Transcriptome analysis reveals differentially expressed transcripts in rat adrenal zona glomerulosa and zona fasciculata.

In mammals, aldosterone is produced in the zona glomerulosa (zG), the outermost layer of the adrenal cortex, whereas glucocorticoids are produced in adjacent zona fasciculata (zF). However, the cellular mechanisms controlling the zonal development and the differential hormone production (i.e. functional zonation) are poorly understood. To explore the mechanisms, we defined zone-specific transcripts in this study. Eleven-week-old male rats were used and adrenal tissues were collected from zG and zF using laser-capture microdissection. RNA was isolated, biotin labeled, amplified, and hybridized to Illumina microarray chips. The microarray data were compared by fold change calculations. In zG, 235 transcripts showed more than a 2-fold up-regulation compared to zF with statistical significance. Similarly, 231 transcripts showed up-regulation in zF. The microarray findings were validated using quantitative RT-PCR and immunohistochemical staining on selected transcripts, including Cyp11b2 (zG/zF: 214.2x), Rgs4 (68.4x), Smoc2 (49.3x), and Mia1 (43.1x) in zG as well as Ddah1 (zF/zG 16.2x), Cidea (15.5x), Frzb (9.5x), and Hsd11b2 (8.3x) in zF. The lists of transcripts obtained in the current study will be an invaluable tool for the elucidation of cellular mechanisms leading to zG and zF functional zonation.

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.

Effects of spironolactone on cerebral vessel structure in rats with sustained hypertension.

BACKGROUND: Spironolactone prevents eutrophic middle cerebral artery (MCA) remodeling in young stroke-prone spontaneously hypertensive rats (SHRSP). Clinically, it is more relevant to identify treatments that improve vessel structure after hypertension and remodeling has developed. We hypothesized that spironolactone would increase the MCA lumen diameter and reduce the wall/lumen ratio in SHRSP treated from 12 to 18 weeks of age. METHODS: Twelve-week-old male SHRSP were treated with spironolactone (SHRSP + spir: 25 mg/kg/day) for 6 weeks and were compared at 18 weeks to age matched untreated SHRSP and Wistar Kyoto (WKY) rats. MCA structure was assessed by pressure myography. The WKY rats were included to provide an indication of the magnitude of the hypertensive MCA remodeling. RESULTS: Spironolactone had no effect on blood pressure as measured by telemetry. MCA myogenic tone was enhanced in the SHRSP + spir. Spironolactone increased the MCA lumen diameter (SHRSP: 223.3 ± 9.7 µm, SHRSP + spir: 283.7 ± 10.1 µm, WKY: 319.5 ± 8.8 µm, analysis of variance (ANOVA) P < 0.05) and reduced the wall/lumen ratio (SHRSP: 0.107 ± 0.007, SHRSP + spir: 0.078 ± 0.006, WKY: 0.047 ± 0.002, ANOVA P < 0.05). Vessel wall stiffness was unchanged by spironolactone. Collagen 1 and 4 mRNA expression was increased in cerebral vessels from SHRSP compared to WKY rats; collagen 1 was reduced by spironolactone. Western blot analysis showed that active matrix metalloproteinase (MMP)-13 expression was increased by spironolactone treatment. The expression of intercellular adhesion molecule 1 (ICAM-1), a marker of inflammation, was increased in SHRSP and reduced by spironolactone. CONCLUSIONS: These studies provide evidence that chronic mineralocorticoid receptor (MR) antagonism improves cerebral vessel structure after remodeling has developed in a model of human essential hypertension.

RhoA/Rho-kinase and vascular diseases: what is the link?

RhoA/Rho-kinase pathway plays an important role in many pathological conditions. RhoA participates in the regulation of smooth muscle tone and activates many downstream kinases. The best characterized are the serine/threonine kinase isoforms (Rho-kinase or ROCK), ROCKα/ROCK2 and ROCKß/ROCK1. ROCK is necessary for diverse functions such as local blood flow, arterial/pulmonary blood pressure, airway resistance and intestinal peristalsis. ROCK activation permits actin/myosin interactions and smooth muscle cells contraction by maintaining the activity of myosin light-chain kinase, independently of the free cytosolic calcium level. The sensitization of smooth muscle myofilaments to calcium has been implicated in many pathological states, such as hypertension, diabetes, heart attack, stroke, pulmonary hypertension, erectile dysfunction, and cancer. The focus of this review is on the involvement of RhoA/Rho-kinase in diseases. We will briefly describe the ROCK isoforms and the role of RhoA/Rho-kinase in the vasculature, before exploring the most recent findings regarding this pathway and various diseases.

O-GlcNAcylation: a novel post-translational mechanism to alter vascular cellular signaling in health and disease: focus on hypertension.

O-Linked attachment of beta-N-acetyl-glucosamine (O-GlcNAc) on serine and threonine residues of nuclear and cytoplasmic proteins is a highly dynamic posttranslational modification that plays a key role in signal transduction pathways. Preliminary data show that O-GlcNAcylation may represent a key regulatory mechanism in the vasculature, modulating contractile and relaxant responses. Proteins with an important role in vascular function, such as endothelial nitric oxide synthase, sarcoplasmic reticulum Ca(2+)-ATPase, protein kinase C, mitogen-activated protein kinases, and proteins involved in cytoskeleton regulation and microtubule assembly are targets for O-GlcNAcylation, indicating that this posttranslational modification may play an important role in vascular reactivity. Here, we will focus on a few specific pathways that contribute to vascular function and cardiovascular disease-associated vascular dysfunction, and the implications of their modification by O-GlcNAc. New chemical tools have been developed to detect and study O-GlcNAcylation, including inhibitors of O-GlcNAc enzymes, chemoenzymatic tagging methods, and quantitative proteomics strategies; these will also be briefly addressed. An exciting challenge in the future will be to better understand the cellular dynamics of this posttranslational modification, as well as the signaling pathways and mechanisms by which O-GlcNAc is regulated on specific proteins in the vasculature in health and disease.

Dietary potassium supplementation improves vascular structure and ameliorates the damage caused by cerebral ischemia in normotensive rats.

BACKGROUND: Dietary potassium supplementation in hypertensive rats is cardioprotective. This protection includes a blood pressure independent reduction in the amount of damage caused by cerebral ischemia. Therefore, we hypothesized that dietary potassium supplementation would improve the outcome of ischemic stroke by improving cerebral vessel structure in normotensive rats. METHODS: Wistar Kyoto (WKY) rats were fed a high (HK) or low potassium (LK) diet for six weeks from six weeks of age. At the end of treatment, cerebral ischemia was induced by middle cerebral artery (MCA) occlusion and the resultant infarct was quantified and expressed as a percentage of the hemisphere infarcted (%HI). MCA structure was assessed in an additional group of rats using a pressurized arteriograph. RESULTS: The cerebral infarct was significantly smaller in rats fed the HK diet, compared to rats fed the LK diet (21 +/- 5.4 vs 33.5 +/- 4.8 %HI HK vs LK p < 0.05). Vessel structure was improved in WKY rats fed the HK diet as indicated by an increase in the MCA lumen (298 +/- 6.3 vs 276 +/- 3.9 mum HK vs LK p < 0.05) and outer diameters (322 +/- 7.6 vs 305 +/- 4.8 mum HK vs LK p < 0.05). Wall thickness and area were unchanged, suggesting an outward euthrophic remodelling process. The HK diet had no effect on body weight or telemetry blood pressure. CONCLUSION: These studies are the first to show a beneficial effect of dietary potassium in rats with normal blood pressure.

Is the mineralocorticoid receptor a potential target for stroke prevention?

In recent years, it has become increasingly clear that the extra-renal effects of aldosterone play an important role in the pathogenesis of cardiovascular disease. Stroke is one of the leading causes of death in the Western world, and MR (mineralocorticoid receptor) antagonism is a potential preventative therapy for patients at risk of both ischaemic and haemorrhagic strokes. This protective effect of MR antagonism appears to occur at the level of the cerebral vasculature and may be related to the expression and activation of the EGFR (epidermal growth factor receptor) and the degree of vessel wall collagen deposition.

Intact female stroke-prone hypertensive rats lack responsiveness to mineralocorticoid receptor antagonists.

Data from the Framingham Heart Study suggest that women may be more sensitive to the deleterious cardiovascular remodeling effects of aldosterone. Previous studies from our laboratory have shown that chronic treatment with spironolactone, a mineralocorticoid receptor (MR) antagonist, decreases ischemic cerebral infarct size and prevents remodeling of the middle cerebral artery (MCA) in male spontaneously hypertensive stroke-prone rats (SHRSP). Therefore, we hypothesized that MR antagonism would reduce ischemic infarct size and prevent MCA remodeling in female SHRSP. Six-week-old female SHRSP were treated for 6 wk with spironolactone (25 or 50 mg.kg(-1).day(-1)) or eplerenone (100 mg.kg(-1).day(-1)) and compared with untreated controls. At 12 wk, cerebral ischemia was induced for 18 h using the intraluminal suture occlusion technique, or the MCA was isolated for analysis of passive structure using a pressurized arteriograph. MR antagonism had no effect on infarct size or passive MCA structure in female SHRSP. To study the potential effects of estrogen, the above experiments were repeated in bilaterally ovariectomized (OVX) female SHRSP treated with spironolactone (25 mg.kg(-1).day(-1)). Infarct size and vessel structure in OVX SHRSP were not different from control SHRSP. Spironolactone had no effect on infarct size in OVX SHRSP. However, MCA lumen and outer diameters were increased in spironolactone-treated OVX SHRSP, suggesting an effect of estrogen. Cerebral artery MR expression, assessed by Western blotting, was increased in female, compared with male, SHRSP. These studies highlight an apparent sexual dimorphism of MR expression and activity in the cerebral vasculature from hypertensive rats.

Spironolactone improves structure and increases tone in the cerebral vasculature of male spontaneously hypertensive stroke-prone rats.

BACKGROUND: Previous studies show that ischemic cerebral infarct size is related to cerebral vessel structure. Spironolactone, a mineralocorticoid receptor antagonist, decreases ischemic cerebral infarct size in male spontaneously hypertensive stroke-prone rats (SHRSP). Therefore, we hypothesized that chronic spironolactone treatment would improve cerebral artery structure in the SHRSP. METHODS: Six-week-old male SHRSP were treated with spironolactone (2.5 mg/day) for 6 weeks and were compared to untreated control SHRSP and normotensive Wistar Kyoto (WKY) rats. Using a pressurized arteriograph, structural measurements of the middle cerebral artery (MCA) were taken under passive (calcium-free), zero-flow conditions. Myogenic tone was calculated from active and passive measurements taken at 75 and 125 mmHg. Mean arterial pressure was measured using radiotelemetry. RESULTS: Myogenic tone was increased only at 75 mmHg in the spironolactone-treated SHRSP compared to control rats. The MCA lumen and outer diameters were increased in the spironolactone-treated SHRSP compared to control SHRSP, but were not different from WKY rats, indicating a decrease in vascular remodeling. There was no effect of spironolactone on blood pressure, suggesting that this is a blood pressure-independent effect. CONCLUSION: Increased myogenic tone and lumen diameter in the spironolactone-treated SHRSP may be responsible for the protective role of spironolactone in ischemic stroke.

Aldosterone: good guy or bad guy in cerebrovascular disease?

Stroke is a leading cause of disability in the Western world, yet the choices for therapeutic intervention are few. The complex role played by aldosterone in the pathogenesis of stroke is beginning to emerge. Chronic mineralocorticoid receptor (MR) blockade reduces the incidence of hemorrhagic strokes and the severity of damage caused by ischemic strokes. This appears to be a vascular phenomenon because MR blockade increases vessel lumen diameter, which presumably increases blood flow and perfusion of the tissue to reduce ischemic damage. However, the vascular protection afforded by MR antagonism is at odds with the results seen within the brain, where MR activation is required for neuronal survival. Both of these divergent effects have possible therapeutic implications for stroke.