Life Sciences 73 (2003) 1253 – 1263 www.elsevier.com/locate/lifescie Change in lipid profile and impairment of endothelium-dependent relaxation of blood vessels in rats after bile duct ligation Hong Ji a, Jenny Y. Jiang a, Zhaolin Xu a, Edwin A. Kroeger a, Samuel S. Lee b, Hongqun Liu b, Hong Shen a, Manna Zhang a, Gerald Y. Minuk a, Patrick C. Choy a,*, Yuewen Gong a a Department of Internal Medicine, Biochemistry and Medical Genetics, Faculty of Medicine, University of Manitoba, A108 Chown Bulding, 753 McDermot Avenue, Winnipeg, Manitoba, Canada R3E 0W3 b Department of Liver Unit, University of Calgary, Calgary, Canada Received 14 November 2002; accepted 12 February 2003 Abstract Hyperlipidemia, a condition normally observed in cholestatic liver disease, is also a risk factor for the development of atherosclerosis. The relationship between the elevation of lipoproteins in cholestatic liver diseases and atherosclerosis formation has not been elucidated. In this study, we propose that the impairment of endothelium-dependent relaxation (EDR) of blood vessels in cholestatic liver diseases may lead to the development of atherosclerosis. Using bile duct ligation (BDL) in rats as a model, we examined the liver function, serum lipid profile, EDR and morphologic change of the aorta from both sham operated and BDL rats. Significant increases in liver and spleen weights, serum alanine transaminase (ALT) and aspartate transaminase (AST) activities and the bilirubin level were observed in BDL rats. Upon bile duct ligation, the total and low-density lipoprotein cholesterol levels were increased but the high-density lipoprotein cholesterol and triglyceride levels were reduced. Less contractility and lowered response to acetylcholine-induced relaxation were found in aorta segments. In addition, the acetylcholine-induced relaxation was blocked by both L-NAME and 15 mM KCl. Our results suggest that both nitric oxide and endothelium-derived hyperpolarizing factor are important elements for the impairment of the EDR in BDL rats. In addition, a mild atrophy of the media of the aorta was detected in BDL rats. We conclude that the alterations of lipid profile and the mild atrophy of the media may lead to the impairment of EDR in the aorta in BDL rats, and these factors may potentiate the development of atherosclerosis. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Aorta; Endothelium-dependent relaxation; Cholestatic diseases; Atherosclerosis * Corresponding author. Tel.: +1-204-789-3236; fax: +1-204-789-3942. E-mail address: pchoy@ms.umanitoba.ca (P.C. Choy). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00423-5 1254 H. Ji et al. / Life Sciences 73 (2003) 1253–1263 Introduction Hyperlipidemia is normally observed in patients with cholestatic liver diseases due to the regurgitation of both phospholipids and cholesterol from the biliary tree into the blood. Events leading to the elevation of cholesterol in hepatobiliary conditions seem to be different from other hyperlipidemic states (Harry et al., 1985; McIntyre et al., 1975; Sabesin, 1982). In the early stages of the disease, levels of very low-density lipoprotein (VHDL) and low-density lipoprotein (LDL) are mildly elevated, but the level of high-density lipoprotein (HDL) is significantly increased. Patients with advanced cholestatic diseases have markedly elevated LDL and decreased HDL (Jahn et al., 1985). It is not clear, however, whether these changes are directly associated with an increased risk for atherosclerosis development. The plasma level of LDL in hyperlipidemic patients and the impairment of endothelial function in arteries are events directly associated with the occurrence of atherosclerosis (Farmer and Gotto, 2002; Jayakody et al., 1988, 1987, 1985). This relationship is confirmed by studies in rabbit model fed with a high cholesterol diet (Jayakody et al., 1988, 1987, 1985) and other diet-induced animal models (Lewis et al., 1999). It is clear that the impairment of endothelium-dependent relaxation is one of the early events during development of atherosclerosis. Bile duct ligation (BDL) in rats has been employed for study of cirrhosis and changes of systemic hemodynamics in advanced liver diseases. In experimental animals and patients with cirrhosis and ascites, hyperdynamic circulation with low arterial pressure, hypervolemia, high cardiac output and reduced peripheral vascular resistance have been well-documented. Hence, the primary aim of this study was to determine whether the elevation of cholesterol in bile duct ligated rats could be associated with the impairment of endothelium-dependent relaxation. Subsequent to the seminal observation of Furchgott and Zawadzki (Furchgott and Zawadzki, 1980) on the involvement of nitric oxide in the endothelium-dependent relaxation, numerous studies have shown that the release of endothelium-derived nitric oxide (EDNO) can be induced by acetylcholine (Ach), which acts on muscarinic receptors of the endothelial cells (Palmer et al., 1987). Other relaxant factors, including endothelium-derived hyperpolarizing factor (EDHF) (Chen et al., 1988), have also been shown to play an important role in the endothelium-dependent relaxation of blood vessels. The over-production of NO has been implicated in the impaired responsiveness to endogenous vasoconstrictors and endothelium-derived vasoactive factors in the BDL rats (Castro et al., 1993; Hartleb et al., 1994). Therefore, this study also aimed to evaluate the relative contribution of EDNO and EDHF to the endothelium-dependent (Ach-induced) relaxation of the aorta in BDL rats. Methods Material Phenylephrine hydrochloride, NG-nitro-L-arginine methyl ester L-NAME), acetylcholine and all other chemicals were purchased from Sigma Co. (St. Louis, MO). Mature male Sprague-Dawley rats (250–350 g) were obtained from the Animal Facility of the University of Manitoba. All rats H. Ji et al. / Life Sciences 73 (2003) 1253–1263 1255 were maintained under 12-hour light/dark cycles with food and water ad libitum. In conducting the research described in this report, all animals received humane care in compliance with institutional guidelines, which is in accordance with criteria set by the Canadian Council on Animal Care. Bile duct ligation Twenty-eight adult male Sprague-Dawley rats were divided in two groups. Bile duct ligation was performed in one group (Lee et al., 1996). Briefly the animal was put under light ether anesthesia and the common bile duct was exposed by median laparotomy and occluded by double ligature with a non-resorbable suture (7-0 silk). The first tie was made below the junction of the hepatic ducts and the second was made above the entrance of the pancreatic ducts. The common bile duct was then resected between the two ligatures, and the abdominal incision was closed. A second group included sham-operated rats. Blood samples were taken from rats every two weeks from the saphenous vein. The animals were sacrificed at 6 weeks after bile duct ligation and assessment of endothelium-dependent relaxation in aortic rings was performed in both animal groups. Sections of the aortic rings were collected for morphological examination. Assessment of endothelium-dependent relaxation in aortic rings: After intraperitoneal administration of an anticoagulant and sedative (800 units of heparin and 0.65 mg diazepam), rats were sacrificed by cervical dislocation. The thoracic aorta was removed and each aortic ring (3 mm segment) was suspended isometrically between two horizontal steel wires in an organ bath (Chen et al., 1997). A resting tension of 2.0 g was applied to the aortic ring and changes in tension were measured with a Grass FT.03 force-displacement transducer connected to a Gould Brush 2400 chart recorder. Aortic rings were equilibrated for 90 minutes with tension being adjusted periodically until the resting tension remained stable at 2.0 g. The organ bath contained 15 ml of Krebs-Henseleit solution which was maintained at 37 jC and saturated with a gas mixture containing 95% O2 and 5% CO2. After an equilibration period of 90 minutes the aortic ring was precontracted with phenylephrine (10 6 M). When the contraction became stable, acetylcholine (10 8 –10 5 M) was added cumulatively to produce endothelium-dependent relaxation. The relaxation produced by acetylcholine was expressed as percentage of the phenylephrine induced contraction. Criteria for acceptability of the endothelium vascular ring preparation included phenylephrine-induced active contraction of >1.5 g and acetylcholine-induced relaxation of >80%. After washout, the preparation was incubated with the test compounds and contraction/relaxation cycle was repeated for comparison. Laboratory procedures Blood samples were from rats at 0, 2, 4, and 6 weeks after sham or BDL procedure. Alanine transaminase (ALT), aspartate transaminase (AST), total cholesterols (TC), triglycerides (TG) and the cholesterol content high-density lipoprotein (HDL) were determined by Roche–Hitachi 917 Clinical Chemistry Analyzer with Roche application. The cholesterol content in low-density lipoprotein (LDL) was calculated by the Friedewald equation (Friedewald et al., 1972). Total bilirubin level was determined by commercial total bilirubin kit from Sigma CO. 1256 H. Ji et al. / Life Sciences 73 (2003) 1253–1263 Histological examination of aortic rings Aortic rings were fixed in 10% paraformaldehyde solution, embedded in paraffin and processed according to conventional histologic techniques for subsequent sectioning and staining in hematoxylin and eosin (HE). Statistical analyses To analyze differences in the treatment groups, we performed the ANOVA and Fisher’s PLSD test as Post hoc test using StatView (version 5.0) software (SAS Institute Inc. Cary, NC). Differences with p values below 0.05 were deemed to be significant. Results Body weights of the two animal groups were monitored weekly. There was no significant difference between the body weights of these two groups throughout the experimental period. A comparison at the six-week interval was as follows: 507.9 F 13.0 g for sham-operated vs 462.9 F 15.4 g for BDL. The wet weights of the liver and spleen of these two animal groups were also compared at the six-week interval. Both the liver and spleen from BDL rats were significantly enlarged, as reflected by their wet weights. Average organ weights between the sham-operated vs BDL rats were: 19.6 F 0.6 g vs 32.1 F 2.2 g for the liver; 0.83 F 0.04 g vs 3.50 F 0.15 g for the spleen. Analysis of plasma enzymes revealed that bile duct ligation caused elevations in alanine transaminase (ALT) and aspartate transaminase (AST) activities as well as the level of bilirubin (Fig. 1). A 2.5 fold increase in ALT and a 4.5 fold increase in AST were observed after the first two weeks of bile duct ligation, and the activities of these two enzymes remained elevated throughout the experimental period. Similar results on the bilirubin levels were also obtained. The complete lipid profiles in the plasma of the two animal groups were determined (Fig. 2). No significant change in levels of total cholesterol was detected in the sera of both the control and BDL rats during the course of bile duct ligation. Comparison between the two experimental groups revealed that there was a significant difference in total cholesterol in BDL rats at week 6 of the ligation (Fig. 2A). This difference was resulted from a decrease in total cholesterol in control rats, rather than an increase in total cholesterol in BDL rats. The obstruction of bile duct caused a small decrease and subsequent increase in the triglyceride levels in the control rats, but the change was not statistically significant. Interestingly, it also resulted in a significant decrease and subsequent increase in the triglyceride levels of the BDL rats (Fig. 2B). Comparison between the two experimental groups indicated that there were significant differences at week 4 and 6 after bile duct ligation. We conclude from this study that the total cholesterol level in the BDL rats was higher than the controls at week 6, whereas the triglyceride level in BDL rats was lower than the controls at week 4 and 6. The levels of HDL in the BDL group were found to be decreasing throughout the course of the ligation (Fig. 2C), whereas no significant change were found in the control group. Comparison between the two experimental groups indicated that there were significant differences at week 4 and 6. In contrast, the level of LDL was markedly increased in the BDL group at the two-, four- and six-week periods (Fig. 2D), whereas the no significant increase was detected in the control group. H. Ji et al. / Life Sciences 73 (2003) 1253–1263 1257 Fig. 1. Liver functions between sham and BDL rats. Sera were prepared from blood samples taken from the saphenous vein at 0, 2, and 4 weeks and by cardiac puncture at 6 week. ALT and AST were determined as described in materials and methods. The bilirubin level was determined by a commercial kit from Sigma Co. The data were obtained from fourteen rats for each group and are depicted as Mean F SE. * indicates p < 0.05. The effect of bile duct ligation on the endothelium-dependent relaxation (EDR) of the aorta was determined. As depicted in Fig. 3A, the contractility of the aorta segment from BDL rats, as determined by the tension study, was inferior to that obtained from the control group. When acetylcholine was added progressively to the bathing solution, the relaxation of the aortic ring from the BDL group was less responsive than the control group (Fig. 3B). The difference in relaxation became more prominent at higher levels of acetylcholine. At 10 6 M of acetylcholine, a significant difference in relaxation between the BDL and control groups was obtained. Since several endothelium-derived mediators might be involved in the relaxation of blood vessels, our first approach was to determine whether EDNO might account for the impairment of the Achinduced relaxation. Aorta rings were incubated for 30 minutes with L-NAME at a concentration (6.8 1258 H. Ji et al. / Life Sciences 73 (2003) 1253–1263 Fig. 2. Lipid profiles in the sera of sham and BDL rats. Sera were prepared from blood samples taken from the saphenous vein at 0, 2, and 4 weeks and by cardiac puncture at 6 week. Total cholesterol (TC), triglyceride (TG), high-density cholesterols (HDLs) and low-density cholesterols (LDLs) were determined as described in materials and methods. The data were obtained from fourteen rats for each group and are depicted as Mean F SE. * p < 0.05. AM). At this concentration, L-NAME has been shown to be effective in blocking the nitric oxidemediated relaxation in vascular smooth muscles (Paskvalin and Khatter, 1997; You et al., 1997). It is clear from this study (Fig. 3C) that the relaxation of the aorta ring was not completely blocked by LNAME. In addition, L-NAME appeared to also impair EDR in aorta rings isolated from the control group (Fig. 3C). Hence, factors other than nitric oxide might be involved to produce the EDR in the aortic rings of these two animal groups. The involvement of the endothelium-derived hyperpolarizing factor (EDHF) for the relaxation of the aortic rings was investigated. The aortic rings were pre-equilibrated in Krebs-Henseleit solution containing 15 mM KCl, which would prevent EDHF-induced hyperpolarization (Adeagbo and Triggle, 1993), but would not alter the basal tone or the contractile response to phenylephrine. In the presence of the elevated level of K+, EDR of the aortic ring from the control group was significantly inhibited (Fig. 3D). A higher degree of inhibition was observed in the aortic rings obtained from the BDL group. H. Ji et al. / Life Sciences 73 (2003) 1253–1263 1259 Fig. 3. Contraction and endothelium-dependent relaxation of aorta rings from sham and BDL rats. A 3 mm segment of the aorta ring was prepared at 6 week after sham or BDL operation. Panel A represents contraction of aorta rings induced by 10 6 M phenylephrine. Panel B displays endothelium-dependent relaxation of aorta rings from sham and BDL rats respectively. Panel C shows L-NAME inhibition of endothelium-dependent relaxation of aorta rings from sham and BDL rats. Panel D displays KCl inhibition of endothelium-dependent relaxation of the aorta rings from sham and BDL rats. The data were obtained from fourteen rats for each group and are depicted as Mean F SE. * indicates p < 0.05. Changes in the contractility and endothelial dependent relaxation of the aortic ring might be caused by morphological changes resulting from bile duct ligation. Hence, aortic rings from both animal groups were examined for morphological damages and histologic changes. No gross morphological change was identified between these two animal groups. Histologic examination of the aortic ring was conducted on routine HE slides. Since changes in the intima represent the first step in atherosclerosis development, the structure of the intima was examined carefully. No detectable pathological changes in the intima were observed, although some alterations were detected in the media of the aortic rings in the BDL group. In this group, the media of the aorta 1260 H. Ji et al. / Life Sciences 73 (2003) 1253–1263 Fig. 4. Histologic examination of the aorta rings from sham and BDL rats (400 ). The aorta rings from sham and BDL rats were fixed in 10% paraformaldehyde solution, embedded in paraffin and stained with hematoxylin and eosin (HE). was relatively thin and the spaces between elastic filaments of the media especially close to the intima were narrower than those in sham-operated rats (Fig. 4). In addition, there were fewer nuclei in the media of the aorta than the sham-operated group. These changes likely represent mild media atrophy in BDL rats. Discussion The presence of hyperlipidemia in patients with cholestatic liver diseases has been well-documented (Quarfordt et al., 1973). The biliary cholestasis, however, accounts for only one third of the increased plasma cholesterol (Byers and Friedman, 1952; Weis and Baas, 1972). In addition to lipid reflux into the blood, an increase in hepatic cholesterol synthesis may also be responsible for the increase in serum cholesterol levels in cholestatic diseases in rats (Frederickson et al., 1954). Although similar changes have not been well defined in human subjects, an altered lipoprotein profile has been documented in several clinical retrospective studies (Crippin et al., 1992; Jahn et al., 1985). The altered lipoprotein profile features higher total cholesterol at week 6 of the ligation, lower triglyceride and HDL-cholesterol levels at week 4 and 6 of the ligation. More importantly, a markedly increase in LDL-cholesterol levels within two weeks of the liver ligation. The change of triglyceride level in cholestasis is still controversial, with one paper documenting a marked elevation (Jahn et al., 1985) and another paper reporting no significant change (Crippin et al., 1992). We have shown conclusively in our study that four weeks after bile duct ligation, the LDL-cholesterol was highly elevated and the level of triglyceride was significantly reduced. Consistent with previous finding, the level of HDL-cholesterol was also reduced at later stages of the bile duct ligation. H. Ji et al. / Life Sciences 73 (2003) 1253–1263 1261 In patients and experimental animals with cirrhosis and ascites, hyperdynamic circulation with low arterial pressure, hypervolemia, high cardiac output and reduced peripheral vascular resistance have been documented (Jimenez and Rodes, 1994). In addition, these patients have impaired vascular sensitivity to endogenous vasopressor agents such as angiotensin II and norepinephrine (Ames et al., 1965; Laragh et al., 1963). At present, the mechanism involved in the impairment of vascular response in these patients remains undefined. In rat models of cirrhosis, the overproduction of NO has been implicated as a key factor for the vascular hyporesponsiveness to angiotensin II or endothelin 1 (Castro et al., 1993; Hartleb et al., 1994). We have demonstrated in this study that the BDL rats displayed an impaired vasoconstriction of the aorta rings by phenylephrine. The result of our study is consistent with a previous report that the impairment of acetylcholine-induced relaxation of aorta rings is time dependent in bile duct ligated rats (Rastegar et al., 2001). The time interval to obtain maximum impairment of acetylcholine-induced relaxation obtained by those authors, however, is different from the current study. Those authors reported that the reduction of EDR reached a plateau at seven days after bile duct ligation (Rastegar et al., 2001), whereas our study indicated that such reduction was not diminished even after 6 weeks of bile duct ligation. One explanation on the discrepancies between these two studies is that the reduction of EDR of the aortic rings may be related to the change of lipid profiles rather than the status of cirrhosis. We have shown that a high level of LDL, coupled with the enhanced ability to generate long-chain species of lysophosphatidylcholine in hyperlipidemic patients, are important factors contributing to the development of atherosclerosis. Indeed, the lysophosphatidylcholine generated during LDL modification has been identified as a factor for the impairment of endothelium-dependent relaxation in vascular preparations (Chen et al., 1997). The mechanism leading to the impairment of endothelium-dependent relaxation by an elevated level of LDL in cholestasis produced in the BDL model would be an interesting topic for future studies. The identification of NO as a physiological mediator was seminal to our understanding of the mechanism leading to endothelium-dependent relaxation and the control of vascular tone (Palmer et al., 1987). Subsequently, other endothelium-derived factors that control fine tone of blood vessels have been discovered (Chen et al., 1988; Thorin and Atkinson, 1994). It is important to evaluate the role of NO on regulation of endothelium-dependent relaxation since the elevation of NO has been demonstrated in patients or animals with cirrhosis (Claria et al., 1992; Guarner et al., 1993; Hori et al., 1996). In view that more than one endothelium-derived factors were involved during cholestatic development, it is imperative for us to evaluate the contribution of factors beyond NO. Quantitative analysis of EDR in the presence of L-NAME, a specific antagonist of nitric oxide synthase, confirmed the role of EDNO in Ach-induced endothelium-dependent relaxation, and incomplete inhibition of L-NAME indicated that EDNO might not be the sole factor involved in impairment of EDR in the BDL rats. Hence, the involvement of the EDHF was investigated. At 15 mM K+, we observed a strong inhibition of EDR in the aorta rings from bile duct ligated rats, which suggests a key role of EDHF in control of EDR in cholestatic stage. In our initial hypothesis, we postulated that the functional changes observed in the aortic rings would be associated with some morphological change in the aortic wall. The intimal layer is predominantly affected by the focal lesions of atherosclerosis. In human smooth muscle cells, extracellular matrix components accumulate slowly within the intima during aging, but the structure of the media does not appear to alter with age (Ross and Glomset, 1973). It is possible that the morphological changes observed in the current study may be related to the functional changes in the aortic ring of the BDL rats. We postulate that more pronounced morphological changes would develop if the study could be 1262 H. Ji et al. / Life Sciences 73 (2003) 1253–1263 extended for another several weeks. We were not able, however, to maintain the experimental group of animals beyond 7–8 weeks due to a high mortality rate. Conclusion Alterations of lipid profile and mild atrophy of the media of aorta were observed in BDL rats. These changes might lead to the impairment of EDR in the aorta in BDL rats, which might contribute to the production of atherosclerosis. In addition, both EDNO and EDHF appear to play important roles in the impairment of EDR in the aorta in BDL rats. Acknowledgements The study was supported by grants from the Canadian Institutes of Health Research (PCC & YG). References Adeagbo, A.S., Triggle, C.R., 1993. Varying extracellular [K+]: a functional approach to separating EDHF- and EDNO-related mechanisms in perfused rat mesenteric arterial bed. J Cardiovasc Pharmacol 21 (3), 423 – 429. Ames, R.P., Borkowski, A.J., Sicinski, A.M., Laragh, J.M., 1965. Prolonged infusion of angiotensin II and norepinephrine on blood pressure, electrolyte balance and aldosterone and cortisol secretion in normal man and cirrhosis with ascites. J Clin Invest 44, 1171 – 1186. 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