Russell L. Prewitt

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.

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.

Captopril reduces aortic and microvascular growth in hypertensive and normotensive rats.

This experiment was designed to investigate the effect of converting enzyme inhibition on functional and structural vascular alterations in one-kidney, one clip hypertensive rats and in normotensive rats. Starting 1 day before surgery, 100 mg/kg/day captopril was given chronically to half of the hypertensive and normotensive groups in their drinking water. With use of intravital microscopy in the cremaster muscle, arteriolar dimensions were measured 4 weeks later, both before and after topical application of 10∼3 M adenosine. Mean blood pressure was 124 ±4 mm Hg in control rats and 103 ±5 mm Hg in captopril-treated control rats (p < 0.05). Mean blood pressure was significantly elevated to 183±5 mm Hg in captopril-treated one-kidney, one clip hypertensive rats and 193 ±5 mm Hg in one-kidney, one clip hypertensive rats. With use of histological techniques, a marked reduction of medial-intimal area of the abdominal aorta was found in captopril-treated control rats (24%), and hypertrophy of the aortic wall in one-kidney, one clip hypertensive rats was decreased 26% by captopril. Structural diameter reductions occurred in large arterioles of the captopril-treated control and hypertensive groups and the nontreated hypertensive group. In spite of a significant increase in wall-to-lumen ratio of first-order arterioles in all captopril-treated rats, captopril decreased cross-sectional wall area of these vessels 37% in hypertensive and 20% in control rats, respectively. Measured by stereological techniques, small arteriolar density decreased 30% in captopril-treated hypertensive rats and 17% in captopril-treated control rats. Therefore, smaller arteriolar lumens, decreased aortic and arteriolar cross-sectional wall area, and arteriolar rarefaction after converting enzyme inhibition, in spite of rising or falling blood pressure, are evidence that vascular growth was inhibited in vivo.

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.

Flow-induced arterial remodeling in rat mesenteric vasculature

This study was designed to characterize in vivo arterial remodeling of male Wistar rat small mesenteric arteries exposed to varying levels of elevated blood flow in the presence of normal arterial pressure. Through a series of arterial ligations, respective ileal artery and second-order branch blood flows acutely increased approximately 36 and approximately 170% over basal levels. Their respective diameters increased 12 and 38% and their wall area increased 58 and 120% in a time-dependent fashion between 1 and 7 days postlitigation compared with same-animal control vessels. Medical extracellular connective tissue increased concomitantly with medical wall hypertrophy. Immunostaining for proliferating cell nuclear antigen and nuclear profile analyses suggests that both smooth muscle and endothelial cell hyperplasia contribute to flow-induced vascular remodeling. The initial stimulus in this model is flow-mediated shear stress, with possible augmentation by hoop stress, which is increased approximately 7% by the resultant vasodilation. Stable wall thickness-to-lumen diameter ratios at 1, 3, and 7 days, however, suggest chronic hoop stress is tightly regulated and remains constant. The model described herein allows analyses of two arteries with different degrees of flow elevation within the same animal and demonstrates that the magnitude of vessel remodeling in vivo is directly dependent on the duration of flow elevation after abrupt arterial occlusion.

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.

Pdtc and Mg132, Inhibitors of Nf-κb, Block Endotoxin Induced Vasodilation of Isolated Rat Skeletal Muscle Arterioles

NF-kappaB is a ubiquitous transcription factor that mediates the inflammatory response. Inhibition of NF-kappaB may be of potential therapeutic benefit in the treatment of septic shock. The antioxidant pyrrolidine dithiocarbamate (PDTC) has been shown in previous work to selectively inhibit NF-kappaB activation. Likewise, the proteasome inhibitor MG132 inhibits NF-kappaB formation and degradation of its inhibitor I-kappaB. The goal of this study was to determine if PDTC and MG-132 could inhibit resistance arteriole vasodilation in response to endotoxin and to determine PDTCs site of action in our isolated vessel preparation. Male Sprague-Dawley rats were given an intraperitoneal injection of PDTC, an intravenous injection of MG132, or a sham injection. First-order cremasteric arterioles were isolated, cannulated, and pressurized. A segment of thoracic aorta was then placed in series with the microvascular preparation. Vessels were allowed to achieve spontaneous myogenic tone in a bath of buffer over 1 h (t = 0). Internal vessel diameters were measured and the response to endotoxin (ET) or continued infusion of buffer was measured over 1 h (t = 60). Group 1 (n = 7) was a time-control group. Group 2 (n = 7) was exposed to ET only, Group 3 (n = 5) received PDTC and was exposed to ET, Group 4 (n = 5) received PDTC only, Group 5 (n = 4) received MG132 only, and Group 6 (n = 5) received MG132 and was exposed to ET. To determine the site of action of PDTC, a segment of aorta from an animal treated with PDTC was placed in series with a cremasteric arteriole from an animal receiving a sham injection. The preparation was then exposed to ET, and this is Group 7 (n = 4). Group 8 (n = 4) received ET and was composed of thoracic aorta from an animal receiving a sham injection and a cremasteric arteriole from a PDTC-treated animal. Spontaneous tone was similar in the eight groups at the end of the equilibration period (t = 0). After 1 h (t = 60), Group 2 (vessels exposed to ET only) had significantly less tone (26.1%+/-2.6%; P < 0.01) than Group 1 (39.0%+/-2.4%), Group 3 (39.3%+/-3.1%), Group 4 (41.2%+/-1.6%), Group 5 (39.2%+/-2.9%), Group 6 (41.0%+/-2.7%), Group 7 (45.1%+/-6.5%), and Group 8 (41.1%+/-4.5%). We conclude that PDTC and MG132, inhibitors of NF-kappaB, block ET-induced vasodilation in isolated rat skeletal muscle arterioles. PDTC has effects at both the level of the aortic segment as well as the resistance arteriole. Inhibitors of NF-kappaB may potentially be of therapeutic benefit in the treatment of septic shock.

Src Tyrosine Kinases and Extracellular Signal–Regulated Kinase 1/2 Mitogen-Activated Protein Kinases Mediate Pressure-Induced C-Fos Expression in Cannulated Rat Mesenteric Small Arteries

Chronic hypertension is associated with remodeling of small arteries. There is evidence that the high pressure itself may cause these structural changes, but the responsible mechanisms are not clearly defined. Previously we showed that pressure-induced c-fos expression in intact cannulated rat mesenteric small arteries was inhibited by genistein, a general tyrosine kinase inhibitor. The purpose of this study was to further unravel the underlying signal transduction mechanisms, and we particularly tested the involvement of src tyrosine kinases and extracellular signal–regulated kinase (ERK). Rat mesenteric small arteries were cannulated in a dual-vessel chamber. After a 60-minute equilibration period, the pressure in 1 artery was increased to 140 mm Hg, while the other artery remained at 90 mm Hg. Semiquantitative reverse transcriptase–polymerase chain reaction was used to determine c-fos expression, and Western blotting was used to examine levels of ERK phosphorylation. The involvement of src and ERK was tested with the inhibitors herbimycin A (1 &mgr;mol/L), PP1 (10 &mgr;mol/L), PP2 (10 &mgr;mol/L), and PD98059 (30 &mgr;mol/L). One-hour exposure to 140 mm Hg increased the c-fos/cyclophilin ratio 3.6-fold, from 0.29±0.07 to 1.06±0.25. All the tested inhibitors suppressed the pressure-induced increase of c-fos expression. A 5-minute exposure period to 140 mm Hg increased ERK phosphorylation, and this was abolished in the presence of PP1. The results suggest that pressure-induced c-fos expression in intact cannulated rat mesenteric small arteries may be mediated, at least in part, by src tyrosine kinases and ERK.

Arteriolar changes in developing and chronic stages of two-kidney, one clip hypertension.

Arteriolar internal and external diameters in the cremaster muscle of two-kidney, one clip hypertensive rats (2K1C) were measured in vivo with video microscopy, both before and after the topical application of adenosine (10(-4) M). Arteriolar density was determined by stereologic techniques. Mean arterial blood pressure was significantly elevated in the 2K1C rats, rising to 186 +/- 6 mm Hg by 8 weeks compared with 113 +/- 4 mm Hg in controls. Lumens of larger arterioles showed a structural reduction at 2 weeks of hypertension and remained at the same level through 8 weeks, while arterioles of control rats showed a progressive increase in diameter with age (101 +/- 6 microns in 2K1C vs. 158 +/- 8 microns in controls at 8 weeks after operation). Wall-to-lumen ratios of larger arterioles were significantly increased at 2, 4, and 8 weeks of hypertension, but cross-sectional wall area was significantly reduced at 8 weeks. Medial hypertrophy was not evident at any stage of hypertension. Arteriolar rarefaction of smaller arterioles was functional at 2 weeks and structural at 8 weeks of hypertension. Vascular tone of the smaller arterioles was elevated in the developing and chronic stages of hypertension. At 2 weeks of hypertension when the structural reduction in diameters of larger arterioles was progressing, the increased vasoconstriction and functional rarefaction may have contributed to the elevated resistance. At 8 weeks, the marked diameter reductions of larger arterioles (36% in first-order arterioles and 25% in second-order arterioles) account for most of the increased resistance to flow.