ± 3.5%, and 76.3 ± 2.1%, respectively, and the EC50 value was 156.5 ± 10.9 nmol/L, 128.5 ± 12.7 nmol/L, and 112.3 ± 10.5 nmol/L, respectively.

The concentration-response curves for Ach-evoked endothelium-dependent relaxation (EDR) of rat isolated aortic rings. The rings were incubated in Krebs' buffer with copresence of homocysteine thiolactone (HTL, 30 mM) and captopril (3 to 30 μmol/L) for 90 min. Values are mean ± SD, n = 6. *P < 0.05 versus HTL (30 mM) group.

There was a significant difference (P < 0.05) compared with those in the HTL group (Figure 2). Figure 3 and 4 show that enalaprilat (0.03 mM) and losartan (0.03 mM) partly ameliorated Ach-induced EDR inhibited by HTL (30 mM) (P < 0.05) but not at the concentration of enalaprilat (0.003, 0.01 mM) (P > 0.05). Emax was 53.6 ± 2.9%, 50.2 ± 4.1%, 46.9 ± 3.7%, 44.8 ± 2.2%, respectively. EC50 was 139.6 ± 10.8 nmol/L, 154.6 ± 12.3 nmol/L, 179.8 ± 14.9 nmol/L, and 208.4 ± 13.0 nmol/L, respectively. As shown in Figure 4, the co-incubation of rings with N-acetylcysteine (0.05 mM), SOD (200 U/mL ), apocynin (0.03 mM), and L-arginine (3 mmol/L) in presence of HTL (30 mM) significantly restored Ach-induced EDR inhibited by HTL (30 mM). The protective effects were parallel with captopril at a concentration of 0.03 mM. Above all, drugs did not affect on EDR of rings induced by Ach compared with control group, P > 0.05 (Figure 5). However, L-NAME (0.01 mM) significantly inhibited the Ach-stimulated EDR; Emax changed from 83.9 ± 2.5% to 49.5 ± 2.3%, and the EC50 value changed from 93.9 ± 7.3 nmol/L to 180.3 ± 13.2 nmol/L, P < 0.05 compared with control group (Figure 5).

Concentration-response curves for Ach-evoked endothelium-dependent relaxation (EDR) of rat isolated aortic rings. The rings were incubated in the copresence of homocysteine thiolactone (HTL, 30 mM) and enalaprilat (3 to 30 μmol/L) for 90 min. Values are mean ± SD, n = 6. *P < 0.05 versus the HTL group.
The concentration response curves for ACh-evoked relaxation of rat isolated aortic rings. The rings were incubated in co-presence of homocysteine thiolactone (HTL, 30 mM) and captopril (0.03 mM), enalaprilat (0.03 mM), SOD (200 U/mL), L-arginine (3 mmol/L), N-acetylcysteine (0.05 mM), losartan (0.03 mM), and apocynin (0.03 mM) for 90 min. Values are mean ± SD, n = 6. *P < 0.05 versus HTL (30 mM) group. **P < 0.05 versus the captopril (0.03 mM) group.
Concentration-response curves for Ach-evoked relaxation of rat isolated aortic rings. The rings were incubated in the presence of captopril (0.03 mM), SOD (200 U/mL), enalaprilat (0.03 mM), PHMB (0.05 mM), losartan (0.03 mM), apocynin (0.03 mM), N-acetylcysteine (0.05 mM), and L-NAME (0.01 mM) for 90 min. Values are mean ± SD, n = 6. *P < 0.05 and **P > 0.05 compared with the control group.

Effects of L-NAME and PHMB on Endothelium-Dependent Relaxation of Isolated Rat Aortic Ring

Co-incubation of aortic rings with L-NAME (0.01 mM) or PHMB (0.05 mM) in the presence of HTL (30 mM) and captopril (0.03 mM) for 90 min significantly attenuated protective effect of captopril on HTL-inhibited EDR. In the captopril, captopril + L-NAME, and captopril + PHMB groups, Emax was 76.3 ± 2.2%, 46.87 ± 1.7%, and 57.69 ± 2.0%, respectively, and EC50 was 112.3 ± 10.5 nmol/L, 163.22 ± 12.7 nmol/L, and 147.83 ± 14.3 nmol/L, respectively (n = 6 in each case, P < 0.05 compared with the captopril group) (Figure 6a). Similarly, the protective effects of N-acetylcysteine (0.05 mM) on HTL-inhibited EDR also counteracted by L-NAME (0.01 mM) or PHMB (0.05 mM). In the N-acetylcysteine, N-acetylcysteine + L-NAME, and N-acetylcysteine + PHMB groups, the Emax values were 70.25 ± 2.4%, 45.6 ± 1.4%, and 56.6 ± 2.3%, and EC50 values were 119.9 ± 11.7 nmol/L, 217.8 ± 13.6 nmol/L, and 187.59 ± 14.2 nmol/L, respectively (n = 6 in each case, P < 0.05 compared with N-acetylcysteine group) (Figure 6b). However, the maximum of endothelium-independent relaxation to SNP (3 μM) was not affected by any group (Table 1; P > 0.05).

Concentration-response curves for Ach-evoked relaxation of rat isolated aortic rings. (a) Rings were co-incubated with captopril (0.03 mM), captopril + L-NAME (0.01 mM), or captopril + PHMB(0.05 mM) in the presence of homocysteine thiolactone (HTL, 30 mM) for 90 min. (b) The rings were co-incubated with N-acetylcysteine (0.05 mM), N-acetylcysteine + PHMB (0.05 mM), or N-acetylcysteine + L-NAME (0.01 mM) in the presence of HTL (30 mM) for 90 min. Values are mean ± SD, n = 6. *P < 0.05 and **P < 0.05 compared with the captopril (0.03 mM) and HTL (30 mM) group or the N-acetylcysteine (0.05 mM) plus HTL (30 mM) group.

Effects of Drugs on Biochemical Index in Aortic Segments

Incubation of isolated aortic segments in HTL for 90 minutes resulted in an elevation of MDA content and reduction of NO content in aortic segments. Treatment with 0.03 mM captopril in the HTL (30 mM) group significantly prevented the increase of MDA content, and protected the release of NO in aortic segments (See Figure 7).

Groups 1 and 2: MDA concentration and NO content of isolated aortic segments in the differently treated groups. The rings were incubated in the co-presence of homocysteine thiolactone (HTL, 30 mM) and captopril (0.03 mM), enalaprilat (0.03 mM), SOD (200 U/mL), L-arginine (3 mmol/L), N-acetylcysteine (0.05 mM), losartan (0.03 mM), and apocynin (0.03 mM) for 90 min. Values are mean ± SD, n = 6. *P < 0.05 compared to the HTL (30 mM) group; #P < 0.05 compared to captopril (0.03 mmol/L) group.


The results of the present study demonstrate that HTL dose-dependently inhibited ACh-induced EDR in isolated rat aortic rings, but it cannot affect endothelium-independent relaxation induced by SNP; it has been demonstrated that endothelium dysfunction induced by HTL is related to the release of NO from endothelial cells. We also found that captopril dose-dependently prevented the inhibition of EDR induced by HTL in rat isolated thoracic aorta (Figure 1) and simultaneously maintained NO content and decreased MDA concentration caused by HTL in rat aortic segments (Table 1). In addition, the inhibition of EDR induced by HTL was partly blocked by N-acetylcysteine and completely counteracted by SOD, apocynin, and L-arginine. However, losartan and enalaprilat (0.03 mM) could only partially improve EDR damaged by HTL. Pretreatment with either the NOS inhibitor L-NAME or a sulfhydryl radical blocking agent PHMB blocked amelioration of both 0.03 mM captopril and N-acetylcysteine on HTL-impaired EDR, respectively.

Hyperhomocysteinemia (HHcy) is thought to promote arteriosclerosis and peripheral arterial disease, in part by impairing the function of endothelium. But there are differences between acute and chronic effects of homocysteine on the vascular dilation that is mediated by the endothelium. There are reports that the flow-induced response of arterioles isolated from HHcy rats are not affected by inhibition of NO synthesis but are abolished by inhibition of prostaglandin synthesis, PGH2/TXA2 receptors, or TXA2 synthase. In contrast, arterioles from rats fed a normal diet dilated in response to increases in flow, a response that is mediated by NO and dilator prostaglandins.14

Development of atherosclerosis involves reactive oxygen species (ROS)-induced oxidative stress in vascular cells. Jakubowski has demonstrated that Hcy-induced vascular damage could be due to HTL, an Hcy-reactive product formed in human cells from the enzymatic conversion of Hcy to the corresponding thioester. This hypothesis is supported by in vitro studies that have demonstrated that HTL induces apoptosis and cell death in human endothelial cells.15 Recent studies have suggested that the toxicity of HTL could be related to its ability to homocysteinylate proteins; in fact, alterations of structural and functional properties were observed in Hcy-proteins. Such as the interaction between HTL and amino groups of apo-B lysyl residues of low-density lipoproteins (LDL) induces the formation of homocystamide-LDL adducts (Hcy- LDL).16,17 Previous studies have shown that an increase in the levels of hydroperoxides in incubation of human endothelial cells in the presence of Hcy-LDL compared with cells treated with control LDL.9 These results suggest that the interaction between Hcy-LDL and cells induced an oxidative damage in human endothelial cells. Other studies have demonstrated that Hcy treatment causes a significant elevation of intracellular superoxide anion leading to increased expression of chemokine receptor in monocytes. NADPH oxidase is primarily responsible for superoxide anion production in monocytes. HTL, even at lower concentrations than Hcy, is cytotoxic and induces cell injury and oxidative damage in cultured EC with generation of ROS, formation of superoxide anions and hydrogen peroxide, and increase in the levels in lipid peroxidation products.18 The present study demonstrates that exposure of isolated aortic rings to HTL elicited a significant inhibition of EDR response to acetylcholine in a dose-dependent manner but did not affect the endothelium-independent relaxation response to SNP. We also observed that HTL significantly increased the MDA contents and decreased NO level in rat aortas. Furthermore, apocynin, an NADPH oxidase inhibitor, reversed the inhibitory effect of HTL on acetylcholine-stimulated aortic relaxation, indicating that the source of superoxide was NADPH oxidase. The same results were suggested in others studies in the HHcy animal's coronary arteries. They have suggested that elevation of plasma homocysteine concentration impairs flow-induced dilation of small coronary arteries and relaxation of peripheral vessels by decreasing bioavailability of NO, an effect that can be reversed by administration of superoxide (O2.−) scavengers. Potential vascular sources of O2.− include xanthine oxidase, cyclooxygenase, NOS, and NAD(P)H oxidases, the relative importance of which may vary among vascular beds and disease conditions. Recent studies suggest that NAD(P)H oxidases are the predominant source of O2.− in coronary arteries. Indeed, although inhibition of xanthine oxidase or cyclooxygenase abolished pathological vasoconstriction in skeletal muscle arterioles of HHcy rats, it failed to restore NO-mediated responses in HHcy coronary arteries.19-22 Since reduced NO and increased O2.− formation are associated with the progression of vascular diseases, this interaction may play a part in eliciting the mechanisms of HTL that induce vascular damage. SOD, a scavenger of O2.− anions, is generally accepted as evidence for the involvement of O2.− anion in a particular system. O2.− anions are noted to inhibit endothelium-mediated relaxation by inactivating endothelium-dependent relaxing factor (EDRF).23 NAC, a precursor of reduced glutathione, which is a thiol-containing compound, have been reported to combine with NO to produce more stable nitrosothiol compounds, which are potent relaxants of vascular smooth muscle.24 In addition, it has been suggested that it may also interact with ROS, thus sparing NO and enhancing its action, a free-radical scavenging effect.25 The above results suggest that increasing NO degradation via the production of oxygen-derived free radicals and reducing endothelium-derived NO synthesis may be involved in endothelial dysfunction caused by HTL.

Captopril, an angiotensin-converting enzyme inhibitor, can reportedly scavenge O2.− anion, which is partly attributed to its sulfhydryl group, besides inhibiting production of angiotensin II, which has been widely used as a potent drug in the treatment of cardiovascular diseases such as hypertension and atherosclerosis.26-28 In summary, there is accumulating evidence from both clinical studies and laboratory investigations that the renin-angiotensin II system represents a critical, causal link in the pathogenesis of human atherosclerosis. Angiotensin II is also a very potent factor for both inflammation and oxidative stress by stimulating NADPH oxidase, oxidized LDL, and so on.29 Several investigators also report that captopril promotes endothelium-derived NO production or release and attenuates the impairment of EDR.30 The present study demonstrates that captopril (0.003 to 0.03 mM) protects the impairment of EDR by HTL in rat aortic rings in a dose-dependent manner and decreases MDA content and elevates the NO level. However, only a high dose of enalaprilat (0.03 mM) partially reversed the HTL-induced endothelial dysfunction in this study. Losartan, which is an AT1 receptor blocker, only partially reversed the effect also. In addition, we also found that the beneficial effect of captopril was blocked by following pretreatment with not only the NOS inhibitor L-NAME but also the free sulfhydryl group blocking agent PHMB. Furthermore, L-arginine, which is a precursor of NO, ameliorated the impairment of EDR elicited by HTL in rat aortic rings accompanied with decrease of MDA content and elevation of NO level. On the basis of these results, we speculate that scavenging oxygen free radicals and improving NO production/release may be involved in the protective effects of captopril against endothelial dysfunction induced by HTL in isolated rat aorta strips.

Captopril eliciting this protection may be related to the sulfhydryl group-mediated scavenging activity of free radicals. Sulfhydryl compounds are a major class of protective agents against oxygen radicals generated by radiation.31 These drugs can neutralize oxygen radicals by either hydrogen atom donation or electron transfer reaction. It also appears that the protective effects of sulfhydryl agents correlate with their direct hydroxyl radical (·OH) scavenging abilities than with their antioxidative potency.32,33 These considerations are further supported by the fact that both the non-sulfhydryl ACE inhibitor enalaprilat and AT1 receptor only exhibited partial protection. A number of studies have demonstrated that ACE inhibitors have the effect of scavenging reactive oxygen molecules and antioxidation independently of ACE inhibition; the former is probably due to the sulfhydryl group, and the latter is sulfhydryl independent.34 Our previous study has also shown that captopril's amelioration endothelial dysfunction by high-dose methionine is more effective than enalapril because of its antioxidant effect and scavenging oxygen free radicals due to containing the -SH group.35


In conclusion, captopril protects the impairment of EDR induced by HTL in isolated rat aorta rings, which may be partly due to scavenging oxygen free radicals and increasing NO production and may also be sulfhydryl-dependent.


1. Refsum H, Ueland PM, Nygard O, et al. Homocysteine and cardiovascular disease. Ann Rev Med. 1998;49:31-61.
2. Lawrence AB, Geoff H, Ji Zhou, et al. Hyperhomocysteinemia and its role in the development of atherosclerosis. Clin Biochem. 2003;36:431-441.
3. Jakubowski H. Calcium-dependent human serum homocysteine thiolactone hydrolase. A protective mechanism against protein N-homocysteinylation. J Biol Chem. 2000;75:3957-3962.
4. Jakubowski H. Metabolism of homocysteine thiolactone in human cell cultures: possible mechanism for pathological consequences of elevated homocysteine levels. J Biol Chem. 1997;272:1935-1942.
5. McCully KS, Wilson RB. Homocysteine theory of arteriosclerosis. Atherosclerosis. 1975;22:215-227.
6. Harker LA, Ross R, Slichter SJ, et al. Homocysteine-induced arteriosclerosis. The role of endothelial cell injury and platelet response to its genesis. J Clin Invest. 1976;58:731-741.
7. Jakubowski H. Homocysteine-thiolactone and S-nitroso-homocysteine mediate incorporation of homocysteine into protein in humans. Clin Chem Lab Med. 2003;41:1462-466.
8. Naszewio M, Mirkiew E, Mccully KS. Thiolaction of low-density lipoprotein by homocystene-thiolactone causes increased aggregation and altered interaction with cultured macrophages. Nutr Metab Cardiovasc Dis. 1994;4:70-77.
9. Vigini A, Nanetti L, Ferretti G, et al. Modification induced by homocysteine and low-density lipoprotein on human aortic endothelial cells: an in vitro study. J Clin Endocrinol Metab. 2004;89:4558-4561.
10. Kerkeni M, Tnani M, Chuniaud L, et al. Comparative study on in vitro effects of homocysteine thiolactone and homocysteine on HUVEC cells: Evidence for a stronger proapoptotic and proinflammative homocysteine thiolactone. Mol Cell Biochem. 2006;291:199-126.
11. Huang R, Huang S, Ling B, et al. N-acetylcysteine, vitamin C, and vitamin E diminish homocysteine thiolactone-induced apoptosis in human promyeloid HL-60 cells. J Nutr. 2002;132:2151-2156.
12. Chen SX, Liu LY, Sun XY, et al. Captopril restores endothelium-dependent relaxation induced by advanced oxidation protein products in rat aorta. J Cardiovasc Pharmacol. 2005;46:803-809.
13. Wang SX, Xiong XM, Song T, et al. Protective effects of cariporide on endothelial dysfunction induced by high glucose. Acta Pharmacologica Sinica. 2005;26:329-333.
14. Bagi Z, Ungvari Z, Szollár L, et al. Flow-induced constriction in arterioles of hyperhomocysteinemic rats is due to impaired nitric oxide and enhanced thromboxane A2 mediation. Arterioscler Thromb Vasc Biol. 2001;21:233-237.
15. Jakubowski H. Homocysteine thiolactone and protein homocysteinylation in human endothelial cells: implications for atherosclerosis. Circ Res. 2000;7:87:45-51.
16. Jakubowski H. Protein homocysteinylation: possible mechanism underlying pathological consequences of elevated homocysteine levels. FASEB J. 1999;13:2277-2283.
17. Jakubowski H. Translational incorporation of S-nitroso-homocysteine into protein. J Biol Chem. 2000;275:21813-21816.
18. Zappacosta B, Mordente A, Persichilli S, et al. Is homocysteine a pro-oxidant? Free Radic Res. 2001;35:499-505.
19. Ungvari Z, Csiszar A, Bagi Z, et al. Impaired NO-mediated flow-induced coronary dilation in hyperhomocysteinemia: morphological and functional evidence for increased peroxynitrite formation. Am J Pathol. 2002;161:145-153.
20. Bagi Z, Ungvari Z, Koller A. Xanthine oxidase-derived reactive oxygen species convert flow-induced arteriolar dilation to constriction in hyperhomocysteinemia. Arterioscler Thromb Vasc Biol. 2002;22:28-33.
21. Mohazzab KM, Kaminski PM, Wolin MS. NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium. Am J Physiol. 1994;266:H2568-H2572.
22. Ungvari Z, Sarkadi-Nagy E, Bagi Z, et al. Simultaneously increased TxA2 activity in isolated arterioles and platelets of rats with hyperhomocysteinemia. Arterioscler Thromb Vasc Biol. 2000;20:1203-1208.
23. Mercie P, Garnier O, Lascoste L, et al. Homocysteine thiolactone induces caspase-independent vascular endothelial cell death with apoptotic features. Apoptosis. 2000;5:403-411.
24. Gryglewaki RJ, Palmer RM, Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived relaxing factor. Nature (Lond.). 1986;320:454-456.
25. Sunman W, Hughes AD, Sever PS. Free-radical scavengers, thiol-containing reagents and endothelium-dependent relaxation in isolated rat and human resistance arteries. Clin Sci. 1993;84:287-295.
26. Knight KR, MacPhadyen K, Lepore D, et al. Enhancement of ischaemic rabbit skin flap sur-vival with the antioxidant and free-radical scavenger N-acetylcysteine. Clin Sci. 1991;81:31-36.
27. Hayek T, Attias J, Smith J, et al. Antiatherosclerotic and antioxida- suggested tive effects of captopril in apolipoprotein e deficient mice. J Cardiovasc Pharmacol. 1998;31:540-544.
28. Lonn E. Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers in atherosclerosis. Curr Atheroscler. 2002;4:363-372.
29. Weiss D, Sorescu D, Taylor WR. Angiotensin II and atherosclerosis. Am J Cardiol. 2001;87(Suppl):25C-32C.
30. Napoli C, Sica V, De Nigris F, et al. Sulfhydryl angiotensin-converting enzyme inhibition induces sustained reduction of systemic oxidative stress and improves the nitric oxide pathway in patients with essential hypertension. Am Heart J. 2004;148:e5-e11.
31. Yasutaka O, Kiyotaka K, Seigo S, et al. Impairment of endothelium-dependent relaxation of rabbit aortas by cigarette smoke extract-role of free radicals and attenuation by captopril. Atherosclerosis. 1997;131:195-202.
32. Simic MG. Mechanism of inhibition of free-radical process in mutagenesis and carcinogenesis. Mutat Res. 1988;202:377-386.
33. Mak IT, Freedman AM, Dickens BF, et al. Protective effects of sulfhydryl-containing angiotensin-converting enzyme inhibitors against free radical injury in endothelial cells. Biochem Pharmacol. 1990;40:2169-2175.
34. Chopra M, Beswick H, Clapperton M, et al. Antioxidant effects of angiotensin-converting enzyme (ACE) inhibitors: free radical and oxidant scavenging are sulfhydryl dependent, but lipid peroxidation is inhibited by both sulfhydryl- and nonsulfhydryl-containing ACE inhibitors. J Cardiovasc Pharmacol. 1992;19:330-340.
35. Liu YH, Liu LY, Wu JX, et al. Comparison of captopril and enalapril to study the role of the sulfhydryl-group in improvement of endothelial dysfunction with ACE inhibitors in high dieted methionine mice. J Cardiovasc Pharmacol. 2006;47:82-88.

angiotensin-converting enzyme inhibitors; homocysteine thiolactone; endothelium-dependent relaxation; rat aorta; oxygen free radicals

© 2007 Lippincott Williams & Wilkins, Inc.