Diabetes mellitus is associated with accelerated atherosclerosis and an increased prevalence of cardiovascular diseases, which have become the principal cause of morbidity and mortality in individuals with diabetes. Endothelial dysfunction characterized by impaired nitric oxide (NO)-mediated and endothelium-dependent vasodilatation is an early step in the development of diabetic vascular diseases. Advanced glycated end products (AGEs), the terminal reaction products of nonenzymatic glycation of proteins, have been implicated in cardiovascular complications associated with diabetes.1 Recently, we demonstrated that endothelium-dependent relaxation of thoracic aorta was significantly attenuated in streptozotocin-induced diabetic rats when their plasma levels of glycated serum proteins reached a level of 1.70 mmol/L compared with control rats whose glycated serum protein's serum concentration was 1.10 mmol/L.2 Subsequently, we confirmed in in vitro study that exposure of normal rat aortic rings to exogenous glycated protein at a concentration of 1.70 mmol/L for 60 minutes did induce a significant impairment of endothelium-dependent relaxation, whereas incubation of rat aortic rings with 1.10 mmol/L glycated protein had no significant effect on endothelium-dependent relaxation.3 Similar results were reported by Moore et al that injection of exogenous glycated proteins to normal rats mimicked the defective endothelium-dependent relaxation observed in diabetes mellitus.4 AGEs have been shown to accelerate the inactivation of NO directly or via increasing the production of reactive oxide species through the activation of protein kinase C5 and suppressing the expression of endothelial NO synthase (NOS) with a subsequent reduction in NO production,6 thereby decreasing the bioavailability of NO and leading to endothelial dysfunction.
In addition to the mechanisms mentioned above are involved in the deleterious effects of AGEs on endothelial function, elevated endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) may be another significant factor. ADMA is derived from the hydrolysis of proteins containing methylated arginine residues and may competitively inhibit the activity of NOS and decrease the production of NO in endothelial cells, subsequently impairing NO-mediated and endothelium-dependent vasodilatation. The elevation of serum ADMA levels has been demonstrated to be associated with impaired endothelium-dependent relaxation in animals and patients with diabetes mellitus,7-10 atherosclerosis,11-13 hyperhomocysteinemia,14 as well as in aging.15,16 Recently, we and others have shown that chronic treatment with insulin and metformin normalized serum level of glycated protein,2,10 accompanied by a corresponding decrease of serum ADMA levels and a significant improvement of impaired endothelium-dependent relaxation,2 indicating that the elevation of endogenous ADMA is closely related to the formation of advanced glycated end products and implicated in endothelial dysfunction associated with diabetes. ADMA is largely metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH) to L-citrulline and dimethylamine.17 Therefore, DDAH plays a crucial role in the regulation of endogenous ADMA concentration. Recently, it has been shown that DDAH is regulated in a redox-sensitive fashion.18 Under conditions such as diabetes,8 hyperhomocysteinemia,14 and hypercholesterolemia,19 increased vascular oxidative stress may inhibit DDAH activity and accelerate ADMA accumulation. Therefore, we hypothesized that AGEs-induced impairment of endothelium-dependent relaxation may be secondary to the inhibition of DDAH activity via oxidative stress.
Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors are widely used as hypocholesterol drugs and have been shown to effectively lower serum lipids and significantly decrease morbidity and mortality of cardiovascular diseases. The beneficial effects of statins are generally attributed to their lipid-lowering properties. Recent experimental and clinical evidence indicates that statins have some cholesterol-independent effects such as decreasing oxidative stress, improving endothelial function, enhancing the stability of atherosclerotic plaques, and suppressing vascular inflammation.20 It has been shown that pravastatin, a potent inhibitor of HMG-CoA reductase, improved endothelial function in animals and patients with diabetes and atherosclerosis.21-22 Based on the important role of AGEs in the development of diabetic vascular complications and the beneficial effect of pravastatin on endothelial function, it is very necessary to investigate whether pravastatin can directly protect endothelium against the impairment induced by AGEs and its potential mechanisms. In the present study, we sought to determine whether glycated bovine serum albumin (AGE-BSA) inhibits the activity of DDAH to contribute to its adverse effect on endothelium-dependent relaxation of rat aorta in vitro and to investigate effects of pravastatin on the inhibition of DDAH activity and endothelial dysfunction induced by AGE-BSA.
MATERIALS AND METHODS
Measurement of Vascular Function
The Animal Care and Use Committee of Central South University approved the study protocol. Sprague-Dawley rats of either sex (weighing 220 ± 30 g) were obtained from Central South University Animal Services (Hunan, China) and were anesthetized with sodium pentobarbital (30 mg/kg, i.p.). The thoracic aorta was immediately isolated and placed in 4°C Krebs bicarbonate buffer containing (in mmol/L) NaCl, 118.3; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25.0; and glucose, 11.0, and then cleaned of adherent connective tissue. The aortic segment was cut into rings 3-4 mm in length, with special care taken to avoid stretching and touching the luminal surface. The rings were suspended horizontally between 2 stirrups in organ chambers filled with 5 mL Krebs solution at 37°C and aerated continuously with 95% O2 and 5% CO2. One stirrup was connected to an anchor, and the other was connected to a force transducer for recording isometric tension. The solution in the chambers was changed every 15-20 minutes. Rings were equilibrated for 60 minutes under 2 g resting tension and then challenged with 60 mmol/L KCl at least 3 times until a reproducible maximal contractile response was obtained. After repeated washing and a further equilibration for 30 minutes, the rings were contracted with a submaximal concentration of phenylephrine (1 μmol/L), and relaxation response to cumulative concentration of acetylcholine (0.03-3 μmol/L) was tested at the plateau phase of phenylephrine contraction. Before finishing the experiment, relaxation with sodium nitroprusside (10 μmol/L) at the plateau phase of phenylephrine contraction was also tested. The rings with the maximal relaxation (Emax) to 3 μmol/L acetylcholine more than 80% were considered as endothelium-intact and used in the study.
After each ring was serially washed, aortic rings were incubated with 1.10 mmol/L or 1.70 mmol/L AGE-BSA for 60 minutes in AGE-BSA groups and with 1.70 mmol/L nonglycated bovine serum albumin (BSA) for 60 minutes in BSA group, respectively. The rings of pravastatin-treated groups were preincubated with different concentrations of pravastatin (0.3-3 mmol/L) for 15 minutes and then coincubated with 1.70 mmol/L AGE-BSA for another 60 minutes, respectively. In the pravastatin alone group, aortic rings were incubated with 1 mmol/L pravastatin without AGE-BSA for 75 minutes. Control rings were incubated with Krebs solution for 75 minutes in the absence of AGE-BSA or drugs.
To investigate whether increased superoxide anion and protein kinase C (PKC) activity were involved in the impaired endothelium-dependent relaxation after exposure to AGE-BSA, rings were preincubated with intracellular antioxidant pyrrolidine dithiocarbamate (PDTC, 30 μmol/L) and PKC inhibitor chelerythrine (1 μmol/L) for 15 minutes and then coincubated with 1.70 mmol/L AGE-BSA for 60 minutes.
To determine whether pravastatin exerts its protective effect on the impaired endothelium-dependent relaxation induced by AGE-BSA by increasing the production of prostacyclin or NO in endothelial cells, rings were incubated with indomethacin (10 μmol/L), an inhibitor of cyclooxygenase and NG-nitro-L-arginine methyl ester (L-NAME, 30 μmol/L), an inhibitor of NOS in the presence of 1.0 mmol/L pravastatin for 15 minutes followed by coincubation with AGE-BSA (1.70 mmol/L) for another 60 minutes.
After the above incubations, all rings were washed repeatedly and then recontracted with 1 μmol/L phenylephrine and relaxation responses to acetylcholine (0.03-3 μmol/L) were repeated. Before finishing the experiment, relaxation to sodium nitroprusside (10 μmol/L) at the plateau phase of the phenylephrine contraction was also tested in isolated aortic rings.
Determination of DDAH Activity, Nitrite/Nitrate, and MDA Contents
Thoracic aortas were isolated from sodium pentobarbital-anesthetized Sprague-Dawley rats and then cleaned of residual blood, fat, and loose connective tissue and opened longitudinally. Vessels were divided into 7 groups in the following experiments. In the AGE-BSA group or BSA control group, aortas were incubated with 1.70 mmol/L AGE-BSA or 1.70 mmol/L nonglycated BSA for 60 minutes at 37°C, aerated continuously with 95% O2 and 5% CO2 but without tension challenge. Aortas from the drug-treatment groups were preincubated with pravastatin (1 mmol/L), PDTC (30 μmol/L), and chelerythrine (1 μmol/L) for 15 minutes and then coincubated with 1.70 mmol/L AGE-BSA for 60 minutes, respectively. In addition, aortas were incubated with 1 mmol/L pravastatin alone for 75 minutes to examine the effects of pravastatin per se on DDAH activity. Control aortas were incubated with Krebs solution for 75 minutes in the absence of AGE-BSA or any drugs.
When the incubations finished, aortas were homogenized in ice-cold phosphate buffer solution (0.1 mol/L, pH 6.5). The homogenate was centrifuged at 3500 g for 30 minutes at 4°C, and the supernatant was assayed for DDAH activity, nitrite/nitrate levels, MDA contents, and protein content. DDAH activity was assayed by the conversion of L-citrulline from ADMA. Briefly, the homogenate (50 μL) was incubated with 1mmol/L ADMA and 0.1 mol/L phosphate buffer solution (pH 6.5) in a total volume of 0.5 mL for 120 minutes at 37°C. The reaction was stopped by the addition of equal volume of 10% trichloroacetic acid. Then the supernatant was incubated with diacetyl monoxime (0.8%) and antipyrine (0.5%) at 60°C for 110 minutes. The amounts of L-citrulline formed were determined by spectrophotometric analysis at 466 nm as described previously.23 As the assay blank, the homogenate was subjected to the same determination process of DDAH activity in the absence of ADMA to provide the background values. Genuine DDAH activity was obtained by experimental data subtracting the background values. One unit of the enzyme was defined as the amount that catalyzed the formation of 1 μmol/L L-citrulline from ADMA per minute at 37°C.
The contents of nitrite in the supernatant were determined using the commercial kit to reflect the levels of NO by converting nitrate, the stable end product of NO, into nitrite, catalyzed by nitrate reductase. The total nitrite was measured at 550 nm with a spectrophotometer.
To assess whether the changes in aortic DDAH activity and NO contents as a consequence of AGE-BSA or pravastatin treatment could be related to alterations in oxidative stress, content of MDA, derived from lipid peroxidation in aorta was also determined using the commercial kit.
The protein content of the supernatant was measured by Coomassie brilliant blue assay kit with bovine serum albumin as the standard.
All chemicals were of the highest purity available. Phenylephrine, acetylcholine, sodium nitroprusside, PDTC, chelerythrine, L-NAME, indomethacin, antipyrine, diacetyl monoxime, ADMA, and bovine serum albumin were all purchased from Sigma Chemical Co (St Louis, MO). The kits for measuring NO, malondialdehyde (MDA), and protein content were products of the Nanjing Jiancheng Bioengineer Institute (Jiangsu, China). Pravastatin was kindly contributed by Blue Treasure Pharmaceutical Factory (Guangdon, China).
Relaxation was expressed as percentage of contraction elicited by phenylephrine. The half-maximum effective concentration (EC50) response to acetylcholine was estimated by linear regression from log concentration-effect curves. Results were expressed as mean ± SEM. Differences between groups were tested with ANOVA followed by the Newman-Keuls test. P < 0.05 was considered significant.
Vascular Function Analysis
There were no differences in contractile responses of rat aortic rings to 1 μmol/L phenylephrine among groups in the present experiment (data not shown). When the phenylephrine-induced contraction had reached a plateau, acetylcholine (0.03-3 μmol/L) was cumulatively added into organ chambers to determine the endothelium-dependent relaxation of aortic rings. In aortic rings of the control group, acetylcholine evoked a concentration-dependent relaxation. The maximal response (Emax) reached 93 ± 3%, and the EC50 value was 88.90 ± 1.13 nmol/L. However, exposure of aortic rings to 1.70 mmol/L AGE-BSA for 60 minutes significantly inhibited the endothelium-dependent relaxation in response to acetylcholine, resulting in a lower Emax and higher EC50 value compared with control group (Emax was 52 ± 3% versus 93 ± 3%, and EC50 was 276.69 ± 1.03 versus 88.90 ± 1.13 nmol/L, n = 5, P < 0.01; Fig. 1 and Table 1). By contrast, exposure of aortic rings to 1.10 mmol/L AGE-BSA or 1.70 mmol/L BSA for 60 minutes didn't affect endothelium-dependent relaxation when compared with control group (Fig. 1 and Table 1).
Pretreatment with various concentrations of pravastatin (0.3-3 mmol/L) markedly attenuated the inhibition of endothelium-dependent relaxation caused by AGE-BSA in a concentration-dependent manner. The Emax value was enhanced, and the EC50 value was reduced in aortic rings of pravastatin-treated groups compared with the AGE-BSA group (Fig. 2 and Table 1). But pravastatin per se had no effect on endothelium-dependent relaxation of rat aortic rings compared with control group (Emax was 92 ± 3% versus 93 ± 3%, and EC50 was 87.56 ± 1.53 versus 88.90 ± 1.13 nmol/L, n = 5, P = NS; Fig. 2 and Table 1).
Preincubation of aortic rings with combined pravastatin (1 mmol/L) and indomethacin (10 μmol/L) or L-NAME (30 μmol/L) following exposure to 1.70 mmol/L AGE-BSA, the former combination did not affect the protective effect of pravastatin on endothelial function (Emax was 92 ± 2% versus 90 ± 3%, EC50 was 95.06 ± 1.18 versus 99.80 ± 1.42 nmol/L, n = 5, P = NS), whereas the latter combination completely abolished the protective effect of pravastatin on AGE-BSA-induced impairment of endothelium-dependent relaxation compared with 1 mmol/L pravastatin-treated group (Emax was 60 ± 3% versus 90 ± 3% and EC50 was 220.80 ± 1.07 versus 99.80 ± 1.42 nmol/L, n = 5, P < 0.01, Fig. 3 and Table 1). These results indicate that the protective effect of pravastatin on impaired endothelium-dependent relaxation induced by AGE-BSA is not a result of increased production of prostacyclin but rather of increased NO synthesis in endothelial cells.
In addition, we also observed a significant amelioration of impaired endothelium-dependent relaxation induced by AGE-BSA when aortic rings were pretreated with chelerythrine (1 μmol/L), an inhibitor of PKC. The Emax and the EC50 value had returned to levels similar to control (Fig. 4 and Table 1). This result indicates that activation of PKC may be implicated in the inhibition of AGE-BSA on endothelium-dependent relaxation. In an analogous manner, incubation of aortic rings with the intracellular antioxidant PDTC (30 μmol/L) also showed a significant improvement of impaired endothelium-dependent relaxation by AGE-BSA. The Emax and EC50 value were 92 ± 2% and 91.20 ± 1.27 nmol/L, which were similar to those of 1 mmol/L pravastatin (Fig. 4 and Table 1). By contrast, endothelium-independent relaxation responses to sodium nitroprusside were not different in aortic rings of the groups after exposure to AGE-BSA or treatment with the drugs in the present study (data not shown).
Figure 5 shows the effects of AGE-BSA and drugs on DDAH activity of rat aorta. As expected, decreased DDAH activity was observed after aorta exposure to 1.70 mmol/L AGE-BSA for 60 minutes compared with control (0.032 ± 0.002 versus 0.095 ± 0.003 U/g protein, n = 5, P < 0.01). Pretreatment of aortas with 1.0 mmol/L pravastatin and then exposure to AGE-BSA restored DDAH activity nearly to a normal level compared with AGE-BSA group (0.084 ± 0.002 versus 0.032 ± 0.002 U/g protein, n = 5, P < 0.01). But nonglycated BSA or pravastatin per se did not affect DDAH activity of rat aortas compared with control (0.089 ± 0.002 or 0.100 ± 0.002 versus 0.095 ± 0.003 U/g protein, n = 5, P = NS). Moreover, treatment with 30 μmol/L PDTC or 1 μmol/L chelerythrine also preserved DDAH activity of rat aortas from inhibition of AGE-BSA (0.086 ± 0.006 or 0.067 ± 0.009 versus 0.032 ± 0.002 U/g protein, n = 5, both P < 0.01).
In parallel to the alterations of DDAH activity, aortic nitrite/nitrate contents were also remarkably decreased after exposure to 1.70 mmol/L AGE-BSA for 60 minutes (from 0.84 ± 0.05 μmol/g protein in control down to 0.30 ± 0.03 μmol/g protein in the AGE-BSA group, n = 5, P < 0.01; Fig. 6). Pretreatment with pravastatin increased the nitrite/nitrate levels from 0.30 ± 0.03 μmol/g protein in AGE-BSA group to 0.73 ± 0.03 μmol/g protein in the pravastatin plus AGE-BSA group (Fig. 6). In a similar way, both PDTC (30 μmol/L) and chelerythrine (1 μmol/L) significantly elevated nitrite/nitrate of rat aortas in the presence of 1.70 mmol/L AGE-BSA compared with the AGE-BSA group (both n = 5, P < 0.01; Fig. 6).
On the contrary, aortic contents of MDA, the reflector of lipid peroxidation, were remarkably elevated in rat aortas incubated with AGE-BSA (1.70 mmol/L) for 60 minutes compared with control aortas (1.95 ± 0.14 μmol/g protein versus 0.78 ± 0.04 μmol/g protein, n = 5, P < 0.01; Fig. 7). Pretreatment with pravastatin prevented the elevation of MDA in rat aortas compared with AGE-BSA-treated aortas (0.90 ± 0.04 μmol/g protein versus 1.95 ± 0.14 μmol/g protein, n = 5, P < 0.01; Fig. 7), which was similar to the effect of antioxidant PDTC (30 μmol/L) or protein kinase C inhibitor chelerythrine (1 μmol/L). By contrast, neither nonglycated BSA nor pravastatin per se had a significant effect on nitrite/nitrate or MDA contents of rat aortas compared with control (all P = NS; Figs. 6 and 7).
The present study demonstrated for the first time that AGE-BSA significantly inhibited DDAH activity and concomitantly decreased nitrite contents in isolated rat aorta, and pravastatin treatment not only restored DDAH activity but also improved the impairment of endothelium-dependent relaxation and normalized nitrite levels in rat aorta after exposure to AGE-BSA. These results indicate that the inhibition of DDAH activity induced by AGE-BSA may contribute to the detrimental effects of AGEs on vascular endothelial function, and pravastatin may be an effective pharmacological approach in preserving DDAH activity and endothelial function from the inhibition of AGEs.
It is well documented that AGEs are an important risk factor in the pathogenesis of diabetic vascular complications. Our and others' previous studies demonstrated that direct treatment of normal rat aortic rings with AGE-BSA might mimic the inhibition of endothelium-dependent relaxation observed in diabetic rats3 and that local application of AGE-BSA in rabbit collared carotid artery stimulated intimal hyperplasia.24 In this study, we have further proved that acute exposure of rat aortic rings to exogenous AGE-BSA did induce a significant inhibition of endothelium-dependent relaxation response to acetylcholine in a dose-dependent manner but did not affect the endothelium-independent relaxation response to sodium nitroprusside. Chronic exposure to AGEs might have different or even worse consequences than an in vitro situation, as reviewed by Basta et al.25 It has been reported that the extent of impairment in endothelium-dependent vasorelaxation was positively correlated with serum concentrations of AGEs in diabetic patients.1 These results support the proposal that AGEs play a key role in the development of endothelial dysfunction or diabetic vascular complications.
Although the precise mechanisms underlying the detrimental effects of AGEs on vascular endothelial function are not fully known, increased oxidative inactivation of NO and decreased production of NO in endothelial cells could be implicated in this process. Several studies have demonstrated that AGEs stimulate superoxide anion production by activating PKC in vascular cells, leading to the inactivation of NO by oxidation.5,26 Conversely, PKC inhibitor and antioxidant may improve the impairment of endothelium-dependent relaxation in streptozotocin-induced diabetic animals.27,28 In the present study, we observed that AGE-BSA significantly increased the MDA contents and decreased nitrite levels in addition to inhibiting DDAH activity in rat aortas. The PKC inhibitor chelerythrine or intracellular antioxidant PDTC reversed these effects of AGE-BSA, suggesting that enhanced oxidative stress via PKC activation may be involved in AGEs-induced endothelial dysfunction.
Accumulating evidence has suggested that elevated ADMA, an endogenous competitive inhibitor of NOS, is closely related to the development of endothelial dysfunction.2,7-10 Recent attention has been focused on the regulatory role of DDAH, an enzyme responsible for the degradation of endogenous ADMA, on the concentration of ADMA under various pathologic conditions. It has been reported that pharmacological inhibition of DDAH leads to local accumulation of ADMA, inhibition of NOS activity in endothelial cells, and impairment of endothelium-dependent relaxation in isolated blood vessels,29 whereas overexpression of DDAH in cultured endothelial cells significantly increased NOS activity and NO production.30 Furthermore, DDAH activity has been shown to be inhibited in vessels from streptozotocin-induced diabetic rats8 and hypercholesterolemic rabbits19 and in cultured endothelial cells after incubation with high glucose8 or with homocysteine14 and oxidized LDL.19 In view of these reports, we speculated that the impairment of endothelium-dependent relaxation induced by AGE-BSA may be secondary to the decrease of DDAH activity. This hypothesis was tested in the present study by determining DDAH activity in rat aorta, and it was found that DDAH activity was significantly inhibited after exposure of rat aorta to AGE-BSA, which was accompanied by a corresponding decrease of aortic nitrite/nitrate contents and inhibition of NO-mediated and endothelium-dependent relaxation. Taken together, these results suggest that inhibition of DDAH activity induced by AGE-BSA and subsequent accumulation of endogenous inhibitor of NOS is an important contributor to the AGE-induced endothelium-dependent vasodilator dysfunction.
How AGEs inhibit DDAH activity is not clear. Recently, Leiper and colleagues shed some light on this issue.18 They identified a highly reactive cysteine residue (Cys-249) in the active site of DDAH, and the sulfhydryl group of the cysteine residue predisposes DDAH to easy oxidation or nitrosation to lose its activity. Furthermore, recent studies from other laboratories have reported that antioxidant polyethylene glycol-conjugated superoxide dismutase and PDTC preserved DDAH activity in endothelial cells after exposure to high glucose8 and homocysteine.14 These studies indicate that DDAH could be regulated by cardiovascular risk factors in a reactive oxygen species-sensitive manner. In the present study, we found that PKC inhibitor chelerythrine and intracellular antioxidant PDTC could reverse the adverse effects of AGE-BSA on DDAH activity and endothelial function of rat aorta, possibly by decreasing oxidative stress, as evidenced by the decrease of aortic MDA levels, thereby preserving the sulfhydryl in the active site of DDAH from oxidative attack.
Because endothelial dysfunction is an early stage in developing diabetes, reversal of this condition may prevent the onset or retard the progression of diabetic vascular complications. Pravastatin, a competitive inhibitor of HMG-CoA reductase, has been widely used as potent hypocholesterolemic drug in the treatment of hypercholesterolemia to prevent the development of cardiovascular diseases.31 Furthermore, pravastatin therapy has been shown to preserve endothelial function in aorta of diabetic rats21 or in carotid arteries of insulin-resistant rats.32 Our study provides the first evidence that pravastatin can protect endothelium against injury induced by AGE-BSA in isolated rat aortas. Moreover, we have recently demonstrated that pravastatin could reverse endothelial dysfunction of rat aorta induced by lysophosphatidylcholine in another study.33 These studies suggest that pravastatin may be an effective pharmacological approach to preserving endothelial function and preventing diabetic vascular complications.
The mechanisms responsible for the beneficial effects of pravastatin on endothelium-dependent relaxation are not fully understood. Increasing biosynthesis of endothelial NO may be one of the potential mechanisms. Results from the present study indicate that pravastatin protection of vascular endothelial function was related to increasing synthesis of endothelial NO, as evidenced by increased nitrite/nitrate levels in rat aortas. In addition, we also found that the protection by pravastatin of vascular endothelial function could be completely abolished by the inhibitor of NO synthase L-NAME but not by the cyclooxygenase inhibitor indomethacin. Similar results have been shown in normocholesterolemic subjects with coronary artery disease.20,34 In addition to increasing synthesis of NO, the present study provides important evidence that pravastatin up-regulated vascular DDAH activity. Because DDAH is a key regulator of endogenous NOS inhibitor ADMA levels, increased DDAH activity may accelerate the degradation of endogenous ADMA, thereby enhancing the activity of eNOS and eventually augmenting the synthesis of NO. Pravastatin may up-regulate DDAH activity by its antioxidant property, which could be another potential mechanism responsible for the protective effect of pravastatin on endothelial function. Pravastatin has been found to inhibit superoxide anions production stimulated by phorbol ester, a PKC activator in vascular endothelial cells,35 and improve endothelial function at the therapeutic dosage in patients or animals with diabetes via down-regulation of PKC activity to decrease oxidative stress.21,35,36 In agreement with these reports, our study shown that pravastatin reversed the elevation of MDA and endothelial dysfunction induced by AGE-BSA in vitro, which are similar to those of antioxidant PDTC and of PKC inhibitor chelerythrine, indicating that the antioxidant property of pravastatin is probably achieved by diminishing the production of oxygen free radicals and by inhibiting PKC activation. The antioxidant property of pravastatin may prevent the oxidative degradation of NO and simultaneously preserve DDAH activity from oxidative modification. Therefore, these two pathways together enhance the bioavailability of NO, improving the impairment of endothelium-dependent relaxation induced by AGE-BSA.
However, the concentrations of pravastatin (0.3-3 mmol/L) used in our study are much higher than physiological concentrations, but they are still in the range of the dosages used by other researchers.37 In their study, the authors demonstrated that pravastatin (0.1-2 mmol/L) dose-dependently decreased metalloproteinase-9 release in primary cultured human monocytes stimulated by TNFα without any cytotoxicity. In our study, pravastatin markedly attenuated the inhibition of endothelium-dependent relaxation induced by AGE-BSA but did not decrease endothelium-independent relaxation, indicating that pravastatin had no cytotoxicity on vascular cells in the range of dosages used in our study. Furthermore, the incubation time of pravastatin in vitro is rather shorter than that of the long-duration administration in vivo studies, and so we used higher concentrations to get positive effect of pravastatin on the impairment of endothelium-dependent relaxation induced by AGE-BSA. Although the experiments in vitro can not completely mimic the conditions in vivo, they can exclude the impact of many factors in vivo and are extensively used to investigate the acute effects of drugs per se on some (pathologic) situations and the mechanisms underlying them. As shown in our study, acute exposure of vascular tissue to AGE-BSA resulted in enhancing oxidative stress and subsequently inhibiting vascular DDAH activity and impairing endothelium-dependent relaxation. In a whole body, exposure to AGEs or intracellular formation of AGEs might have different or even worse consequences than in an in vitro situation. In addition to the impairment of endothelial function, chronic exposure to AGEs may also induce alterations of vascular structure and other functions, including vascular stiffness, enhanced extracellular matrix permeability, and diminished antiproliferation, antioxidant, and antiinflammatory properties.25
In conclusion, the present study demonstrated for the first time that exogenous AGE-BSA inhibited DDAH activity of isolated rat aorta, which may be involved in impairment of endothelium-dependent relaxation induced by AGE-BSA, and pravastatin treatment not only restored DDAH activity but also improved the impairment of endothelium-dependent relaxation in rat aorta after exposure to AGE-BSA, which may be related to its antioxidant properties. Therefore, pravastatin is an effective pharmacological approach in preserving DDAH activity and reversing endothelial dysfunction of blood vessels under conditions of high AGE levels such as diabetes and aging.
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