Anca D. Dobrian

Blockade of Interleukin-17A Results in Reduced Atherosclerosis in Apolipoprotein E–Deficient Mice

Background— T cells play an important role during the immune response that accompanies atherosclerosis. To date, the role for interleukin (IL)-17A in atherogenesis is not well defined. Here, we tested the hypothesis that atherosclerosis-prone conditions induce the differentiation of IL-17A–producing T cells, which in turn promote atherosclerosis. Methods and Results— IL-17A was found to be elevated in the plasma and tissues of apolipoprotein E–deficient (Apoe−/−) mice. IL-17A–expressing T cells were significantly increased in the aortas, spleen, and lamina propria of aged Apoe−/− mice compared with age-matched C57BL/6 mice. IL-17A+ T cells resided in both adventitia and aortas of aged Apoe−/− mice fed a chow diet. Elevated levels of IL-17A+ T cells were also detected in the aortas of 21-week-old Apoe−/− mice fed a Western diet for 15 weeks. IL-17A+ T cells were characterized as predominantly CD4+ T helper 17 (Th17) cells and &ggr;&dgr;+ T cells. Blockade of IL-17A in Apoe−/− mice by use of adenovirus-produced IL-17 receptor A reduced plaque burden in Apoe−/− mice fed a Western diet for 15 weeks. In addition, the treatment diminished circulating IL-6 and granulocyte colony-stimulating factor levels and limited CXCL1 expression and macrophage content within the aortas. Conversely, IL-17A treatment of whole aorta isolated from Apoe−/− mice promoted aortic CXCL1 expression and monocyte adhesion in an ex vivo adhesion assay. Conclusions— These results demonstrate that atherosclerosis-prone conditions induce the differentiation of IL-17A–producing T cells. IL-17A plays a proatherogenic inflammatory role during atherogenesis by promoting monocyte/macrophage recruitment into the aortic wall.

Development of Hypertension in a Rat Model of Diet-Induced Obesity

Although obesity is a risk factor for hypertension, the relationship between these 2 conditions is not well understood. Therefore, we examined some parameters of hypertension and cardiovascular disease in a dietary model of obesity. Male Sprague-Dawley rats were provided either a control diet (C) or a diet containing 32% kcal as fat (similar to a Western diet) for 1, 3, or 10 weeks. Rats in the latter group diverged based on body weight gain into obesity-prone (OP) and obesity-resistant (OR) groups. Systolic blood pressure in OP rats was significantly higher after 10 weeks of the diet (149+/-4. 8 mm Hg) compared with both OR and C groups (131+/-3.7 and 129+/-4.5 mm Hg, respectively). The aortic wall area of OP rats was significantly increased, indicating arterial hypertrophy, and a 2-fold increase in plasma renin activity was found in OP rats compared with OR and C rats. The lipid profile showed a significant increase in plasma and VLDL triglycerides of OP versus OR and C groups as early as 3 weeks on the diet. Plasma and LDL-cholesterol levels were increased in the OP group versus the OR and C groups after 3 weeks of the diet, but the difference was blunted after 10 weeks. Lipid peroxidation (thiobarbituric acid-reactive substances) in OP rats was increased 2-fold in LDL and 1.5-fold in aortic wall compared with OR rats, suggesting an increased oxidative stress in these animals. Periodic acid-Schiff staining of the kidney showed mesangial expansion and focal sclerosis that were more prominent in OP rats than in OR rats. The results suggest that hypercholesterolemia, but not hypertriglyceridemia, is linked to the diet; that hypertension and renin-angiotensin system activation are associated with obesity; and that lipid peroxidation and renal damage are the results of both factors.

Oxidative Stress in a Rat Model of Obesity-Induced Hypertension

The mechanisms underlying the development of hypertension in obesity are not yet fully understood. We recently reported the development of hypertension in a rat model of diet-induced obesity. When Sprague-Dawley rats (n=60) are fed a moderately high fat diet (32 kcal% fat) for 10 to 16 weeks, approximately half of them develop obesity (obesity-prone [OP] group) and mild hypertension (158±3.4 mm Hg systolic pressure), whereas the other half (obesity-resistant [OR] group) maintains a body weight equivalent to that of a low fat control group and is normotensive (135.8±3.8 mm Hg). We examined the potential role of oxidative stress in the development of hypertension in this model. Lipid peroxides measured as thiobarbituric acid–reactive substances showed a significant increase in the LDL fraction of OP rats (2.8±0.32 nmol malondialdehyde/mg protein) compared with OR and control rats (0.9±0.3 nmol malondialdehyde/mg protein). Also, aortic and kidney thiobarbituric acid–reactive substances showed a significant (3- and 5- fold) increase in OP rats after 16 weeks of diet. In addition, superoxide generation by aortic rings, measured by lucigenin luminescence, showed a 2-fold increase in the OP group compared with both the OR and control groups. In addition, free isoprostane excretion and nitrotyrosine in the kidney showed an increase in OP rats only. The urine and plasma nitrate/nitrite measured by the LDH method showed a 1.8-fold decrease in OP rats compared with OR rats. However, endothelial NO synthase expression in the kidney cortex and medulla assessed by reverse transcriptase–polymerase chain reaction showed a strong increase in the OP rats versus OR and control rats (endothelial NO synthase/&bgr;-actin ratio 1.3±0.04 in OP rats versus 0.44±0.02 in OR rats), suggesting a possible shift toward superoxide production by the enzyme. Collectively, the data show a decreased NO bioavailability in OP animals that is due in part to the increased oxidative stress.

Functional and pathological roles of the 12- and 15-lipoxygenases

The 12/15-lipoxygenase enzymes react with fatty acids producing active lipid metabolites that are involved in a number of significant disease states. The latter include type 1 and type 2 diabetes (and associated complications), cardiovascular disease, hypertension, renal disease, and the neurological conditions Alzheimers disease and Parkinsons disease. A number of elegant studies over the last thirty years have contributed to unraveling the role that lipoxygenases play in chronic inflammation. The development of animal models with targeted gene deletions has led to a better understanding of the role that lipoxygenases play in various conditions. Selective inhibitors of the different lipoxygenase isoforms are an active area of investigation, and will be both an important research tool and a promising therapeutic target for treating a wide spectrum of human diseases.

Pioglitazone Prevents Hypertension and Reduces Oxidative Stress in Diet-Induced Obesity

Abstract—The objective of this study was to determine the effect of pioglitazone on blood pressure (BP) and oxidative balance in obese, hypertensive, Sprague-Dawley rats and to identify some of the molecular mechanisms involved. After 12 weeks of a moderately high-fat diet, rats diverged into obesity-prone (OP) and obesity-resistant (OR) groups (n=6 per group). At the end of the diet, peroxisome proliferator activated receptor-&ggr; (PPAR&ggr;) mRNA expression and activity in the renal cortex and medulla of OP rats were significantly lower compared with that in OR rats. Pioglitazone treatment increased PPAR&ggr; expression and activity in OP rats, suggesting a possible direct ligand-related effect of pioglitazone. As opposed to the untreated OP group, which showed moderate hypertension (systolic BP=159±5.3 mm Hg) after 12 weeks, pioglitazone-treated rats were normotensive (systolic BP=123.9±2.7 mm Hg). Insulin production was reduced by 2-fold in the OP group treated with pioglitazone. Urinary isoprostanes and renal lipid peroxides were also reduced in OP rats treated with pioglitazone compared with untreated counterparts. Also, expression of p47 phox and gp91 phox, both increased in OP versus OR rats, was reduced in the former by pioglitazone treatment. In addition, pioglitazone treatment increased nitrate/nitrite excretion and expression of renal endothelial and neuronal nitric oxide synthase. Collectively, the results show that pioglitazone treatment prevented hypertension and renal oxidative stress both by reducing free-radical production and by increasing nitric oxide production/availability.

Dipeptidyl peptidase IV inhibitor sitagliptin reduces local inflammation in adipose tissue and in pancreatic islets of obese mice.

Adipose tissue inflammation and reduced pancreatic β-cell function are key issues in the development of cardiovascular disease and progressive metabolic dysfunction in type 2 diabetes mellitus. The aim of this study was to determine the effect of the DPP IV inhibitor sitagliptin on adipose tissue and pancreatic islet inflammation in a diet-induced obesity model. C57Bl/6J mice were placed on a high-fat (60% kcal fat) diet for 12 wk, with or without sitagliptin (4 g/kg) as a food admix. Sitagliptin significantly reduced fasting blood glucose by 21% as well as insulin by ∼25%. Sitagliptin treatment reduced body weight without changes in overall body mass index or in the epididymal and retroperitoneal fat mass. However, sitagliptin treatment led to triple the number of small adipocytes despite reducing the number of the very large adipocytes. Sitagliptin significantly reduced inflammation in the adipose tissue and pancreatic islet. Macrophage infiltration in adipose tissue evaluated by immunostaining for Mac2 was reduced by sitagliptin (P < 0.01), as was the percentage of CD11b+/F4/80+ cells in the stromal vascular fraction (P < 0.02). Sitagliptin also reduced adipocyte mRNA expression of inflammatory genes, including IL-6, TNFα, IL-12(p35), and IL-12(p40), 2.5- to fivefold as well as 12-lipoxygenase protein expression. Pancreatic islets were isolated from animals after treatments. Sitagliptin significantly reduced mRNA expression of the following inflammatory cytokines: MCP-1 (3.3-fold), IL-6 (2-fold), IL-12(p40) (2.2-fold), IL-12(p35) (5-fold, P < 0.01), and IP-10 (2-fold). Collectively, the results indicate that sitagliptin has anti-inflammatory effects in adipose tissue and in pancreatic islets that accompany the insulinotropic effect.

Role of Angiotensin II and Free Radicals in Blood Pressure Regulation in a Rat Model of Renal Hypertension

One-kidney, 1-clip rats (1K1C) or uninephrectomized controls were treated with either the superoxide dismutase mimetic tempol (0.5 mmol · kg−1 · d−1), angiotension type 1 receptor inhibitor losartan (50 mmol · L−1 · kg−1 · d−1), or both (n=6 per group) for 2 weeks. At the end of the study, systolic blood pressure (BP) decreased on average by 21% in tempol-treated and 29% in losartan-treated versus untreated 1K1C (217±4.4 mm Hg) and was normalized in the losartan plus tempol group. Mean BP also decreased from 159±3.7 mm Hg in 1K1C to 93±2.8 mm Hg in the losartan plus tempol group. Also, aortic wall area was reduced by 18% in losartan- or tempol-treated 1K1C and by 30% in losartan plus tempol rats compared with untreated 1K1C. Plasma renin activity was increased from 4.8±0.3 in untreated 1K1C to 15.9±0.9 ng · mL−1 · h−1 in losartan-treated but not tempol-treated 1K1C. Superoxide generation by the isolated aortic rings assessed by lucigenin chemiluminescence was significantly decreased (by ≈40%) in all losartan, tempol, and losartan plus tempol groups compared with untreated 1K1C. Nitrotyrosine ELISA in the kidney displayed a significant reduction, from 59±13 ng/mg of protein in 1K1C to 12.5±5 ng/mg of protein in the losartan plus tempol 1K1C. Western blotting for nNOS in kidney cortex and medulla showed a protein increase in both fractions of 1K1C versus controls and was normalized by losartan plus tempol treatment. Collectively, data show a synergistic effect of losartan and tempol on BP reduction in 1K1C rats. The mechanism may involve reduced superoxide production and nitrotyrosine formation in kidney and decreased kidney neuronal-type NO synthase expression in treated animals. This status in the oxidative balance seems to affect BP in the renal hypertensive rats.

Evidence for activation of inflammatory lipoxygenase pathways in visceral adipose tissue of obese Zucker rats

Central obesity is associated with low-grade inflammation that promotes type 2 diabetes and cardiovascular disease in obese individuals. The 12- and 5-lipoxygenase (12-LO and 5-LO) enzymes have been linked to inflammatory changes, leading to the development of atherosclerosis. 12-LO has also been linked recently to inflammation and insulin resistance in adipocytes. We analyzed the expression of LO and proinflammatory cytokines in adipose tissue and adipocytes in obese Zucker rats, a widely studied genetic model of obesity, insulin resistance, and the metabolic syndrome. mRNA expression of 12-LO, 5-LO, and 5-LO-activating protein (FLAP) was upregulated in adipocytes and adipose tissue from obese Zucker rats compared with those from lean rats. Concomitant with increased LO gene expression, the 12-LO product 12-HETE and the 5-LO products 5-HETE and leukotriene B4 (LTB4) were also increased in adipocytes. Furthermore, upregulation of key proinflammatory markers interleukin (IL)-6, TNFα, and monocyte chemoattractant protein-1 were observed in adipocytes isolated from obese Zucker rats. Immunohistochemistry indicated that the positive 12-LO staining in adipose tissue represents cells in addition to adipocytes. This was confirmed by Western blotting in stromal vascular fractions. These changes were in part reversed by the novel anti-inflammatory drug lisofylline (LSF). LSF also reduced p-STAT4 in visceral adipose tissue from obese Zucker rats and improved the metabolic profile, reducing fasting plasma glucose and increasing insulin sensitivity in obese Zucker rats. In 3T3-L1 adipocytes, LSF abrogated the inflammatory response induced by LO products. Thus, therapeutic agents reducing LO or STAT4 activation may provide novel tools to reduce obesity-induced inflammation.

Adaptation of Resistance Arteries to Increases in Pressure

During the development of hypertension, hypertrophy of smooth muscle cells and deposition of extracellular matrix thicken the walls of large arteries without reducing the size of the lumen. The small arteries and arterioles remodel inwardly through a eutrophic process of rearrangement of the same smooth muscle cells around a smaller lumen. Pressure, through an increase in circumferential wall stress, can account for both hypertrophy and inward, eutrophic remodeling. The small arteries constrict during an elevation of pressure, thus restoring wall stress toward control levels. The large arteries have little vasoactivity and respond to the increase in wall stress by initiating a growth process. Mechanotransduction of the pressure stimulus to a growth response is being studied in small mesenteric arteries. Raising the pressure from 90 to 140 mmHg initiates a signaling process starting with phosphorylation of Src within 1 minute. This is followed by phosphorylation of Erk 1/2 peaking at 5 minutes and expression of c‐fos mRNA within 30 minutes. Gene expression correlates with wall stress and is thus inhibited by a myogenic response. Maintained vasoconstriction in an isolated arteriole results in inward, eutrophic remodeling within 4 days. Thus, the current data support the hypothesis that wall thickness is determined by circumferential wall stress, and lumen size is determined by vascular tone.

In vitro formation of oxidatively-modified and reassembled human low-density lipoproteins: antioxidant effect of albumin.

In early atherogenesis, excess plasma lipoproteins accumulate into the arterial lesion-prone areas as modified and reassembled lipoproteins (MRLp) appearing mostly as lipid droplets and vesicles. In the present study we produced such MRLp, in a cell-free system, devoid of any component of extracellular matrix, by subjecting in vitro human low-density lipoproteins (LDL) to autoxidation or copper-induced oxidation, for up to 96 h. As visualized by negative staining electron microscopy, a large number of lipoprotein particles (Lp) were progressively transformed into aggregates (ALp), fused particles (FLp) and vesicles (VLp). These modifications were paralleled by peroxidation of the samples as revealed by chemical analysis of each MRLp fraction isolated by a three-step purification procedure. LDL peroxidation in the above conditions was inhibited by the presence of albumin as assessed by TBARS and lipid analysis, and by the lack of MRLp formation. This protective effect was independent of albumin source (bovine, human, rabbit) and occurs at an albumin/LDL ratio of 1 when Cu2+ was present, and at a ratio of 0.25 in autoxidative conditions. The results show that: (i) in vitro LDL autoxidation or copper-induced peroxidation in a cell-free system can generate modified and reassembled lipoproteins similar to those detected in vivo in the arterial intima at the inception of atherogenesis; (ii) Lp particles appear to be sequentially transformed in self-aggregates, droplets and vesicles; (iii) serum albumin can completely prevent these LDL alterations.