Anne M. Dorrance

Direct regulation of blood pressure by smooth muscle cell mineralocorticoid receptors

Hypertension is a cardiovascular risk factor present in over two-thirds of people over age 60 in North America; elevated blood pressure correlates with increased risk of heart attack, stroke and progression to heart and kidney failure. Current therapies are insufficient to control blood pressure in almost half of these patients. The mineralocorticoid receptor (MR), acting in the kidney, is known to regulate blood pressure through aldosterone binding and stimulation of sodium retention. However, recent studies support the concept that the MR also has extrarenal actions and that defects in sodium handling alone do not fully explain the development of hypertension and associated cardiovascular mortality. We and others have identified functional MR in human vascular smooth muscle cells (SMCs), suggesting that vascular MR might directly regulate blood pressure. Here we show that mice with SMC-specific deficiency of the MR have decreased blood pressure as they age without defects in renal sodium handling or vascular structure. Aged mice lacking MR in SMCs (SMC-MR) have reduced vascular myogenic tone, agonist-dependent contraction and expression and activity of L-type calcium channels. Moreover, SMC-MR contributes to angiotensin II–induced vascular oxidative stress, vascular contraction and hypertension. This study identifies a new role for vascular MR in blood pressure control and in vascular aging and supports the emerging hypothesis that vascular tone contributes directly to systemic blood pressure.

The effects of hypertension on the cerebral circulation.

Maintenance of brain function depends on a constant blood supply. Deficits in cerebral blood flow are linked to cognitive decline, and they have detrimental effects on the outcome of ischemia. Hypertension causes alterations in cerebral artery structure and function that can impair blood flow, particularly during an ischemic insult or during periods of low arterial pressure. This review will focus on the historical discoveries, novel developments, and knowledge gaps in 1) hypertensive cerebral artery remodeling, 2) vascular function with emphasis on myogenic reactivity and endothelium-dependent dilation, and 3) blood-brain barrier function. Hypertensive artery remodeling results in reduction in the lumen diameter and an increase in the wall-to-lumen ratio in most cerebral arteries; this is linked to reduced blood flow postischemia and increased ischemic damage. Many factors that are increased in hypertension stimulate remodeling; these include the renin-angiotensin-aldosterone system and reactive oxygen species levels. Endothelial function, vital for endothelium-mediated dilation and regulation of myogenic reactivity, is impaired in hypertension. This is a consequence of alterations in vasodilator mechanisms involving nitric oxide, epoxyeicosatrienoic acids, and ion channels, including calcium-activated potassium channels and transient receptor potential vanilloid channel 4. Hypertension causes blood-brain barrier breakdown by mechanisms involving inflammation, oxidative stress, and vasoactive circulating molecules. This exposes neurons to cytotoxic molecules, leading to neuronal loss, cognitive decline, and impaired recovery from ischemia. As the population ages and the incidence of hypertension, stroke, and dementia increases, it is imperative that we gain a better understanding of the control of cerebral artery function in health and disease.

Regulation of gene expression in the mammalian eye and its relevance to eye disease

We used expression quantitative trait locus mapping in the laboratory rat (Rattus norvegicus) to gain a broad perspective of gene regulation in the mammalian eye and to identify genetic variation relevant to human eye disease. Of >31,000 gene probes represented on an Affymetrix expression microarray, 18,976 exhibited sufficient signal for reliable analysis and at least 2-fold variation in expression among 120 F2 rats generated from an SR/JrHsd × SHRSP intercross. Genome-wide linkage analysis with 399 genetic markers revealed significant linkage with at least one marker for 1,300 probes (α = 0.001; estimated empirical false discovery rate = 2%). Both contiguous and noncontiguous loci were found to be important in regulating mammalian eye gene expression. We investigated one locus of each type in greater detail and identified putative transcription-altering variations in both cases. We found an inserted cREL binding sequence in the 5′ flanking sequence of the Abca4 gene associated with an increased expression level of that gene, and we found a mutation of the gene encoding thyroid hormone receptor β2 associated with a decreased expression level of the gene encoding short-wavelength sensitive opsin (Opn1sw). In addition to these positional studies, we performed a pairwise analysis of gene expression to identify genes that are regulated in a coordinated manner and used this approach to validate two previously undescribed genes involved in the human disease Bardet–Biedl syndrome. These data and analytical approaches can be used to facilitate the discovery of additional genes and regulatory elements involved in human eye disease.

An epoxide hydrolase inhibitor, 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA), reduces ischemic cerebral infarct size in stroke-prone spontaneously hypertensive rats

Soluble epoxide hydrolase (sEH) inhibitors have been demonstrated to have cardiovascular protective actions. This hydrolase enzyme converts fatty acid epoxides to their corresponding diols, and this conversion can alter the biologic activity of these metabolites. We hypothesized that 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA), a sEH inhibitor, would protect stroke-prone spontaneously hypertensive rats from cerebral ischemia. AUDA was administered to 6-week-old male rats for 6 weeks, during which blood pressure was measured by telemetry. Cerebral ischemia was induced by middle cerebral artery occlusion, the size of the cerebral infarct was assessed after 6 hours of ischemia, and the results were expressed as a percentage of the hemisphere infarcted (%HI). Vascular structure and function were assessed using a pressurized arteriograph. Plasma levels of AUDA at the end of the treatment period averaged 5.0 ± 0.4 ng/mL, and the urinary excretion rate was 99 ± 21 ng/d. AUDA-treated rats had significantly smaller cerebral infarcts than control rats (36 ± 4% vs 53 ± 4% HI, treated versus control, P < 0.05, n = 6). This difference occurred independently of changes in blood pressure. AUDA treatment increased the passive compliance of the cerebral vessels but had no effect on vascular structure. The results of this study provide novel evidence suggesting that the sEH inhibitor AUDA is a possible therapeutic agent for ischemic stroke.

Downregulation of Renal CYP-Derived Eicosanoid Synthesis in Rats With Diet-Induced Hypertension

Abstract—The incidence of essential hypertension increases with obesity; however, the mechanisms that link obesity with hypertension are unclear. Renal cytochrome P450 (CYP)-derived eicosanoids—hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs)—have been shown to play an important role in the regulation of renal function, vascular tone, and blood pressure. The objective of this study was to examine CYP-derived eicosanoid synthesis in the different renal zones (cortex, medulla, and papilla) of rats fed a high fat diet (HF). Male Sprague-Dawley rats were fed a HF diet or regular rat chow for 10 weeks. After 10 weeks, HF rats showed significantly higher systolic blood pressure, body weight, and fat:body weight ratio. The renal &ohgr;-hydroxylase activity was decreased by 46% in cortex, 43% in medulla, and 46% in papilla of HF rats. The renal epoxygenase activity was decreased by 46% in cortex, 31% in medulla, and 56% in papilla of HF rats. Interestingly, the changes in the rate of 20-HETE and EET formation in different renal zones were consistent with the levels of expression of CYP4A and CYP2C23 proteins, respectively. Furthermore, there were no significant changes in the synthesis of these metabolites in the renal microvessels. These results demonstrate that HF diet causes the downregulation of CYP4A and CYP2C23 in renal tubules, and these proteins are responsible for renal 20-HETE and EET formation. The reduction in the synthesis of these eicosanoids may play an important role in the regulation of renal function and blood pressure in obesity-induced hypertension.

Chemerin Connects Fat to Arterial Contraction

Objective—Obesity and hypertension are comorbid in epidemic proportion, yet their biological connection is largely a mystery. The peptide chemerin is a candidate for connecting fat deposits around the blood vessel (perivascular adipose tissue) to arterial contraction. We presently tested the hypothesis that chemerin is expressed in perivascular adipose tissue and is vasoactive, supporting the existence of a chemerin axis in the vasculature. Approach and Results—Real-time polymerase chain reaction, immunohistochemistry, and Western analyses supported the synthesis and expression of chemerin in perivascular adipose tissue, whereas the primary chemerin receptor ChemR23 was expressed both in the tunica media and endothelial layer. The ChemR23 agonist chemerin-9 caused receptor, concentration-dependent contraction in the isolated rat thoracic aorta, superior mesenteric artery, and mesenteric resistance artery, and contraction was significantly amplified (more than 100%) when nitric oxide synthase was inhibited and the endothelial cell mechanically removed or tone was placed on the arteries. The novel ChemR23 antagonist CCX832 inhibited phenylephrine-induced and prostaglandin F2&agr;-induced contraction (+perivascular adipose tissue), suggesting that endogenous chemerin contributes to contraction. Arteries from animals with dysfunctional endothelium (obese or hypertensive) demonstrated a pronounced contraction to chemerin-9. Finally, mesenteric arteries from obese humans demonstrate amplified contraction to chemerin-9. Conclusions—These data support a new role for chemerin as an endogenous vasoconstrictor that operates through a receptor typically attributed to function only in immune cells.

Increased activation of stromal interaction molecule-1/Orai-1 in aorta from hypertensive rats: a novel insight into vascular dysfunction.

Disturbances in the regulation of cytosolic calcium (Ca2+) concentration play a key role in the vascular dysfunction associated with arterial hypertension. Stromal interaction molecules (STIMs) and Orai proteins represent a novel mechanism to control store-operated Ca2+ entry. Although STIMs act as Ca2+ sensors for the intracellular Ca2+ stores, Orai is the putative pore-forming component of Ca2+ release–activated Ca2+ channels at the plasma membrane. We hypothesized that augmented activation of Ca2+ release–activated Ca2+/Orai-1, through enhanced activity of STIM-1, plays a role in increased basal tonus and vascular reactivity in hypertensive animals. Endothelium-denuded aortic rings from Wistar-Kyoto and stroke-prone spontaneously hypertensive rats were used to evaluate contractions because of Ca2+ influx. Depletion of intracellular Ca2+ stores, which induces Ca2+ release–activated Ca2+ activation, was performed by placing arteries in Ca2+ free-EGTA buffer. The addition of the Ca2+ regular buffer produced greater contractions in aortas from stroke-prone spontaneously hypertensive rats versus Wistar-Kyoto rats. Thapsigargin (10 &mgr;mol/L), an inhibitor of the sarcoplasmic reticulum Ca2+ ATPase, further increased these contractions, especially in stroke-prone spontaneously hypertensive rat aorta. Addition of the Ca2+ release–activated Ca2+ channel inhibitors 2-aminoethoxydiphenyl borate (100 &mgr;mol/L) or gadolinium (100 &mgr;mol/L), as well as neutralizing antibodies to STIM-1 or Orai-1, abolished thapsigargin-increased contraction and the differences in spontaneous tone between the groups. Expression of Orai-1 and STIM-1 proteins was increased in aorta from stroke-prone spontaneously hypertensive rats when compared with Wistar-Kyoto rats. These results support the hypothesis that both Orai-1 and STIM-1 contribute to abnormal vascular function in hypertension. Augmented activation of STIM-1/Orai-1 may represent the mechanism that leads to impaired control of intracellular Ca2+ levels in hypertension.

Mineralocorticoid Receptor Activation Causes Cerebral Vessel Remodeling and Exacerbates the Damage Caused by Cerebral Ischemia

Mineralocorticoid receptor antagonists protect against ischemic cerebrovascular disease; this appears to be caused by changes in cerebral vessel structure that would promote blood flow. Therefore, we hypothesized that mineralocorticoid receptor activation with deoxycorticosterone acetate would cause deleterious remodeling of the cerebral vasculature and exacerbate the damage caused by cerebral ischemia. Six-week-old male Wistar rats were treated with deoxycorticosterone acetate (200 mg/kg) for 6 weeks. At 12 weeks of age, the deoxycorticosterone acetate–treated rats had elevated systolic blood pressure compared with age-matched controls (157±5.9 versus 124±3.1 mm Hg deoxycorticosterone acetate versus control; P<0.05). The area of ischemic damage resulting from middle cerebral artery occlusion was greater in the deoxycorticosterone acetate–treated rats than control (63.5±3.72 versus 46.6±5.52% of the hemisphere infarcted, deoxycorticosterone acetate versus control; P<0.05). Middle cerebral artery structure was assessed using a pressurized arteriograph under calcium-free conditions. Over a range of intralumenal pressures, the lumen and ODs of the middle cerebral arteries were smaller in the deoxycorticosterone acetate–treated rats than the control rats (P<0.05). There was also an increase in the wall thickness and wall:lumen ratio in the vessels from deoxycorticosterone acetate–treated rats (P<0.05). The vessels from the deoxycorticosterone acetate–treated rats were stiffer than those from control rats as evidenced by a leftward shift in the stress/strain curve. These novel data suggest that mineralocorticoid receptor activation without salt loading and nephrectomy is sufficient to elicit deleterious effects on the cerebral vasculature that lead to inward hypertrophic remodeling and an increase in the ischemic damage in the event of a stroke.

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.

Glucocorticoids inhibit tetrahydrobiopterin-dependent endothelial function

Tetrahydrobiopterin (BH4) acts as an important co-factor for endothelial nitric oxide synthase (eNOS). Glucocorticoids have been shown to inhibit expression of the rate-limiting enzyme for tetrahydrobiopterin synthesis, GTP cyclohydrolase, in other cell types. We hypothesized that endothelium-dependent vasodilator responses would be blunted in rats made hypertensive with dexamethasone. Further, we hypothesized that treatment of rat vascular segments with dexamethasone would result in attenuation of endothelial function accompanied by decreased GTP cyclohydrolase expression. We report that endothelium-dependent relaxation responses to the calcium ionophore A23187 are reduced in aortic rings from dexamethasone-hypertensive rats compared with sham values. Dexamethasone incubation abolishes contraction to Nω-nitro-L-arginine (L-NNA, 10–5M) in endothelium-intact aortic rings, and inhibits expression of GTP cyclohydrolase. We conclude that inhibition of BH4 synthesis by glucocorticoid regulation of GTP cyclohydrolase expression may contribute to reduced endothelium-dependent vasodilation characteristic of glucocorticoid-induced hypertension.