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【摘要】
Objective- This work was undertaken to investigate comparative effect of AT1 receptor blocker (ARB), 3-hydroxy-3-methylglutaryl (HMG) coenzymeA (CoA) reductase inhibitor (statin), and their combination on vascular injury of salt-sensitive hypertension.
Methods and Results- Salt-loaded Dahl salt-sensitive hypertensive rats (DS rats) were treated with (1) vehicle, (2) hydralazine (5 mg/kg/d), (3) olmesartan (0.5 mg/kg/d), (4) pravastatin (100 mg/kg/d), and (5) combined olmesartan and pravastatin for 4 weeks. Olmesartan or pravastatin significantly and comparably improved vascular endothelium-dependent relaxation to acetylcholine, coronary arterial remodeling, and eNOS activity of DS rats. Olmesartan prevented vascular eNOS dimer disruption or the downregulation of dihydrofolate reductase (DHFR) more than pravastatin, whereas Akt phosphorylation was enhanced by pravastatin but not olmesartan, indicating differential pleiotropic effects between olmesartan and pravastatin. Add-on pravastatin significantly enhanced the improvement of vascular endothelial dysfunction and remodeling by olmesartan in DS rats. Moreover, pravastatin enhanced the increase in eNOS activity by olmesartan, being associated with additive effects of pravastatin on phosphorylation of Akt and eNOS.
Conclusions- Olmesartan and pravastatin exerted beneficial vascular effects in salt-sensitive hypertension, via differential pleiotropic effects. Pravastatin enhanced vascular protective effects of olmesartan. Thus, the combination of ARB with statin may be the potential therapeutic strategy for vascular diseases of salt-sensitive hypertension.
Olmesartan, pravastatin, and their combination were compared on vascular effects in salt-sensitive hypertensive rats (SS rats). Olmesartan or pravastatin improved vascular injury of SS rats, via different pleiotropic effects. Pravastatin enhanced vascular effect of olmesartan, via Akt and eNOS. ARB combined with statin is useful for treatment of salt-sensitive hypertension.
【关键词】 eNOS dimers DHFR oxidative stress vascular injury combined ARB and statin
Introduction
Accumulating evidence indicates that renin-angiotensin system (RAS) plays a crucial role in the pathophysiology of cardiovascular diseases in hypertension, and that RAS blockers, including angiotensin-converting enzyme inhibitors and AT1 receptor blockers (ARB), are the useful therapeutic agents for hypertensive cardiovascular diseases. 1 As hypertension is often accompanied by dyslipidemia in the same patients, their treatment frequently involves the combination of RAS blockers with 3-hydroxy-3-methylglutaryl coenzymeA (HMG-CoA) reductase inhibitors (statins), potent inhibitors of cholesterol biosynthesis. Clinical evidence show that statins improve endothelial dysfunction and reduce the incidence of atherosclerotic events, 2-5 and these vascular protective effects by statins are at least partially attributed to their pleiotropic vascular effects beyond lowering of plasma cholesterol. 4-6 However, the difference in vascular pleiotropic effects between RAS blockers and statins remains to be fully understood. Moreover, the significance and the advantage of their combination therapy in hypertension, particularly salt-sensitive hypertension, are not defined.
Clinically, salt-sensitive hypertensive patients are more prone to cardiovascular diseases than their salt-insensitive counterparts. 7,8 Therefore, it is a key clinical issue to elucidate the effect of RAS blockers and statins in salt-sensitive hypertension. Moreover, it is not clear at all whether add-on statin treatment enhances vascular protective effects of RAS blockers in salt-sensitive hypertension. Dahl salt-sensitive hypertensive rats (DS rats) constitute a paradigm of salt-sensitive hypertension in humans and therefore are the useful animal model to investigate the mechanism underlying cardiovascular injury in salt-sensitive hypertension.
The aim of our present work was to compare the impact of olmesartan (an ARB), pravastatin (a statin), and their combination on vascular diseases in DS rats and to examine the relative role of reactive oxygen species (ROS) and eNOS in their pleiotropic effects. We obtained the evidence that olmesartan and pravastatin improved vascular injury of DS rats, via different pleiotropic effects on ROS and eNOS, and pravastatin significantly enhanced vascular effects of olmesartan in DS rats.
Methods
Experimental Animals
All procedures were in accordance with institutional guidelines for animal research. DS rats (Japan SLC Inc, Shizuoka, Japan) were used in the present study. At 7 weeks of age, the diet of DS rats was switched from a 0.3% NaCl (low-salt) to an 8% NaCl (high-salt) diet. 9 Control DS rats were fed a 0.3% NaCl diet, throughout the experiments.
Experiment I: Time Course of Impact of Sodium Loading
The first experiments were performed to examine time-dependent effect of high-salt loading on vascular endothelial function, NADPH oxidase activity, superoxide, eNOS activity and phosphorylation, disruption of eNOS dimers, plasma NO 2 /NO 3, Akt phosphorylation, nitrotyrosine, dihydrofolate reductase (DHFR), and coronary arterial thickening in DS rats. DS rats, fed low-salt diet throughout the experiment, served as control. Blood pressure (BP) was periodically measured by tail-cuff plethysmography (BP-98A; Softron Co). Eight-, 12-, and 16-week-old DS rats, subjected to high-salt diet for 1, 5, and 9 weeks, respectively, were anesthetized with ether, arterial blood was immediately collected by cardiac puncture, and plasma was collected by centrifugation and stored at -80°C until use. Then, the carotid artery and the thoracic aorta were immediately excised to estimate vascular endothelial function and the above mentioned biochemical parameters, and the heart was also removed to examine coronary arterial thickening.
Experiment II: Comparative Effect of an ARB, a Statin, and Their Combination
The second experiments were carried out to compare the effect of olmesartan (Sankyo Co. Ltd), pravastatin (Sankyo Co. Ltd), their combination, or hydralazine on DS rats fed high-salt diet. Twelve-week-old DS rats, which had fed a high-salt diet from 7 weeks of age, were orally given olmesartan (0.5 mg/kg/d), pravastatin (100 mg/kg/d), combined olmesartan (0.5 mg/kg/d) and pravastatin (100 mg/kg/d), or hydralazine (5 mg/kg/d) for 4 weeks. Olmesartan and pravastatin were suspended in 0.5% carboxymethyl cellulose (CMC), and were given to rats by gastric gavage once a day. Hydralazine was given to rats as the drinking water. It is well known that oral administration of 100 mg/kg/d of pravastatin to rats yields plasma pravastatin concentration similar to that seen in patients taking clinical doses of pravastatin 10,11 and does not significantly change plasma cholesterol levels in DS rats. Furthermore, in preliminary experiments, we found that olmesartan, pravastatin, their combination, and hydralazine at the above mentioned dose exerted similar hypotensive effects in DS rats. Therefore, this experimental protocol allowed us to elucidate the lipid-independent and blood pressure-independent effect of each therapy on DS rats. BP and heart rate were measured in conscious rats at 3 to 5 hours after oral dosing, every week. After 4 weeks of treatment, DS rats were anesthetized with ether, then the carotid artery, the thoracic aorta, and the heart were immediately excised to compare the effect of each treatment on vascular endothelial function, NADPH oxidase activity, p22phox, superoxide, eNOS activity, eNOS dimers, phosphorylation of eNOS and Akt, DHFR, extracellular signal regulated-kinase (ERK), nitrotyrosine, phospho-MEK, coronary arterial thickening, perivascular fibrosis, cardiac hypertrophy, and cardiac fibrosis and macrophage infiltration.
A detailed description of the Methods is available in the online data supplement at http://atvb.ahajournals.org.
Results
Time Course of BP, Vascular Endothelial Function, and Coronary Remodeling in DS Rats Fed High-Salt Diet
DS rats were fed high-salt (8% NaCl) diet from 7 weeks of age. We measured BP, endothelial function, and coronary arterial thickening in 8-, 12-, and 16-week-old DS rats, fed high-salt diet for 1, 5, and 9 weeks, respectively (supplemental Figure I). Compared with control DS rats fed low-salt diet, BP in salt-loaded DS rats progressively increased with time. As shown by vascular relaxing response to acetylcholine (Ach) (supplemental Figure I-B), vascular endothelium-dependent relaxation in salt-loaded DS rats was already slightly but significantly impaired at 8 weeks of age ( P <0.05) and was thereafter progressively impaired with time, which was followed by the significant coronary arterial thickening at 16 weeks of age.
Time Course of Vascular NADPH Oxidase, Superoxide, eNOS Activity, and Plasma NO 2 /NO 3
As shown in Figure 1A and 1 B, vascular NADPH oxidase activity and superoxide of DS rats were already significantly increased by 1 week of high-salt loading ( P <0.05) (at 8 weeks of age) and remained increased throughout the treatment of high salt. On the other hand, vascular eNOS activity of salt-loaded DS rats was not altered at 8 or 12 weeks of age, but was significantly reduced by 53% at 16 weeks of age ( P <0.01), compared with low-salt diet ( Figure 1 C). As in vascular eNOS activity, plasma NO 2 /NO 3 concentrations were significantly decreased only at 16 weeks of age ( Figure 1 D).
Figure 1. Time course of vascular NADPH oxidase (A), superoxide (B), eNOS activity (C), and plasma NO 2 /NO 3 (D) in DS rats. High Na, 8% NaCl diet; Low Na, 0.3% NaCl diet. Values are mean±SEM (n=5).
Time Course of Phospho-Akt, Phospho-eNOS, and the Disruption of eNOS Dimers
As shown in supplemental Figure II, 1, 5, or 9 weeks of high-salt diet did not affect phosphorylation of vascular Akt throughout the treatment. On the other hand, vascular phospho-eNOS levels were significantly reduced in 16-week-old DS rats subjected to 9 weeks of high-salt intake ( P <0.05).
Supplemental Figure III shows the detection of vascular eNOS protein dimers and monomers by low-temperature SDS-PAGE followed by Western blot analysis. In low-salt fed DS rats, eNOS existed exclusively as the dimers, whereas in high-salt fed 16-week-old DS rats, eNOS was present predominantly in the monomers and eNOS dimers were nearly absent, indicating the significant disruption of eNOS protein dimers by 9 weeks of salt loading. No significant disruption of vascular eNOS dimers was found in 8- and 12-week-old salt-loaded DS rats (data not shown).
Effect of Olmesartan, Pravastatin, Combined Olmesartan and Pravastatin, and Hydralazine on BP and Plasma Lipids of DS Rats Fed High-Salt Diet
Each drug was given 12-week-old DS rats which had been already fed high-salt diet for 5 weeks, and drug treatment was carried out for 4 weeks. As shown in supplemental Figure IV, olmesartan, pravastatin, combined olmesartan and pravastatin, and hydralazine slightly reduced BP of DS rats to a comparable degree throughout the treatment, except for greater reduction of BP by hydralazine than the other drug treatments at 13 weeks of age.
After 4 weeks of treatment, plasma cholesterol concentrations in vehicle-, olmesartan-, pravastatin-, combined olmesartan and pravastatin-, and hydralazine-treated salt-loaded DS rats, and low-salt DS rats were 77±8, 76±6, 78±6, 77±8, 74±5, and 71±4 mg/dL, respectively, and plasma triglyceride were 140±20, 124±18, 144±19, 108±9, 119±5, and 136±14 mg/dL, respectively. There was no significant difference in plasma cholesterol or triglyceride levels, among all groups of DS rats.
Comparative Effect on Serum Creatinine and Plasma Thiobarbituric Acid Reactive Substances
As shown in supplemental Figure V, serum creatinine and plasma thiobarbituric acid reactive substances (T-BARS; a marker of lipid peroxidation) were significantly higher in 16-week-old salt-loaded DS rats compared with control. Olmesartan, pravastatin, and their combination almost normalized serum creatinine. Olmesartan and pravastatin alone significantly and comparably reduced plasma T-BARS, and their combination reduced it more than either monotherapy.
Comparative Effect on Vascular Endothelial Function and Coronary Remodeling of DS Rats Fed High-Salt Diet
As shown in Figure 2, either olmesartan or pravastatin, but not hydralazine, significantly prevented the impairment of vascular endothelium-dependent relaxation to Ach and also prevented the progression of coronary arterial thickening and perivascular fibrosis, to a comparable degree. The combination of olmesartan with pravastatin prevented vascular endothelial dysfunction more than monotherapy with either agent alone and prevented coronary arterial thickening and perivascular fibrosis more than pravastatin alone, despite no difference in BP and plasma lipids among all drug treatment groups.
Figure 2. Effect of vehicle (V), olmesartan (O), pravastatin (P), combined olmesartan and pravastatin (O+P), and hydralazine (H) on acetylcholine-induced vascular relaxation (A), coronary arterial thickening (B), and perivascular fibrosis (C) of DS rats fed high-salt diet. A, Each plot represents mean±SEM (n=8 to 9 per group). Right top panels show representative images of light micrographs of LV coronary arterial thickening and perivascular fibrosis. Original magnification, x 200. Each bar represents mean±SEM (n=4 to 8 per group). L indicates DS rats fed low-salt diet; NS, not significant.
As shown in supplemental Figure VI, the phosphorylation of vascular MEK and ERK was significantly enhanced in 16-week-old salt-loaded DS rats compared with control. Olmesartan and pravastatin alone similarly prevented the phosphorylation of vascular MEK and ERK compared with control, and their combination decreased the phosphorylation of MEK and ERK more than pravastatin alone.
Comparative Effect on Vascular NADPH Oxidase, p22phox, Superoxide, and eNOS Activity of DS Rats Fed High-Salt Diet
As shown in Figure 3, either olmesartan or pravastatin alone, but not hydralazine, significantly ameliorated the increase in vascular NADPH oxidase activity, the increase in p22phox protein expression, the increase in vascular superoxide, or the decrease in vascular eNOS activity of salt-loaded DS rats. The inhibitory effect of olmesartan, regarding NADPH oxidase, p22phox, and superoxide, was greater than that of pravastatin. On the other hand, no significant difference was noted in the beneficial effect on vascular eNOS activity between olmesartan and pravastatin. Add-on pravastatin significantly enhanced the improvement of superoxide ( P <0.05) and eNOS activity ( P <0.01) by olmesartan alone, whereas pravastatin failed to enhance the suppression of NADPH oxidase and p22phox by olmesartan.
Figure 3. Effect of each treatment on vascular NADPH oxidase (A), p22phox (B), superoxide (C), and eNOS activity (D) of DS rats fed high-salt diet. Top panels in B and C show representative Western blot and fluorescence photomicrographs, respectively, in each group. # P <0.05, * P <0.01 vs V. Each bar represents mean±SEM. A: L, n=9; V, n=9; H, n=4; O, n=9; P, n=9; O+P, n=8. B: L, n=9; V, n=9; H, n=5; O, n=9; P, n=9; O+P, n=8. C: n=5 per group. D: n=4 to 5 per group. Abbreviations are the same as Figure 2. L indicates DS rats fed low-salt diet.
Comparative Effect on Vascular Phospho-Akt, Phospho-eNOS of DS Rats Fed High-Salt Diet
In contrast to no significant effect of olmesartan on vascular Akt phosphorylation, pravastatin alone or olmesartan combined with pravastatin significantly and comparably increased phosphorylation of Akt in DS rats ( Figure 4 ). However, there was no significant difference in vascular total Akt levels in each group. Either olmesartan or pravastatin alone significantly upregulated vascular phospho-eNOS levels and total eNOS, and add-on pravastatin significantly enhanced the upregulation of phospho-eNOS and total eNOS by olmesartan ( P <0.05).
Figure 4. Effect of each treatment on phospho-akt (p-akt), total akt, phospho-eNOS (p-eNOS), and total eNOS of DS rats fed high-salt diet. Top panels show representative Western blot in each group. Each bar represents mean±SEM (n=5 per group). Abbreviations are the same as Figure 2. L indicates DS rats fed low-salt diet.
Comparative Effect on Vascular eNOS Dimers and DHFR Protein Expression of DS Rats Fed High-Salt Diet
Either olmesartan or pravastatin alone significantly prevented the disruption of vascular eNOS protein dimers in salt-loaded DS rats, but olmesartan prevented it more than pravastatin ( P <0.01; Figure 5 A). Add-on pravastatin did not significantly enhance the suppressive effect of olmesartan on eNOS dimer disruption. As shown in Figure 5 B, vascular DHFR protein levels were significantly downregulated in 16-week-old salt-loaded DS rats compared with control DS rats ( P <0.01), although no significant difference in DHFR was noted between salt-loaded and control DS rats at the age of 8 and 12 weeks. Olmesartan, but not pravastatin, significantly prevented the downregulation of DHFR in salt-loaded DS rats ( P <0.01). Add-on pravastatin did not significantly enhance the upregulation of DHFR by olmesartan.
Figure 5. Effect of each treatment on eNOS dimer disruption (A) and DHFR protein levels (B) of DS rats fed high-salt diet. Top panels in A and B show representative Western blot in each group. Each bar represents mean±SEM (n=5 per group). Abbreviations are the same as Figure 2. L indicates DS rats fed low-salt diet.
Comparative Effect on Vascular Nitrotyrosine of DS Rats Fed High-Salt Diet
As shown by Western blot analysis and immunohistochemistry in Figure 6, vascular nitrotyrosine levels were significantly increased in 16-week-old salt-loaded DS rats compared with control DS rats ( P <0.01), although no significant difference in nitrotyrosine was noted between salt-loaded and control DS rats at the age of 8 and 12 weeks. Olmesartan and pravastatin significantly reduced vascular nitrotyrosine. The combination of olmesartan with pravastatin did not significantly enhance the suppressive effect of nitrotyrosine by olmesartan, while significantly enhanced the reduction of nitrotyrosine by pravastatin.
Figure 6. Effect of each treatment on vascular nitrotyrosine of DS rats fed high-salt diet. Vascular nitrotyrosine was determined by immunohistochemistry (A) and Western blot analysis (B). Each bar represents mean±SEM (n=5 per group). Abbreviations are the same as Figure 2. L indicates DS rats fed low-salt diet.
Comparative Effect on Cardiac Hypertrophy, Fibrosis, and Macrophage Infiltration of DS Rats Fed High-Salt Diet
As shown in supplemental Figure VII, olmesartan and pravastatin alone significantly prevented cardiac hypertrophy and fibrosis in salt-loaded DS rats, and their combination prevented them more than either monotherapy. Cardiac macrophage infiltration was significantly and comparably prevented by olmesartan and pravastatin, and their combination prevented it more than pravastatin monotherapy (supplemental Figure VIII). These results suggest that the combination of pravastatin and olmesartan may be useful for the treatment of cardiac remodeling in salt-sensitive hypertension.
Discussion
It is well established that atherosclerosis is closely associated with the impairment of vascular endothelial function, whose hallmark is an impairment of endothelium-dependent vasorelaxation induced by reduced bioavailability of NO. 12-14 A decline in NO bioavailability is mainly caused by the reduction of NO synthesis by eNOS and the increased inactivation of NO by superoxide. 12-14 Thus, the dysfunction of eNOS and the activation of NADPH oxidase which generates superoxide play a major role in vascular endothelial dysfunction and vascular remodeling. Previous works 15-17 on salt-loaded DS rats show that vascular NADPH oxidase and superoxide were significantly increased whereas eNOS activity was inversely significantly decreased in DS rats suffering from severe endothelial dysfunction and vascular hypertrophy. However, in previous works, 15-17 time course of these parameters in DS rats has not been examined and therefore the relative relationship among NADPH oxidase, superoxide, and eNOS, regarding the role in vascular injury, is not elucidated. In our present work, we found that slight but significant impairment of vascular endothelium-dependent relaxation occurred by 1 week after start of salt loading (supplemental Figure I), being associated with the early increase in NADPH oxidase activity and superoxide ( Figure 1 ). These results, taken together with no change in eNOS activity at this early stage of salt loading, showed that NADPH oxidase-generated superoxide, but not eNOS, was involved in the early onset of vascular endothelial dysfunction in salt-sensitive hypertension. Notably, much later than the onset of increase in NADPH oxidase activity, the remarkable reduction of vascular eNOS activity occurred only in 16-week-old DS rats fed high-salt diet for 9 weeks ( Figure 1 ). At 16 weeks of age, DS rats had much more severe endothelial dysfunction and remarkable coronary arterial remodeling than at the earlier age (supplemental Figure I). Therefore, in contrast to the importance of NADPH oxidase at the early phase of salt loading, vascular eNOS seems to be mainly involved in the exacerbation of vascular endothelial dysfunction and vascular structural changes of salt-sensitive hypertension, indicating differential role between NADPH oxidase and eNOS in the progression of vascular injury in salt-sensitive hypertension.
eNOS activity is regulated by not only its phosphorylation but also multiple factors. In this study, to elucidate the underlying mechanism of the decreased eNOS activity in 16-week-old salt-loaded DS rats, we examined the phosphorylation of vascular Akt and eNOS, the disruption of eNOS dimers, peroxynitrite, and DHFR expression in DS rats. Akt appears to be the principle kinase phosphorylating eNOS, leading to endothelium-dependent vasodilatation. 18 Previous studies show that the formation of eNOS protein homodimers is necessary for enzymatic activity, and peroxynitrite can disrupt eNOS protein dimers through oxidation and displacement of the zinc metal ion, leading to the reduction of eNOS activity. 19,20 DHFR is a key enzyme synthesizing tetrahydrobiopterin, eNOS cofactor, and very recent in vitro study on cultured vascular endothelial cells 21 indicate that downregulation of DHFR causes the uncoupling of eNOS, leading to the decrease in enzymatic activity. 21 However, the in vivo role of Akt, peroxynitrite, disruption of eNOS dimers, and DHFR in hypertension remains unknown. In the present work, Akt phosphorylation was not altered throughout salt loading, indicating the minor role of Akt in the decreased eNOS activity in 16-week-old DS rats. On the other hand, eNOS protein dimers were markedly diminished in 16-week-old DS rats (supplemental Figure III), indicating that the disruption of eNOS dimers significantly occurred in DS rats. Furthermore, this disruption of eNOS dimers was associated with the increase in nitrotyrosine (an indicator of peroxynitrite) and the downregulation of DHFR. Therefore, we obtained the first evidence that the disruption of eNOS dimers was involved in the reduction of eNOS activity in salt-sensitive hypertension, and eNOS dimer disruption might be attributed to the increased peroxynitrite and the downregulation of DHFR.
Although previous studies indicate that monotherapy with ARB (candesartan) 16 or statin (atorvastatin) 15 improves vascular endothelial dysfunction and hypertrophy, reduces superoxide, and increases eNOS activity in salt-loaded DS rats, previous studies did not rule out the possible involvement of blood pressure lowering or plasma lipid lowering in these vascular effects. In our present study, we have obtained the evidence that olmesartan and pravastatin improved vascular injury of salt-sensitive hypertension, independently of blood pressure or plasma lipid. Notably, despite similar beneficial effects between olmesartan and pravastatin on vascular endothelial function, coronary remodeling, and eNOS activity in DS rats, olmesartan normalized vascular NADPH oxidase activity, p22phox, superoxide, nitrotyrosine (peroxynitrite), and the disruption of eNOS dimers to a greater extent than pravastatin. Olmesartan significantly enhanced DHFR expression, in contrast to no effect of pravastatin on DHFR. Taken together with the fact that the increase in peroxynitrite 19,20 or DHFR downregulation 21 leads to the disruption of eNOS dimers, our present data support the notion that the restoration of eNOS activity by olmesartan was mediated by the suppression of eNOS dimer disruption, which was at least partially mediated by the amelioration of peroxynitrite and DHFR downregulation. On the other hand, pravastatin significantly enhanced the phosphorylation of Akt, differing from no effect of olmesartan on Akt. Akt phosphorylation is well known to enhance eNOS activity. 20,22,23 Accordingly, the restoration of eNOS activity by pravastatin might be at least in part attributed to the enhancement of Akt phosphorylation. Thus, the mechanism responsible for the restoration of eNOS activity in salt-sensitive hypertension seems to differ between olmesartan and pravastatin.
The therapeutic significance of the combination therapy of RAS blocker with statin remains obscure, and previous clinical and experimental reports are rare and contradictory. 24-32 Particularly, the significance of addition of statin to RAS blocker is undefined in salt-sensitive hypertension, 32 which encouraged us to investigate whether add-on pravastatin enhances the beneficial effect of olmesartan in DS rats. Of note are the observations that pravastatin significantly enhanced the improvement of endothelium-dependent vascular relaxation and coronary remodeling by olmesartan in DS rats, despite no additive effect of pravastatin on blood pressure and plasma lipid. Therefore, our work provided the first evidence that pravastatin has the potential to enhance vascular protective effect of olmesartan in salt-sensitive hypertension, independent of blood pressure and lipid. Interestingly, pravastatin significantly enhanced the normalization of vascular eNOS activity by olmesartan. In contrast to no effect of olmesartan alone on Akt phosphorylation, pravastatin significantly increased Akt phosphorylation ( Figure 4 ). Therefore, the addition of pravastatin to olmesartan increased the phosphorylation of Akt, as well as pravastatin alone. Moreover, add-on pravastatin significantly enhanced the phosphorylation of eNOS by olmesartan, and this enhancement by pravastatin might be at least in part mediated by the enhancement of the increase in total eNOS protein levels by olmesartan ( Figure 4 ). Thus, the increase in Akt phosphorylation and eNOS phosphorylation seems to be responsible for the increase in eNOS activity by add-on pravastatin.
In the present study, we found no statistically significant additive effect of pravastatin on olmesartan-treated DS rats, with regard to eNOS dimers, DHFR, or ERK. As described above, the formation of eNOS protein homodimers is necessary for eNOS enzymatic activity, and is known to be disrupted mainly by the increase in peroxynitrite 19,20 and the reduction of DHFR. 21 In this study, we found that vascular peroxynitrite was significantly increased in salt-sensitive hypertension, and that olmesartan and pravastatin alone significantly reduced vascular peroxynitrite. The combination of olmesartan with pravastatin did not significantly enhance the reduction of peroxynitrite by olmesartan. On the other hand, very importantly, the combination of olmesartan with pravastatin more potently inhibited vascular peroxynitrite production more than pravastatin monotherapy, indicating that inhibitory effect of pravastatin on peroxynitrite production was weaker than that of olmesartan. These findings can account for no significant effect of add-on pravastatin on peroxynitrite. Taken together with the fact that peroxynitrite and DHFR are key regulators of eNOS dimer formation, no significant effect of add-on pravastatin on eNOS dimers can be explained by no significant effect of add-on pravastatin on peroxynitrite and DHFR.
Recent in vitro study using cultured vascular endothelial cells indicated that angiotensin II downregulates DHFR. 21 However, it remains to be determined whether or not the downregulation of DHFR by angiotensin II can apply to in vivo situation. We have obtained the first evidence that the downregulation of DHFR occurred in salt-sensitive hypertension and olmesartan, but not pravastatin, significantly prevented the downregulation of DHFR in salt-sensitive hypertension. Taken together with the findings that renin-angiotensin system is involved in vascular diseases of salt-sensitive hypertension, our present work demonstrated that angiotensin II is implicated in the downregulation of vascular DHFR in salt-sensitive hypertension.
As in the case of ERK, add-on pravastatin did not significantly enhance the inhibition of vascular MEK phosphorylation by olmesartan (supplemental Figure VI). Therefore, no effect of add-on pravastatin on ERK phosphorylation can be explained by no effect of add-on pravastatin on MEK phosphorylation, because MEK is the major upstream activator of ERK.
Study limitation
Pravastatin is a hydrophilic statin, and the direct vascular effects of pravastatin remain to be fully understood. Taken together with previous findings 10,11 our present results show the direct vascular pleiotropic effects of pravastatin, and this notion is also supported by the findings of MEGA Study 33 that a low dose of pravastatin suppresses primary cardiovascular events in Japanese hypercholesterolemic patients as much as hydrophobic statins. However, our present in vivo study did not allow us to elucidate the detailed molecular mechanism underlying the effect of pravastatin on vascular injury in salt-sensitive hypertension, because the study on the detailed molecular mechanism was out of scope. Further study is needed to elucidate more detailed molecular mechanism responsible for the effect of olmesartan, pravastatin, and their combination on vascular injury of salt-sensitive hypertension.
In conclusion, we obtained the evidence that olmesartan and pravastatin exerts beneficial effect on vascular endothelial function and remodeling of salt-loaded DS rats, via differential multiple pleiotropic effects, and that add-on pravastatin treatment significantly enhanced the improvement of vascular injury by olmesartan via the phosphorylation of Akt and eNOS. We propose that combination of ARB with statin may be potentially promising therapeutic strategy of salt-sensitive hypertensive patients with hyperlipidemia, beyond blood pressure and lipid control.
Acknowledgments
Sources of Funding
This work was supported in part by Grants-in-Aid for Scientific Research (16590202) from the Ministry of Education, Culture, Sports, Science, and Technology.
Disclosures
None.
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作者单位：Departments of Pharmacology and Molecular Therapeutics (E.Y., T.Y., T.T., K.K., Y.T., Z.-F.L., Y.-F.D., S.M., S.K.-M.) and Cardiovascular Medicine (H.O.), Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan.