Hannah Garver

Doxycycline, a matrix metalloprotease inhibitor, reduces vascular remodeling and damage after cerebral ischemia in stroke-prone spontaneously hypertensive rats.

Matrix metalloproteases (MMPs) are a family of zinc peptidases involved in extracellular matrix turnover. There is evidence that increased MMP activity is involved in remodeling of resistance vessels in chronic hypertension. Thus we hypothesized that inhibition of MMP activity with doxycycline (DOX) would attenuate vascular remodeling. Six-week-old male stroke-prone spontaneously hypertensive rats (SHRSP) were treated with DOX (50 mg·kg(-1)·day(-1) in the drinking water) for 6 wk. Untreated SHRSP were controls. Blood pressure was measured by telemetry during the last week. Middle cerebral artery (MCA) and mesenteric resistance artery (MRA) passive structures were assessed by pressure myography. MMP-2 expression in aortas was measured by Western blot. All results are means ± SE. DOX caused a small increase in mean arterial pressure (SHRSP, 154 ± 1; SHRSP + DOX, 159 ± 3 mmHg; P < 0.001). Active MMP-2 expression was reduced in aorta from SHRSP + DOX (0.21 ± 0.06 vs. 0.49 ± 0.13 arbitrary units; P < 0.05). In the MCA, at 80 mmHg, DOX treatment increased the lumen (273.2 ± 4.7 vs. 238.3 ± 6.3 μm; P < 0.05) and the outer diameter (321 ± 5.3 vs. 290 ± 7.6 μm; P < 0.05) and reduced the wall-to-lumen ratio (0.09 ± 0.002 vs. 0.11 ± 0.003; P < 0.05). Damage after transient cerebral ischemia (transient MCA occlusion) was reduced in SHRSP + DOX (20.7 ± 4 vs. 45.5 ± 5% of hemisphere infarcted; P < 0.05). In the MRA, at 90 mmHg DOX, reduced wall thickness (29 ± 1 vs. 22 ± 1 μm; P < 0.001) and wall-to-lumen ratio (0.08 ± 0.004 vs. 0.11 ± 0.008; P < 0.05) without changing lumen diameter. These results suggest that MMPs are involved in hypertensive vascular remodeling in both the peripheral and cerebral vasculature and that DOX reduced brain damage after cerebral ischemia.

Large-conductance Ca2+-activated K+ channel β1-subunit knockout mice are not hypertensive

Large-conductance Ca2+-activated K+ (BK) channels are composed of pore-forming α-subunits and accessory β1-subunits that modulate Ca2+ sensitivity. BK channels regulate arterial myogenic tone and renal Na+ clearance/K+ reabsorption. Previous studies using indirect or short-term blood pressure measurements found that BK channel β1-subunit knockout (BK β1-KO) mice were hypertensive. We evaluated 24-h mean arterial pressure (MAP) and heart rate in BK β1-KO mice using radiotelemetry. BK β1-KO mice did not have a higher 24-h average MAP when compared with wild-type (WT) mice, although MAP was ∼10 mmHg higher at night. The dose-dependent peak declines in MAP by nifedipine were only slightly larger in BK β1-KO mice. In BK β1-KO mice, giving 1% NaCl to mice to drink for 7 days caused a transient (5 days) elevation of MAP (∼5 mmHg); MAP returned to pre-saline levels by day 6. BK β1-KO mesenteric arteries in vitro demonstrated diminished contractile responses to paxilline, increased reactivity to Bay K 8644 and norepinephrine (NE), and maintained relaxation to isoproterenol. Paxilline and Bay K 8644 did not constrict WT or BK β1-KO mesenteric veins (MV). BK β1-subunits are not expressed in MV. The results indicate that BK β1-KO mice are not hypertensive on normal or high-salt intake. BK channel deficiency increases arterial reactivity to NE and L-type Ca2+ channel function in vitro, but the L-type Ca2+ channel modulation of MAP is not altered in BK β1-KO mice. BK and L-type Ca(2+) channels do not modulate murine venous tone. It appears that selective loss of BK channel function in arteries only is not sufficient to cause sustained hypertension.

The Development of Hypertension and Hyperaldosteronism in a Rodent Model of Life-Long Obesity

Aldosterone has been linked to the deleterious cardiovascular effects of obesity in humans. The association of aldosterone with obesity in rodents is less well defined, particularly in models of diet-induced obesity. We hypothesized that adrenal aldosterone production and aldosterone synthase expression would be increased in rats with obesity-induced hypertension. Male Sprague Dawley rats were fed a high-fat (HF: 36% fat) or control diet from 3 wk of age, and mean arterial pressure (MAP) was measured by telemetry. MAP was increased after 4 wk of HF diet; this was 6 wk before changes in body weight. Mineralocorticoid receptor antagonism did not prevent the HF-induced increase in MAP. After 17 wk on the diets, HF rats had increased body and fat weights (abdominal and epididymal) and were insulin resistant (Homeostasis Model Assessment index: 3.53 ± 0.43 vs. 8.52 ± 1.77; control vs. HF, P < 0.05). Plasma aldosterone levels were increased in the HF rats (64.14 ± 14.96 vs. 206.25 ± 47.55 pg/ml; control vs. HF, P < 0.05). This occurred independently of plasma renin activity (4.8 ± 0.92 vs. 4.73 ± 0.66 ng/ml/h, control vs. HF). The increase in aldosterone was accompanied by a 2-fold increase in adrenal aldosterone synthase mRNA expression and zona glomerulosa hypertrophy. Rats were also studied after 8 wk of HF diet, a time when MAP, but not body weight, was increased. At this time plasma aldosterone was unchanged but plasma renin activity was increased (4.4 ± 0.5 vs. 8.1 ± 1.3 ng/ml/h; control vs. HF, P < 0.05). These studies suggest that rats fed a HF diet from weaning may be a useful model for studying obesity-associated hyperaldosteronism.

Chronic estradiol-17β exposure increases superoxide production in the rostral ventrolateral medulla and causes hypertension: Reversal by resveratrol

Women are exposed to estrogen in several forms, such as oral contraceptive pills and hormone replacement therapy. Although estrogen was believed to be cardioprotective, lately, its beneficial effects are being questioned. Recent studies indicate that oxidative stress in the rostral ventrolateral medulla (RVLM) may play a role in the development of hypertension. Therefore, we hypothesized that chronic exposure to low levels of estradiol-17β (E(2)) leads to hypertension in adult-cycling female Sprague Dawley (SD) rats potentially through generation of superoxide in the RVLM. To test this hypothesis, young adult (3 or 4 mo old) female SD rats were either sham-implanted or implanted (subcutaneously) with slow-release E(2) pellets (20 ng/day) for 90 days. A group of control and E(2)-treated animals were fed lab chow or chow containing resveratrol (0.84 g/kg of chow), an antioxidant. Rats were implanted with telemeters to continuously monitor blood pressure (BP) and heart rate (HR). At the end of treatment, the RVLM was isolated for measurements of superoxide. E(2) treatment significantly increased mean arterial pressure (mmHg) and HR (beats/min) compared with sham rats (119.6 ± 0.8 vs. 105.1 ± 0.7 mmHg and 371.7 ± 1.5 vs. 354.4 ± 1.3 beats/min, respectively; P < 0.0001). Diastolic and systolic BP were significantly increased in E(2)-treated rats compared with control animals. Superoxide levels in the RVLM increased significantly in the E(2)-treated group (0.833 ± 0.11 nmol/min·mg) compared with control (0.532 ± 0.04 nmol/min·mg; P < 0.05). Treatment with resveratrol reversed the E(2)-induced increases in BP and superoxide levels in the RVLM. In conclusion, these findings support the hypothesis that chronic exposure to low levels of E(2) induces hypertension and increases superoxide levels in the RVLM and that this effect can be reversed by resveratrol treatment.

Aging is associated with changes to the biomechanical properties of the posterior cerebral artery and parenchymal arterioles.

Artery remodeling, described as a change in artery structure, may be responsible for the increased risk of cardiovascular disease with aging. Although the risk for stroke is known to increase with age, relatively young animals have been used in most stroke studies. Therefore, more information is needed on how aging alters the biomechanical properties of cerebral arteries. Posterior cerebral arteries (PCAs) and parenchymal arterioles (PAs) are important in controlling brain perfusion. We hypothesized that aged (22-24 mo old) C57bl/6 mice would have stiffer PCAs and PAs than young (3-5 mo old) mice. The biomechanical properties of the PCAs and PAs were assessed by pressure myography. Data are presented as means ± SE of young vs. old. In the PCA, older mice had increased outer (155.6 ± 3.2 vs. 169.9 ± 3.2 μm) and lumen (116.4 ± 3.6 vs. 137.1 ± 4.7 μm) diameters. Wall stress (375.6 ± 35.4 vs. 504.7 ± 60.0 dyn/cm(2)) and artery stiffness (β-coefficient: 5.2 ± 0.3 vs. 7.6 ± 0.9) were also increased. However, wall strain (0.8 ± 0.1 vs. 0.6 ± 0.1) was reduced with age. In the PAs from old mice, wall thickness (3.9 ± 0.3 vs. 5.1 ± 0.2 μm) and area (591.1 ± 95.4 vs. 852.8 ± 100 μm(2)) were increased while stress (758.1 ± 100.0 vs. 587.2 ± 35.1 dyn/cm(2)) was reduced. Aging also increased mean arterial and pulse pressures. We conclude that age-associated remodeling occurs in large cerebral arteries and arterioles and may increase the risk of cerebrovascular disease.

Vascular BK channel deficiency exacerbates organ damage and mortality in endotoxemic mice

Abstract We determined the contribution of vascular BK channels to endotoxin (lipopolysaccharide, LPS)-induced hypotension, organ damage, and mortality using smooth muscle BK channel deficiency (BK channel &bgr;1-subunit knockout, BK &bgr;1-KO) mice. BK &bgr;1-KO mice were more sensitive to LPS-induced mortality compared with wild-type mice. After LPS (20 mg/kg, intraperitoneally), BK &bgr;1-KO mice had a more rapid fall in heart rate and blood pressure (measured by radiotelemetry), shorter latency to mortality, and higher mortality rate than wild-type mice. Twenty-two hours after LPS treatment, wild-type and BK &bgr;1-KO mice had reduced norepinephrine reactivity and impaired constrictor responses to the BK channel blocker paxilline in mesenteric arteries in vitro and higher iNOS expression in the heart, but not in mesenteric arteries. Endotoxemic BK &bgr;1-KO mice also showed more severe lung and intestinal injury, higher myeloperoxidase activity and polymorphonuclear neutrophil infiltration in lung and liver. Endotoxemic BK &bgr;1-KO mice had higher plasma tumor necrosis factor &agr; and interleukin 6 levels at 22 hours, but not 6 hours post-LPS. Exaggerated mortality in BK &bgr;1-KO mice also occurred in the cecal ligation/puncture model of septic shock. Reduced vascular BK channel function does not protect against hypotension in the early stage of septic shock; in the later stage, smooth muscle BK channel deficiency enhances organ damage and mortality.

Long-Term Inhibition of Xanthine Oxidase by Febuxostat Does Not Decrease Blood Pressure in Deoxycorticosterone Acetate (DOCA)-Salt Hypertensive Rats

Xanthine oxidase and its products, uric acid and ROS, have been implicated in the pathogenesis of cardiovascular disease, such as hypertension. We have previously reported that allopurinol inhibition of XO does not alter the progression of deoxycorticosterone acetate (DOCA)-salt hypertension in rats. However other researchers have observed a reduction in blood pressure after allopurinol treatment in the same model. To resolve this controversy, in this study we used the newer and more effective XO inhibitor febuxostat, and hypothesized that a more complete XO blockade might impair hypertension development and its end-organ consequences. We used DOCA-salt hypertensive rats and administered vehicle (salt water) or febuxostat (orally, 5 mg/kg/day in salt water) in a short-term “reversal” experiment (2 weeks of treatment 3 weeks after DOCA-salt beginning) and a long-term “prevention” experiment (treatment throughout 4 weeks of DOCA-salt). We confirmed XO inhibition by febuxostat by measuring circulating and tissue levels of XO metabolites. We found an overall increase in hypoxanthine (XO substrate) and decrease in uric acid (XO product) levels following febuxostat treatment. However, despite a trend for reduced blood pressure in the last week of long-term febuxostat treatment, no statistically significant difference in hemodynamic parameters was observed in either study. Additionally, no change was observed in relative heart and kidney weight. Aortic media/lumen ratio was minimally improved by long-term febuxostat treatment. Additionally, febuxostat incubation in vitro did not modify contraction of aorta or vena cava to norepinephrine, angiotensin II or endothelin-1. We conclude that XO inhibition is insufficient to attenuate hypertension in the rat DOCA-salt model, although beneficial vascular effects are possible.

One-month serotonin infusion results in a prolonged fall in blood pressure in the deoxycorticosterone acetate (DOCA) salt hypertensive rat.

A 7-day infusion of serotonin (5-hydroxytryptamine, 5-HT) causes a sustained fall in elevated blood pressure in the male deoxycorticosterone acetate (DOCA)-salt rat. As hypertension is a long-term disease, we presently test the hypothesis that a longer (30 day) 5-HT infusion could cause a sustained fall in blood pressure in the established hypertensive DOCA-salt rat. This time period (∼4 weeks) was also sufficient to test whether 5-HT could attenuate the development of DOCA-salt hypertension. 5-HT (25 μg/kg/min; sc) or vehicle (Veh) was delivered via osmotic pump to (1) established DOCA-salt rats for one month, (2) Sprague-Dawley rats prior to DOCA-salt administration for one month, and blood pressure and heart rate measured telemetrically. On the final day of 5-HT infusion, free platelet poor plasma 5-HT concentrations were significantly higher in 5-HT versus Veh-infused rats, and mean arterial pressure was significantly lower in 5-HT-infused (135 ± 4 mmHg vs Veh-infused 151 ± 7 mmHg) established DOCA-salt rats. By contrast, 5-HT-infusion did not prevent the development of DOCA-salt hypertension (144 ± 7 mmHg vs Veh = 156 ± 6 mmHg). Isometric contraction of aortic strips was measured, and neither the potency nor maximum contraction to the alpha adrenergic receptor agonist phenylephrine (PE) or 5-HT were modified by infusion of 5-HT (established or preventative infusion), and maximum aortic relaxation to acetylcholine (ACh) was modestly but not significantly enhanced (∼15% improvement). This study demonstrates 5-HT is capable of lowering blood pressure in established DOCA-salt hypertensive rats over the course of one month in a mechanism that does not significantly modify or is dependent on modified vascular responsiveness. This finding opens the possibility that elevation of 5-HT levels could be useful in the treatment of hypertension.

BK channel β1-subunit deficiency exacerbates vascular fibrosis and remodelling but does not promote hypertension in high-fat fed obesity in mice.

Objective: Reduced expression or increased degradation of BK (large conductance Ca2+-activated K+) channel &bgr;1-subunits has been associated with increased vascular tone and hypertension in some metabolic diseases. The contribution of BK channel function to control of blood pressure (BP), heart rate (HR) and vascular function/structure was determined in wild-type and BK channel &bgr;1-subunit knockout mice fed a high-fat or control diet. Methods and results: After 24 weeks of high-fat diet, wild-type and BK &bgr;1-knockout mice were obese, diabetic, but normotensive. High-fat-BK &bgr;1-knockout mice had decreased HR, while high-fat-wild-type mice had increased HR compared with mice on the control diet. Ganglion blockade caused a greater fall in BP and HR in mice on a high-fat diet than in mice on the control diet. &bgr;1-adrenergic receptor blockade reduced BP and HR equally in all groups. &agr;1-adrenergic receptor blockade decreased BP in high-fat-BK &bgr;1-knockout mice only. Echocardiographic evaluation revealed left ventricular hypertrophy in high-fat-BK &bgr;1-knockout mice. Although under anaesthesia, mice on a high-fat diet had higher absolute stroke volume and cardiac output, these measures were similar to control mice when adjusted for body weight. Mesenteric arteries from high-fat-BK &bgr;1-knockout mice had higher norepinephrine reactivity, greater wall thickness and collagen accumulation than high-fat-wild-type mesenteric arteries. Compared with control-wild-type mesenteric arteries, high-fat-wild-type mesenteric arteries had blunted contractile responses to a BK channel blocker, although BK &agr;-subunit (protein) and &bgr;1-subunit (mRNA) expression were unchanged. Conclusion: BK channel deficiency promotes increased sympathetic control of BP, and vascular dysfunction, remodelling and fibrosis, but does not cause hypertension in high-fat fed mice.

Chronic exposure to low doses of estradiol-17ß increases blood pressure in young female rats: A possible role for central Endothelin-1

Previously, we demonstrated that chronic exposure to low levels of estradiol-17β (E2) increases mean arterial pressure (MAP) in young female Sprague-Dawley (SD) rats, however, the underlying mechanisms are unclear. Since endothelin-1 (ET-1) is implicated in blood pressure (BP) regulation, we hypothesized that E2’s effects on MAP are mediated through central ET-1. To test this, young female SD rats were either sham implanted or implanted s.c. with slow-release E2 pellets (20 ng/day for 90 days). BP was monitored by telemetry. After 75 days of E2 exposure, ETA antagonist or vehicle was administered i.c.v. After 90 days of E2 exposure, rats were sacrificed, and the paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) were microdissected for gene expression and protein analysis of ET-1 and its receptors. E2 exposure increased MAP after pellet implantation. Gene expression of ET-1 and ETA but not ETB receptors were upregulated in the PVN and RVLM of E2 treated animals. Further, the protein levels of ETA receptor were also increased in the PVN of E2 treated animals. However, i.c.v. infusion of the ETA antagonist did not completely block the increase in blood pressure. Our results suggest that increases in central ET-1 activity could possibly play a role in chronic E2-induced increase in BP but further studies are needed to completely understand the contribution of ET-1 in this phenomenon.