Vascular Angiotensin Type 1 Receptor Expression Is Associated with Vascular Dysfunction, Oxidative Stress and Inflammation in Fructose-Fed Rats

451 Hypertens Res Vol.30 (2007) No.5 p.451-457 Original Article Vascular Angiotensin Type 1 Receptor Expression Is Associated with Vascular Dysfunction, Oxidative Stress and Inflammation in Fructose-Fed Rats Michael D. NYBY1), Karolin ABEDI2), Victoria SMUTKO2), Pirooz ESLAMI1), and Michael L. TUCK1),2) This study determined whether or not oxidative stress and vascular dysfunction in fructose-induced hyperinsulinemic rats are associated with activation of the vascular renin-angiotensin system (RAS). Four groups of rats were used. CONT rats were fed normal rat chow, CONT + CAP were fed normal rat chow and given 500 mg/L captopril in their drinking water, fructose-fed rats (FFR) were fed a high-fructose diet and FFR+CAP were fed the high-fructose diet plus captopril in water. After 8 weeks, the vascular reactivity of mesenteric artery segments was measured. Blood was analyzed for insulin, glucose, hydrogen peroxide and 8-isoprostane. Aortic and heart tissue were used for subjected to quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis. Systolic blood pressure was significantly higher in FFR (p < 0.05), and captopril treatment inhibited the blood pressure increase. Mesenteric artery dose-response curves to acetylcholine were shifted to the right in FFR (p < 0.05) and were normal in FFR + CAP. Plasma insulin (p < 0.05), hydrogen peroxide (p < 0.02) and 8-isoprostane (p < 0.05) were increased in FFR. Captopril treatment reducd hydrogen peroxide and 8-isoprostane concentrations. Aortic tissue mRNA expression levels were increased for angiotensin-converting enzyme (ACE, p < 0.05), angiotensin type 1 receptor (AT1R, p < 0.02), NOX4 (p < 0.02) and VCAM-1 (p < 0.05) in FFR aortic samples. Captopril treatment reduced AT1R, NOX4 and VCAM-1 expression in FFR to levels not different from CONT. Similar changes in heart tissue mRNA expression for angiotensinogen, AT1R and NOX4 were also observed. These results demonstrate that vascular RAS is upregulated in FFR and support the hypothesis that vascular RAS mediates vascular dysfunction and vascular oxidative stress in FFR. (Hypertens Res 2007; 30: 451–457) Key Words: angiotensin, hypertension, oxidative stress, vascular, insulin resistance Introduction Feeding normal rats a high-fructose diet induces insulin resistance, hypertension, dyslipidemia and vascular dysfunction (1, 2). Although the fructose-fed rat (FFR) model exhibits many metabolic disorders, the cause of hypertension in these animals is not known. Other studies show that reactive oxy- gen species (ROS) are elevated in FFR (3–5) and can interfere with nitric oxide (NO) production, which maintains vascular relaxation in resistance arteries (6). Nitric oxide synthase (NOS) activity itself is also reduced in FFR aortas (7). Thus, a reduction of NOS activity and an increase of ROS production in FFR together could lead to decreased NO bioavailability, resulting in the increased vascular contraction and cardiovascular risk seen in this model of From the 1)David Geffen School of Medicine at UCLA, Los Angeles, USA; and 2)Department of Medicine, VA Greater Los Angeles Healthcare System, Sepulveda, USA. This study was supported by Department of Veterans Affairs Merit Review Funds (to M.L.T.). Address for Reprints: Michael D. Nyby, VA Greater Los Angels Healthcare System, Sepulveda (111E), 16111 Plummer St. Sepulveda, CA 91343, USA. E-mail: Received September 8, 2006; Accepted in revised form December 20, 2006. 452 Hypertens Res Vol. 30, No. 5 (2007) the metabolic syndrome. Recent studies have shown that the renin-angiotensin system (RAS) may be involved in the vascular derangements that occur in patients with type 2 diabetes or the metabolic syndrome (8). Previous studies from our laboratory have shown that high insulin concentrations can stimulate the RAS and subsequent production of angiotensin II in cultured vascular smooth muscle cells (9, 10). A fructose-fed rat study has shown that RAS inhibition with enalapril increases vascular endothelial nitric oxide synthase (eNOS) activity (11), and it can be argued that the angiotensin type 1 receptor (AT1R) is responsible for hypertension (12). However, it has yet to be demonstrated that gene expression of local vascular components of the RAS is increased in cardiovascular tissue from FFR, and this would be a necessary step in the implication of its role in hypertension. Elevated expression of the AT1R gene, but not that of any other RAS component, has been found in aortas of fructose-fed mice, but no heart tissue gene expression data are available (4). Further, it has yet to be shown that gene expression of components of oxidative stress and inflammation in FFR cardiovascular tissue can be inhibited by angiotensin-converting enzyme (ACE) inhibition. The present study was undertaken to determine whether or not hypertension, the production of vascular ROS and endothelial dysfunction in FFR are associated with increased expression of the cardiovascular RAS and whether or not any of these would form a mechanism for the induction of hypertension. Fig. 1. Systolic blood pressure changes during the 60% fructose vs. control dietary study in male CD:IGS rats (n= 6 in each group). Blood pressure increased in both FFR and FFR + CAP groups until week 4, when captopril treatment was initiated (indicated by arrow). At weeks 7 and 8, blood pressure was significantly lower in FFR + CAP than in FFR (*p< 0.05). diets, the animals were euthanized, and blood, heart, aorta and mesenteric artery samples were removed for study. Vascular Reactivity Studies Methods Animals The animal protocols were approved by the Institutional Animal Care and Use Committee of the VA Greater Los Angeles Healthcare System, and the animals were housed in an AAALAC-approved animal research facility. Male CD:IGS rats (Charles River Laboratories, Wilmington, USA) weighing 250–300 g were divided into four groups. The control (CONT) and control with captopril (CONT+CAP) groups were fed normal rat chow (Purina, Richmond, USA). The FFR and fructose-fed with captopril (FFR+CAP) groups were fed a high-fructose diet (60% fructose by weight, Harlan-Teklad, Madison, USA). All rats were given regular drinking water for the first 4 weeks. The CONT+CAP and FFR +CAP were thereafter given drinking water containing 500 mg/L captopril (ICN Chemicals, Costa Mesa, USA). Systolic blood pressure was measured weekly using a tail-cuff method with an optical sensor (IITC, Woodland Hills, USA) as previously described (2, 13). Measures were taken to reduce stress in the rats while their blood pressures were determined. To accustom the rats to the restraints used for blood pressure measurement, they were kept in regular shoebox cages with a 14 cm length of 10 cm diameter plastic pipe. The IITC system we use to measure blood pressure requires minimal heating of the animals (29°C). After 8 weeks on the Proximal sections of the duodenum and intact mesenteric vessel a (...truncated)