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CARDIOVASCULAR
Department of Experimental Pharmacology, Chair of Pharmacology, Jagiellonian University Medical College, Cracow, Poland (B.L., M.B., J.J., S.C.); and Department of Human Nutrition, Faculty of Food Technology, Agricultural University, Cracow, Poland (M.F., R.B.K., P.M.P.)
Received November 18, 2005; accepted March 16, 2006.
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Abstract |
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; Chlopicki and Gryglewski, 2004). In particular, hypertension and hypercholesterolemia alone impaired endothelium-dependent vasodilation, and their combination led to additive impairment of the NO-dependent endothelial function (Bonetti et al., 2003).
The endothelial function was also impaired in hypercholesterolemic rabbits (Verbeuren et al., 1990) and mice (d'Uscio et al., 2001) as well as in other species (Shaul, 2003). It has repeatedly been shown that a high-cholesterol diet in nonhuman primates, swine, rabbits, mice, and pigeons led to intimal lesions and to the development of atherosclerotic plaques (Faggiotto et al., 1984; Jerome and Lewis, 1985; Rosenfeld et al., 1987). Although the precise mechanisms of plaque development in response to hypercholesterolemia are still not clear, the involvement of platelets, monocytes and lymphocytes, endothelial dysfunction, and systemic inflammatory response are all well documented (Hansson et al., 2002).
Interestingly, in numerous studies rats were resistant to the development of hypercholesterolemia-induced atherosclerosis (Nakamura et al., 1989; Moghadasian, 2002; Pisulewski et al., 2005). For example, electron microscopic studies revealed subtle endothelial changes in rat aorta only after they had been fed a high-fat diet for 12 months (Still and O'Neal, 1962). In fact, normotensive rats fed with a high-cholesterol diet did not develop endothelial dysfunction (Pisulewski et al., 2005).
In contrast to the apparent resistance of rats to hypercholesterolemia-induced endothelial dysfunction and atherosclerosis, hypertension in rats is linked to endothelial dysfunction, as is the case in many other species. Indeed, the impairment of NO-dependent endothelial function as well as endothelial inflammation was repeatedly detected in Salt-Dahl rats, deoxycorticosterone acetate-salt hypertensive rats, and spontaneously hypertensive rats (SHR), as well as in one-kidney, one-clip model of hypertension (Ortiz and Garvin, 2001). Importantly, lowering blood pressure in hypertensive rats by various pharmacological interventions normalized endothelial function (Hoshino et al., 1994). Furthermore, nonhypotensive drugs endowed with endothelial action such as antioxidants, a xanthine oxidase inhibitor (allopurinol), or statins improved endothelial function in rats with hypertension (Carneado et al., 2002; Ulker et al., 2003).
In humans (Bonetti et al., 2003) and in some species of laboratory animals, coexistence of hypertension and hypercholesterolemia leads to additive effects on endothelium. For example, in hypertensive Watanabe hypercholestrolemic rabbits, vascular lesions were greater than in normotensive rabbits with hypercholesterolemia (Chobanian et al., 1989).
That is why in the present study, we tested whether preexisting hypertension would facilitate the development of endothelial dysfunction in hypercholesterolemic rats. Therefore, SHR and normotensive controls (WKY) were fed with a hypercholesterolemic diet for 12 weeks, after which the functional activity of endothelial NO and PGI2 was assessed in isolated aortic rings. In addition, the intensity of hypercholesterolemia-induced inflammation was assessed by the measurement of monocyte chemoattractant protein 1 (MCP-1) in plasma. Aortic root tissue was also examined for the presence of early atherosclerotic plaques.
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Materials and Methods |
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Animals. Twelve-month-old SHR (n = 20) and WKY (n = 20) male rats, weighing 350 to 550 g, were obtained from the Animal Laboratory of Polish Mother's Research Institute Hospital in Lodz, Poland. The rats were housed individually in stainless steel, wire-bottomed cages in an isolated room at controlled temperature and ambient humidity. Lights were maintained on a 12-h light/dark cycle. The animals were acclimatized to these conditions for 1 week and given free access to water and the basal semipurified AIN-93M diet (Faculty of Food Technology, Agricultural University, Cracow, Poland). After the adaptation period, the SHR and WKY rats were randomly divided into four groups to receive one of the following types of diet for the 12-week period: the control diet (basal dry rat AIN-93M diet) or the hypercholesterolemic diet (basal AIN-93M diet + 1 g/100 g of cholesterol + 0.5 g/100 g of cholic acid + 20 g/100 g of butter) (Reeves, 1997). The final composition of both types of diet is given in Table 1.
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Indirect blood pressure (BP) was monitored by the tail-cuff method at the starting point of the experiment and at the end of the experiment after 12 weeks of the respective diet. Mean BP at the beginning of the experiment was significantly higher in the SHR than in the control WKY (189 ± 6.7 versus 119 ± 10.4 mm Hg, respectively). The type of diet did not influence BP significantly in SHR or WKY rats.
Determination of Lipid Profile in the SHR and WKY Rat. After decapitation blood samples for lipid analyses were collected in test tubes and centrifuged (4000g, 10 min) to obtain serum. Samples of serum were immediately frozen (–20°C) and stored until assayed. Using commercially available kits, the serum samples were analyzed for total cholesterol and triglycerides (CORMAY, Lublin, Poland) and for HDL cholesterol and LDL cholesterol (Olympus Diagnostica GmbH, Hamburg, Germany).
Determination of MCP-1 Level in SHR and WKY Rat Serum. For determination of levels of MCP-1, blood samples were collected and centrifuged (4000g, 10 min) to obtain serum. Samples were stored at –70°C until assayed using the commercially available enzyme immunoassay kits (BioSource, Camarillo, CA). The level of MCP-1 in serum was expressed in picograms per milliliter.
Protocol of Experiments in Isolated Aortic Rings. Rats were anesthetized with thiopental-sodium (120–150 mg kg–1 body weight) (Biochemie GmbH, Kundl-Rakusko, Austria), and the thoracic aorta was quickly removed by central sternotomy, after which it was gently washed through the lumen with ice-cold saline and placed in ice-cold Krebs-Henseleit buffer of the following composition: 118 mM NaCl, 2.52 mM CaCl2, 1.64 mM MgSO4, 24.88 mM NaHCO3, 1.18 mM K2PO4, 4.7 mM KCl, and 10.0 mM glucose. Loose connective tissue was removed, and each aorta was cut into six rings 3 to 4 mm in length. Vascular rings were then transferred to organ chambers filled with 5 ml of Krebs-Henseleit solution maintained at 37°C, pH 7.4, and gassed with carbogen. Rings were mounted between two hooks attached to an isometric force transducer (Bie-gastab K30 type 351; Hugo Sachs, March-Hugstetten, Germany) with continuous recording of tension (Graphtec WR3320; Graphtec GB Limited, Wrexham, UK). After mounting the rings, the resting tension was stepwise increased to reach a final 4 g, after which the rings were incubated to equilibrate for 30 min. The viability of the tissue was documented by a contractile response to potassium chloride (KCl) at a final concentration of 3 x 10–2 to 9 x 10–2 M). Next, the aortic rings were precontracted with phenylephrine (final concentration of 10–8–3 x 10–7 M) to obtain submaximal contraction (60–80% of KCl-induced maximum response), and after obtaining a stable plateau phase of contraction, the integrity of the endothelium was assessed with acetylcholine (ACh; 10–6 M). Then relaxation to cumulative concentrations of ACh (10–9–10–5 M) or histamine (10–8–3 x 10–4 M) was induced. The endothelium-independent vasorelaxation was evoked by S-nitroso-N-acetyl-penicillamine (SNAP; 10–9–10–5 M). To test the involvement of NO in acetylcholine or histamine-induced responses, these responses were repeated in the presence of an inhibitor of nitric oxide synthesis—L-NG-nitroarginine methyl ester (L-NAME; 3 x 10–4 M). Rings were incubated with L-NAME for at least 15 min before eliciting vasodilator responses. The plateau of L-NAME-induced contraction (after phenylephrine precontraction) was seen approximately 10 min after the addition of L-NAME. Thus, a 15-min incubation period with L-NAME was chosen for experiments. The relaxation or contraction response was expressed as a percentage of the contraction induced by phenylephrine (10–8–3 x 10–7 M) or KCl (9 x 10–3 M).
Basal production of NO by aortic rings was determined on the basis of the magnitude of contraction induced by L-NAME. In this experiment, rings were precontracted with phenylephrine (10–8–5 x 10–8 M to obtain 10 to 30% precontraction of maximal KCl-induced contraction), and then L-NAME (3 x 10–4 M) was added. To see a vasoconstrictor response to L-NAME, vessels needed to be precontracted with phenylephrine (by 10 –30%). The plateau phase of L-NAME-induced contraction was determined and expressed as a percentage of the maximum KCl-induced contraction.
Determination of Basal Prostacyclin Production in Aortic Rings. PGI2 release by aortic rings was analyzed by measuring its stable hydrolysis product 6-keto-PGF1
in an organ bath medium. Commercially available enzyme immunoassay kits (Cayman Chemical Company, Ann Arbor, MI) were used. Aortic rings were washed with fresh Krebs-Henseleit solution and then incubated for 30 min under a 4-g stretch as for the functional experiments. Samples of supernatant were collected after 20 min of incubation and then processed for levels of 6-keto-PGF1 according to the manufacturer's instructions. Aorta rings were collected after the experiments, dried for 48 h in 56°C, and weighed. PGI2 production was expressed as pg/ml/mg of dry weight of aortic rings. The enzymatic source of PGI2 was assessed using nonselective COX or selective COX-2 inhibitors such as indomethacin (5 x 10–6 M) or rofecoxib (10–6 M), respectively. The rings were preincubated with inhibitors for at least 20 min.
Oil Red O Staining of Aortic Roots and Liver. The heart and ascending aorta were snap-frozen in OCT (Optimal Cutting Temperature) gel (Tissue-Tek; Cell Path, Oxford, UK) and stored in –80°C. Cryosections were cut in a Leica 3050 cryostat (Leica Microsystems, Nussloch, Germany) using a standardized protocol. Briefly, serial cryostat sections were cut from the aortic root upward from the point, where all three cusps were visible. Then, 10 sections were collected at every 100 µm over a segment of approximately 5 to 10 mm of the aortic root before being fixed with formaldehyde. Livers from the rats were also taken to OCT, and cryosections were prepared by standard procedure. Sections of aorta and liver were stained with oil red O (ORO) and mounted in water-soluble mounting media (Kaiser's glycerol gelatin). For each section, images were captured by digital camera and analyzed.
Statistical Analysis. Results are expressed as means ± S.E.M. Comparison of means was assessed by analysis of variance and post hoc Scheffe's test. P < 0.05 was considered significant.
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Effects of the Hypercholesterolemic Diet on Endothelium-Dependent and Endothelium-Independent Vasodilation in Aorta in SHR and WKY Rats. In all four experimental groups (SHR control, SHR hypercholesterolemia, WKY control, and WKY hypercholesterolemia) ACh induced a concentration-dependent vasodilation in aortic rings with an intact endothelium (Fig. 2A). The maximum vasodilation induced by ACh (10–5 M) did not differ between the four experimental groups. The lack of effect of the hypercholesterolemic diet in either SHR or WKY rats on endothelium-mediated response was also noted for another endothelium-dependent vasodilator, histamine (Fig. 2B). Maximum vasodilation induced by histamine was obtained with 3 x 10–4 M histamine and did not differ between the four experimental groups (Fig. 2B).
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Effect of L-NAME on Endothelium-Dependent Responses in SHR and WKY Rats Fed with Control and Hypercholesterolemic Diets. The addition of L-NAME to aortic rings slightly precontracted with phenylephrine induced further vasoconstriction. The magnitude of L-NAME-induced response was not diminished but slightly increased in SHR and WKY rats fed with the hypercholesterolemic diet compared with their counterparts fed with the control diet. However, only in SHR rats did this difference reached statistical significance (p < 0.05) (Fig. 3).
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Effects of the Hypercholesterolemic Diet on Basal Prostacyclin Production by Aortic Rings. The basal release of prostacyclin in aortic rings was approximately 4-fold higher in SHR then in WKY rats (p < 0.001) (Fig. 5). However, neither in SHR nor in WKY rats did the hypercholesterolemic diet modify the basal level of prostacyclin production compared with their counterparts fed with the control diet (Fig. 5). In the presence of indomethacin (5 x 10–6 M) or rofecoxib (10–6 M), basal prostacyclin production by aortic rings was markedly decreased in SHR and in WKY rats irrespective of the type of diet (Fig. 5).
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ORO Staining of Aortic Roots and Liver. The oil red O staining of aortic sections did not reveal lipid accumulation in aortic roots from SHR or WKY rats fed with the hypercholesterolemic diet (Fig. 7). In contrast, lipid deposits were abundant in the liver of both the SHR and the WKY rats fed with the hypercholesterolemic diet (Fig. 8).
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). These findings stay in line with the known resistance of normotensive rats to developing hypercholesterolemia-induced atherosclerosis (Nakamura et al., 1989; Moghadasian, 2002). Here, using SHR rats, we comprehensively demonstrated that even in the presence of pre-existing hypertension, the implementation of hypercholesterolemia still remained without a significant effect on the endothelial function. Indeed, 12-week long hypercholesterolemia did not affect the vasodilator activity of NO that was assessed on the basis of the magnitude of L-NAME-induced vasoconstriction and the NO-dependent vasodilation induced by acetylcholine and histamine. Twelve-week long hypercholesterolemia in SHR rats was also without an effect on the basal activity of PGI2 as well as on the functional activity of the vasoconstrictor response to acetylcholine, which could be attributed to the endothelium-derived contracting factor (EDCF). Furthermore, hypercholesterolemia was not associated with a robust inflammatory response but only with mildly elevated MCP-1 levels and was not associated with atherosclerotic plaque formation as evidenced by a lack of fatty-streak formation in the aortic roots. Therefore, together with our previous report (Pisulewski et al., 2005), we claim that, irrespective of the presence or absence of hypertension, hypercholesterolemia in rats does not impair the activity of endothelial NO and PGI2 and does not induce a significant inflammatory response of the endothelium that could lead to the development of atherosclerosis.
The NO-dependent function was slightly impaired in the SHR and the WKY rats, and the 12-week long hypercholesterolemia did not modify it further. The lack of effect of hypercholesterolemia on the NO-dependent function in the SHR rats was also found by Lindberg et al. (1995). In contrast, Cappelli-Bigazzi et al. (1997) showed mild impairment of acetylcholine-induced vasorelaxation in SHR rats fed with the hypercholesterolemic diet and more profound impairment of acetylcholine-induced and nitroglycerin-induced vasorelaxation in SHR rats fed with an atherogenic diet (1% cholesterol and vitamin D2). The reason for this discrepancy is not clear. However, there are differences in experimental design between the study of Cappelli-Bigazzi et al. (1997) and our own. For example, Cappelli-Bigazzi et al. (1997) used 6-week-old rats, whereas in our study 12-month-old rats were used. Moreover, Cappelli-Bigazzi et al. (1997) analyzed endothelium-dependent responses in the presence of indomethacin. Whether the elimination of COX-derived products by indomethacin could uncover the impairment of the NOdependent function in hypercholesterolemia remains to be elucidated.
In contrast to Cappelli-Bigazzi et al. (1997), we also assessed effects of hypercholesterolemia on basal PGI2 production and on the magnitude of vasoconstrictor response to acetylcholine that could be attributed to EDCF. Neither of them was changed by hypercholesterolemia.
Indeed, basal PGI2 production was not affected by hypercholesterolemia either in the SHR or in the WKY rats. Interestingly, PGI2 production in aorta was higher in the SHR rats compared with the WKY rats, although in both strains of rats it was markedly inhibited by rofecoxib. Thus, the major source of vascular PGI2 in the aorta from the SHR and WKY rats is COX-2 and not COX-1 (Hocherl et al., 2002). Previously, it was found that COX-1 and PGI2 synthase in SHR thoracic aorta are significantly higher than in WKY rats (Numaguchi et al., 1999). Whether COX-2 is also overexpressed in SHR rats remains to be tested. In rabbits, a hypercholesterolemic diet as short as 4 weeks led to the substantial inhibition of PGI2 production (Dembinska-Kiec et al., 1977). In SHR or in WKY rats, a hypercholesterolemic diet lasting 12 weeks did not influence vascular PGI2 production. These results further support the notion that hypercholesterolemia in SHR rats does not lead to the development of endothelial dysfunction. Indeed, the impairment of the NO-dependent function goes along with the impairment of COX-1-derived PGI2 production (Frein et al., 2005) or compensatory overproduction of COX-2-derived PGI2 (Belton et al., 2000). Neither of these responses was noted in our study.
It was repeatedly shown that the impairment of endothelial activity of PGI2 and NO is associated with a concomitant inflammatory activation of endothelium reflected, e.g., in an increased release of soluble adhesion molecules (e.g., soluble intercellular adhesion molecule 1, soluble vascular cell adhesion molecule 1) or chemokines (e.g., MCP-1). Indeed, PGI2 as well as NO inhibits the inflammatory response of endothelium, so their down-regulation may importantly accelerate the inflammation of endothelium (Chlopicki and Gryglewski, 2004). It is noteworthy that MCP-1 was shown to be down-regulated by exogenous NO (Zeiher et al., 1995). Although we found a moderate increase in MCP-1 plasma levels by hypercholesterolemia, it reached significance in the WKY rats but not in the SHR rats. Furthermore, the increase in MCP-1 levels was much less pronounced than in other species of animals fed with a hypercholesterolemic diet (Kowala et al., 2000).
In various experimental settings, the impairment of NO-dependent function was demonstrated to be associated with enhanced activity of the EDCF (Vanhoutte et al., 2005). It was also shown in the SHR rats that in the presence of L-NAME, acetylcholine caused vasoconstriction that was mediated by thromboxane receptors (Gluais et al., 2005). This response was attributed to EDCF (Vanhoutte et al., 2005). In our experiments, a vasoconstrictor response to acetylcholine in the presence of L-NAME could also be blocked by a thromboxane receptor antagonist (SQ 29548) both in SHR and WKY rats (data not shown). We showed that the magnitude of this response, which could be attributed to EDCF, was not influenced by the hypercholesterolemic diet either in the SHR or in the WKY rats.
Taken together, we demonstrated that a 12-week-long period of hypercholesterolemia did not influence the activity of NO, PGI2, EDCF, and MCP-1 either in the SHR or in the WKY rats significantly. It was therefore not surprising that we did not find signs of atherosclerotic plaque development in aortic roots, which is believed to be a primary place for plaque development in hypercholesterolemia-induced atherosclerosis in rodents (Bonthu et al., 1997).
We did not investigate mechanisms that could be involved in keeping the endothelial function insensitive to hypercholesterolemia in normotensive or hypertensive rats. However, the fact that in rats a diet enriched with 1% cholesterol and 20% butter led only to a modest increase of total and LDL cholesterol should be taken into account. Such an increase could be just insufficient to induce alterations in vascular function. Indeed, in our experiments as well as in studies of other authors (Cappelli-Bigazzi et al., 1997; Ren et al., 2001), the total cholesterol and LDL cholesterol in rats fed with a high-cholesterol and high-fat diet rose to approximately 6 and 7 mM, respectively, whereas in rabbits, a similar type of diet resulted in a total cholesterol level of above 25 mM (Chiba et al., 1997; Zulli et al., 2003). In addition, in apoE–/–or apoE/low-density lipoprotein receptor–/– mice that developed spontaneous hypercholesterolemia, total cholesterol levels reached 15 or 25 mM, respectively. When apoE–/– mice were fed with a Western-type diet, the total cholesterol levels amounted to approximately 40 mM (Plump et al., 1992; Breslow, 1996). Thus, it could well be that endothelial dysfunction and the progression of atherosclerosis develop in response to a much higher level of cholesterol than that achieved in rats. Therefore, it could well be that cholesterol transport through the vascular wall and reverse cholesterol transport is so efficient in rats that it prevents the development of severe hypercholesterolemia and lipid accumulation in the vascular wall. In our study, oil red staining was negative and did not reveal lipid deposits in the subendothelial space of the aortic roots of the hypercholesterolemic SHR or WKY rats. On the other hand, the liver in the SHR or WKY rats fed with the hypercholesterolemic diet was heavily overloaded with lipids. The adventitia of the descending aorta was also overloaded with lipids (data not shown). These results could support the hypothesis of the highly efficient cholesterol transport through the vascular wall in rats. Interestingly, a hypercholesterolemic diet in normotensive Wistar rats led to the increased density of adventitial vasa vasorum (Kai et al., 2002), which could also facilitate outflow of cholesterol from the vessel wall.
HDL plays a role in the reverse cholesterol transport and possesses direct antiatherosclerotic effects, including the stimulation of NO and PGI2 release from the endothelium (Nofer et al., 2002). In our experiments, the level of HDL was not modified by a hypercholesterolemic diet. Thus, it still remains to be determined what is the contribution of the reverse cholesterol transport, protective properties of HDL per se, and other mechanisms to the resistance of the rat aorta to lipid accumulation in the subendothelial space.
In summary, in spontaneously hypertensive rats, hypercholesterolemia failed to modify the NO-dependent and PGI2-dependent endothelial function and did not induce a robust inflammatory response. Therefore, we postulate that irrespective of the presence or absence of hypertension, rats are resistant to hypercholesterolemia-induced atherosclerosis because hypercholesterolemia in this species does not lead to the impairment of activity of endothelial NO and PGI2 and the substantial accumulation of lipids in subendothelial space. Further studies are needed to elucidate the mechanisms involved.
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Footnotes |
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ABBREVIATIONS: NO, nitric oxide; SHR, spontaneously hypertensive rats; WKY, Wistar-Kyoto rats; PGI2, prostacyclin; MCP-1, monocyte chemoattractant protein 1; BP, blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; ACh, acetylcholine; SNAP, S-nitroso-N-acetyl-penicillamine; L-NAME, L-NG-nitroarginine methyl ester; 6-keto-PGF1, 6-keto-prostaglandin F1; EDCF, endothelium-derived contracting factor; COX, cyclooxygenase; ORO, oil red O; SQ 29548, [1S(1,2(Z),3,4)]-7-[3-[[2-[(phenylamino) carbonyl]hydrazino]methyl]-7-oxabicy-clo[2.2.1] hept-2-yl]-5-heptenoic acid; apoE, apolipoprotein E.
Address correspondence to: Dr. Stefan Chlopicki, Professor, Department of Experimental Pharmacology, Chair of Pharmacology, Jagiellonian University, ul Grzegorzecka 16, 31-531 Krakow, Poland. E-mail: s.chlopicki{at}cyfronet.krakow.pl
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