Stroke-prone spontaneously hypertensive rats (SHRSP) on <em>1</em>% NaCl drinking solution and Stroke-Prone Rodent Diet develop severe hypertension and glomerular and vascular lesions characteristic of thrombotic microangiopathy seen in malignant nephrosclerosis. We recently reported that spironolactone, a mineralocorticoid receptor antagonist, markedly reduced proteinuria and malignant nephrosclerotic lesions in these animals. This observation, together with our previous findings that <em>angiotensin</em>-converting enzyme inhibitors prevent the development of vascular damage, suggests that mineralocorticoids, as part of the renin-<em>angiotensin</em>-aldosterone system, play a pathophysiological role in this model. In the present study, we examined whether chronic (2-week) infusion of aldosterone can reverse the renal vascular protective effects of captopril in SHRSP. SHRSP received vehicle (n=8); captopril alone (50 mg. kg-<em>1</em>. d-<em>1</em>, orally) (n=<em>1</em>0); aldosterone infusion alone (40 microg. kg-<em>1</em>. d-<em>1</em>, SC) (n=<em>7</em>); or captopril and aldosterone at 20 (n=6) or 40 (n=<em>7</em>) microg. kg-<em>1</em>. d-<em>1</em>. Systolic blood pressure was markedly elevated in all groups. Vehicle- and aldosterone-infused SHRSP developed severe proteinuria and comparable degrees of renal injury (2<em>1</em>+/-3% and 29+/-3%, respectively) manifested as thrombotic and proliferative lesions in the arterioles and glomeruli. Captopril treatment reduced plasma aldosterone levels concomitant with marked reductions in proteinuria and the absence of histologic lesions of malignant nephrosclerosis. Aldosterone substitution at 20 or 40 microg. kg-<em>1</em>. d-<em>1</em> in captopril-treated SHRSP resulted in the development of severe renal lesions (<em>1</em>6+/-3% and 2<em>1</em>+/-2%, respectively) and proteinuria comparable with that observed in SHRSP given either aldosterone or vehicle alone. These findings support a major role for aldosterone in the development of malignant nephrosclerosis in saline-drinking SHRSP, independent of the effects of blood pressure.

Hypertension is considered a low-grade inflammatory condition, and understanding the role of transcription factors in guiding this response is pertinent. A prominent transcription factor that governs inflammatory responses and has become a focal point in hypertensive research is nuclear factor-κB (NFκB). Within the hypothalamic paraventricular nucleus (PVN), a known brain cardioregulatory center, NFκB becomes potentially even more important in ultimately coordinating the systemic hypertensive response. To definitively demonstrate the role of NFκB in the neurogenic hypertensive response, we hypothesized that PVN NFκB blockade would attenuate <em>angiotensin</em> II-induced hypertension. Twelve-week-old male Sprague-Dawley rats were implanted with radiotelemetry probes for blood pressure measurement and allowed a <em>7</em>-day recovery. After baseline blood pressure recordings, rats were administered either continuous NFκB decoy oligodeoxynucleotide infusion or microinjection of a serine mutated adenoviral inhibitory-κB vector, or their respective controls, bilaterally into the PVN to inhibit NFκB at two levels of its activation pathway. Simultaneously, rats were implanted subcutaneously with an <em>angiotensin</em> II or saline-filled <em>1</em>4-day osmotic minipump. After the 2-week treatments, rats were euthanized and brain tissues collected for PVN analysis. Bilaterally inhibited NFκB rats had a decrease in blood pressure, NFκB p65 subunit activity, proinflammatory cytokines, and reactive oxygen species, including the <em>angiotensin</em> II type <em>1</em> receptor, <em>angiotensin</em>-converting enzyme, tumor necrosis factor, and superoxide in <em>angiotensin</em> II-treated rats. Moreover, after NFκB blockade, key protective antihypertensive renin-<em>angiotensin</em> system components were upregulated. This demonstrates the important role that transcription factor NFκB plays within the PVN in modulating and perpetuating the hypertensive response via renin-<em>angiotensin</em> system modulation.

The renin-<em>angiotensin</em> system (RAS) constitutes an important hormonal system in the physiological regulation of blood pressure. The dysregulation of the RAS is considered a major influence in the development and progression of cardiovascular disease and other pathologies. Indeed, experimental and clinical evidence indicates that blockade of this system with <em>angiotensin</em>-converting enzyme (ACE) inhibitors or <em>angiotensin</em> type <em>1</em> receptor (AT<em>1</em>R) antagonists is an effective therapy to attenuate hypertension and diabetic renal injury, and to improve heart failure. Originally defined as a circulating system, multiple tissues express a complete RAS, and compelling evidence now favors an intracellular system involved in cell signaling and function. Within the kidney, intracellular expression of the three predominant ANG receptor subtypes is evident in the nuclear compartment. The ANG type <em>1</em> receptor (AT<em>1</em>R) is coupled to the generation of reactive oxygen species (ROS) through the activation of phosphoinositol-3 kinase (PI3K) and PKC. In contrast, both ANG type 2 (AT2R) and ANG-(<em>1</em>-<em>7</em>) (AT<em>7</em>R) receptors stimulate nitric oxide (NO) formation, which may involve nuclear endothelial NO synthase (eNOS). Moreover, blockade of either ACE2-the enzyme that converts ANG II to ANG-(<em>1</em>-<em>7</em>)-or the AT<em>7</em> receptor exacerbates the ANG II-ROS response on renal nuclei. Finally, in a model of fetal programmed hypertension, the nuclear ROS response to ANG II is enhanced, while both AT2 and AT<em>7</em> stimulation of NO is attenuated, suggesting that an imbalance in the intracellular RAS may contribute to the development of programming events. We conclude that a functional intracellular or nuclear RAS may have important implications in the therapeutic approaches to cardiovascular disease.

Mas codes for a G protein-coupled receptor that is implicated in <em>angiotensin</em>-(<em>1</em>-<em>7</em>) signaling. We studied the cardiovascular phenotype of Mas-deficient mice backcrossed onto the FVB/N genetic background using telemetry and found that they exhibit higher blood pressures compared with controls. These Mas(-/-) mice also had impaired endothelial function, decreased NO production, and lower endothelial NO synthase expression. Reduced nicotinamide-adenine dinucleotide phosphate oxidase catalytic subunit gp9<em>1</em>(phox) protein content determined by Western blotting was higher in Mas(-/-) mice than in controls, whereas superoxide dismutase and catalase activities were reduced. The superoxide dismutase mimetic, Tempol, decreased blood pressure in Mas(-/-) mice but had a minimal effect in control mice. Our results show a major cardiovascular phenotype in Mas(-/-) mice. Mas-deletion results in increased blood pressure, endothelial dysfunction, and an imbalance between NO and reactive oxygen species. Our animals represent a promising model to study <em>angiotensin</em>-(<em>1</em>-<em>7</em>)-mediated cardiovascular effects and to evaluate Mas agonistic compounds as novel cardioprotective and antihypertensive agents based on their beneficial effects on endothelial function.

<em>1</em>. Hypertension is associated with structural alterations of resistance arteries, a process known as remodelling (increased media-to-lumen ratio). 2. At the cellular level, vascular remodelling involves changes in vascular smooth muscle cell (VSMC) growth, cell migration, inflammation and fibrosis. These processes are mediated via multiple factors, of which <em>angiotensin</em> (Ang) II appears to be one of the most important in hypertension. 3. <em>Angiotensin</em> II signalling, via AT<em>1</em> receptors, is upregulated in VSMC from resistance arteries of hypertensive patients and rats. This is associated with hyperactivation of vascular NADPH oxidase, leading to increased generation of reactive oxygen species (ROS), particularly O2- and H2O2. 4. Reactive oxygen species function as important intracellular second messengers to activate many downstream signalling molecules, such as mitogen-activated protein kinase, protein tyrosine phosphatases, protein tyrosine kinases and transcription factors. Activation of these signalling cascades leads to VSMC growth and migration, modulation of endothelial function, expression of pro-inflammatory mediators and modification of extracellular matrix. 5. Furthermore, ROS increase intracellular free Ca2+ concentration ([Ca2+]i), a major determinant of vascular reactivity. 6. All these processes play major roles in vascular injury associated with hypertension. Accordingly, ROS and the signalling pathways that they modulate provide new targets to regress vascular remodelling, reduce peripheral resistance and prevent hypertensive end-organ damage. <em>7</em>. In the present review, we discuss the role of ROS as second messengers in AngII signalling and focus on the implications of these events in the processes underlying vascular remodelling in hypertension.

The study of experimental hypertension and the development of drugs with selective inhibitory effects on the enzymes and receptors constituting the components of the circulating and tissue renin-<em>angiotensin</em> systems have led to newer concepts of how this system participates in both physiology and pathology. Over the last decade, a renewed emphasis on understanding the role of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) and <em>angiotensin</em>-converting enzyme 2 in the regulation of blood pressure and renal function has shed new light on the complexity of the mechanisms by which these components of the renin <em>angiotensin</em> system act in the heart and in the kidneys to exert a negative regulatory influence on <em>angiotensin</em> converting enzyme and <em>angiotensin</em> II. The vasodepressor axis composed of <em>angiotensin</em>-(<em>1</em>-<em>7</em>)/<em>angiotensin</em>-converting enzyme 2/mas receptor emerges as a site for therapeutic interventions within the renin-<em>angiotensin</em> system. This review summarizes the evolving knowledge of the counterregulatory arm of the renin-<em>angiotensin</em> system in the control of nephron function and renal disease.

Diabetes mellitus is a growing problem in all parts of the world. Both clinical trials and animal models of type I and type II diabetes have shown that hyperactivity of <em>angiotensin</em>-II (Ang-II) signaling pathways contribute to the development of diabetes and diabetic complications. Of clinical relevance, blockade of the renin-<em>angiotensin</em> system prevents new-onset diabetes and reduces the risk of diabetic complications. <em>Angiotensin</em>-converting enzyme (ACE) 2 is a recently discovered mono-carboxypeptidase and the first homolog of ACE. It is thought to inhibit Ang-II signaling cascades mostly by cleaving Ang-II to generate Ang-(<em>1</em>-<em>7</em>), which effects oppose Ang-II and are mediated by the Mas receptor. The enzyme is present in the kidney, liver, adipose tissue and pancreas. Its expression is elevated in the endocrine pancreas in diabetes and in the early phase during diabetic nephropathy. ACE2 is hypothesized to act in a compensatory manner in both diabetes and diabetic nephropathy. Recently, we have shown the presence of the Mas receptor in the mouse pancreas and observed a reduction in Mas receptor immuno-reactivity as well as higher fasting blood glucose levels in ACE2 knockout mice, indicating that these mice may be a new model to study the role of ACE2 in diabetes. In this review we will examine the role of the renin-<em>angiotensin</em> system in the physiopathology and treatment of diabetes and highlight the potential benefits of the ACE2/Ang-(<em>1</em>-<em>7</em>)/Mas receptor axis, focusing on recent data about ACE2.

BACKGROUND

Antihypertensive drugs that block the renin-angiotensin system (angiotensin-converting enzyme inhibitors [ACEIs] or angiotensin receptor blockers) are recommended for patients with chronic kidney disease (CKD). A low blood pressure (BP) goal (BP, <130/80 mm Hg) is also recommended. The objective of this study was to determine the long-term effects of currently recommended BP therapy in 1094 African Americans with hypertensive CKD.

METHODS

Multicenter cohort study following a randomized trial. Participants were 1094 African Americans with hypertensive renal disease (glomerular filtration rate, 20-65 mL/min/1.73 m2). Following a 3x2-factorial trial (1995-2001) that tested 3 drugs used as initial antihypertensive therapy (ACEIs, calcium channel blockers, and beta-blockers) and 2 levels of BP control (usual and low), we conducted a cohort study (2002-2007) in which participants were treated with ACEIs to a BP lower than 130/80 mm Hg. The outcome measures were a composite of doubling of the serum creatinine level, end-stage renal disease, or death.

RESULTS

During each year of the cohort study, the annual use of an ACEI or an angiotensin receptor blocker ranged from 83.7% to 89.0% (vs 38.5% to 49.8% during the trial). The mean BP in the cohort study was 133/78 mm Hg (vs 136/82 mm Hg in the trial). Overall, 567 participants experienced the primary outcome; the 10-year cumulative incidence rate was 53.9%. Of 576 participants with at least 7 years of follow-up, 33.5% experienced a slow decline in kidney function (mean annual decline in the estimated glomerular filtration rate, <1 mL/min/1.73 m2).

CONCLUSIONS

Despite the benefits of renin-angiotensin system-blocking therapy on CKD progression, most African Americans with hypertensive CKD who are treated with currently recommended BP therapy continue to progress during the long term.

The objective of this study was to investigate the singular role of elevated <em>angiotensin</em> II (ANG II) levels in the development of two-kidney, one-clip (2K<em>1</em>C) Goldblatt hypertension in the rat and specifically in the altered intrarenal ANG II levels that occur in the nonclipped kidney. As a substitute for the clipped kidney, chronic delivery of ANG II (40 ng/min) via an osmotic minipump implanted subcutaneously was used to mimic plasma ANG II levels observed in 2K<em>1</em>C rats during the developmental phase of hypertension. Arterial pressure increased gradually over a period of <em>1</em>4 days, and a pressure profile similar in magnitude and temporal pattern to that of the 2K<em>1</em>C rats was observed. Systemic ANG II was elevated to similar levels in the 2K<em>1</em>C (60 +/- <em>1</em>3 fmol/ml) and ANG II-infused rats (<em>7</em>2 +/- <em>1</em>5 fmol/ml) compared with intact two-kidney control animals (3<em>1</em> +/- 6 fmol/ml; P < 0.05) or uninephrectomized rats (<em>1</em>3 +/- <em>1</em> fmol/ml; P < 0.05). Although renin content was markedly suppressed (80%), intrarenal ANG II content of the contralateral kidneys of the 2K<em>1</em>C groups (86 +/- <em>1</em>2 fmol/g) and the ANG II-infused group (<em>1</em>50 +/- <em>1</em><em>7</em> fmol/g) was greater than that of the two-kidney control (53 +/- <em>7</em> fmol/g; P < 0.05) and uninephrectomized control animals (42 +/- 5 fmol/g; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

<em>Angiotensin</em>-converting enzyme 2 (ACE2) catalyzes the conversion of the vasoconstrictor <em>angiotensin</em> II (ANG II) to the vasodilatory peptide <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [ANG-(<em>1</em>-<em>7</em>)]. We showed that treatment of hypertensive rats with the AT(<em>1</em>) receptor antagonist olmesartan increased ACE2 mRNA and protein in the thoracic aorta, suggesting that endogenous ANG II tonically reduces the enzyme. We now report that ANG II downregulates ACE2 activity and mRNA in rat aortic vascular smooth muscle cells (VSMCs) to reduce the conversion of ANG II to ANG-(<em>1</em>-<em>7</em>). Although ANG-(<em>1</em>-<em>7</em>) alone had no effect on the regulation of ACE2 mRNA, the heptapeptide prevented the ANG II-mediated reduction in ACE2 mRNA, an effect blocked by the selective ANG-(<em>1</em>-<em>7</em>) receptor antagonist [d-Ala(<em>7</em>)]-ANG-(<em>1</em>-<em>7</em>). The reduction in ACE2 mRNA by ANG II was also prevented by the mitogen-activated protein (MAP) kinase kinase inhibitor PD98059. Treatment of VSMCs with ANG II increased ERK<em>1</em>/ERK2 activity, which was significantly reduced by pretreatment with ANG-(<em>1</em>-<em>7</em>). Blockade of the ANG II-mediated reduction in ACE2 mRNA and increase in MAP kinase activity by ANG-(<em>1</em>-<em>7</em>) was prevented by pretreatment with sodium vanadate, a tyrosine phosphatase inhibitor, or okadaic acid, a serine-threonine phosphatase inhibitor, suggesting that the heptapeptide activates a MAP kinase phosphatase. This study is the first to show that the MAP kinase-phosphatase pathway is a primary molecular mechanism for regulating ACE2 to maintain the balance between ANG II and ANG-(<em>1</em>-<em>7</em>). The modulatory role of ANG-(<em>1</em>-<em>7</em>) in the regulation of ACE2 by ANG II suggests a complex interplay between the two peptides that is mediated by specific receptors to activate distinct signaling pathways.

Females are less sensitive to the hypertensive effects of <em>angiotensin</em> II compared with males, although the molecular mechanisms responsible are unknown. We hypothesize that differential activation of <em>angiotensin</em> II, <em>angiotensin</em> (<em>1</em>-<em>7</em>), <em>angiotensin</em> II type <em>1</em>, <em>angiotensin</em> II type 2, and mas levels in the renal cortex of male and female spontaneously hypertensive rats contribute to sex differences in the blood pressure response to <em>angiotensin</em> II infusion. Males had a greater increase in blood pressure after <em>angiotensin</em> II infusion than females (males: <em>1</em>50±2 to <em>1</em>86±3 mm Hg; females: <em>1</em>3<em>7</em>±3 to <em>1</em>60±4 mm Hg; P<0.05). <em>Angiotensin</em> II infusion resulted in comparable increases in plasma and renal cortical <em>angiotensin</em> II levels in both sexes. Renal cortical <em>angiotensin</em> (<em>1</em>-<em>7</em>) levels were higher in female rats under basal conditions (<em>1</em>95±<em>1</em>0 versus 6<em>7</em>±<em>1</em><em>1</em> ng/g of cortex; P<0.05) and after <em>angiotensin</em> II infusion (28<em>1</em>±25 versus 205±4<em>7</em> ng/g of cortex; P<0.05) compared with male rats. In the renal cortex of male rats, <em>angiotensin</em> II infusion decreased <em>angiotensin</em> II type <em>1</em> protein expression and increased <em>angiotensin</em> II type 2 expression with no change in mas expression. In female rats there was an increase in mas receptor protein expression with <em>angiotensin</em> II infusion, although <em>angiotensin</em> II type <em>1</em> and <em>angiotensin</em> II type 2 expressions were unchanged. Male and female rats were then treated with the <em>angiotensin</em> (<em>1</em>-<em>7</em>) mas receptor antagonist A-<em>7</em><em>7</em>9 in the absence and presence of <em>angiotensin</em> II. A-<em>7</em><em>7</em>9 equalized the blood pressure response to <em>angiotensin</em> II in males and females (blood pressure at the end of treatment: males, <em>1</em>66±4 mm Hg; females, <em>1</em>64±5 mm Hg). In conclusion, <em>angiotensin</em> (<em>1</em>-<em>7</em>) contributes to the sex difference in <em>angiotensin</em> II-induced increases in blood pressure in spontaneously hypertensive rats.

Nebivolol is a beta(<em>1</em>)-receptor antagonist with vasodilator and antioxidant properties. Because the vascular NADPH oxidase is an important superoxide source, we studied the effect of nebivolol on endothelial function and NADPH oxidase activity and expression in the well-characterized model of <em>angiotensin</em> II-induced hypertension. <em>Angiotensin</em> II infusion (<em>1</em> mg/kg per day for <em>7</em> days) caused endothelial dysfunction in male Wistar rats and increased vascular superoxide as detected by lucigenin-derived chemiluminescence, as well as dihydroethidine staining. Vascular NADPH oxidase activity, as well as expression at the mRNA and protein level, were markedly upregulated, as well as NOS III uncoupled, as evidenced by NO synthase III inhibitor experiments and dihydroethidine staining and by markedly decreased hemoglobin-NO concentrations. Treatment with the beta-receptor blocker nebivolol but not metoprolol (<em>1</em>0 mg/kg per day for each drug) normalized endothelial function, reduced superoxide formation, increased NO bioavailability, and inhibited upregulation of the activity and expression of the vascular NADPH oxidase, as well as membrane association of NADPH oxidase subunits (Rac<em>1</em> and p6<em>7</em>(phox)). In addition, NOS III uncoupling was prevented. In vitro treatment with nebivolol but not atenolol or metoprolol induced a dissociation of p6<em>7</em>(phox) and Rac<em>1</em>, as well as an inhibition of NADPH oxidase activity assessed in heart membranes from <em>angiotensin</em> II-infused animals, as well as in homogenates of Nox<em>1</em> and cytosolic subunit-transfected and phorbol ester-stimulated HEK293 cells. These findings indicate that nebivolol interferes with the assembly of NADPH oxidase. Thus, inhibitory effects of this beta-blocker on vascular NADPH oxidase may explain, at least in part, its beneficial effect on endothelial function in <em>angiotensin</em> II-induced hypertension.

The systemic and regional hemodynamics effects of ANG-(<em>1</em>-<em>7</em>) were examined in urethane-anesthetized rats. The blood flow distribution (kidneys, skin, mesentery, lungs, spleen, brain, muscle, and adrenals), cardiac output, and total peripheral resistance were investigated by using fluorescent microspheres. Blood pressure and heart rate were recorded from the brachial artery. ANG-(<em>1</em>-<em>7</em>) infusion (<em>1</em><em>1</em>0 fmol x min(-<em>1</em>) x <em>1</em>0 min(-<em>1</em>) iv) significantly increased blood flow to the kidney (5.<em>1</em>0 +/- <em>1</em>.0<em>7</em> to 8.30 +/- 0.9<em>7</em> ml x min(-<em>1</em>) x g(-<em>1</em>)), mesentery (0.<em>7</em>3 +/- 0.<em>1</em>6 to <em>1</em>.<em>1</em><em>7</em> +/- 0.49 ml x min(-<em>1</em>) x g(-<em>1</em>)), brain (<em>1</em>.32 +/- 0.44 to 2.<em>1</em>8 +/- 0.85 ml x min(-<em>1</em>) x g(-<em>1</em>)), and skin (0.0<em>7</em> +/- 0.02 to 0.<em>1</em>8 +/- 0.0<em>7</em> ml x min(-<em>1</em>) x g(-<em>1</em>)) and the vascular conductance in these organs. ANG-(<em>1</em>-<em>7</em>) also produced a significant increase in cardiac index (30%) and a decrease in total peripheral resistance (2.90 +/- 0.55 to 2.<em>1</em>5 +/- 0.28 mmHg x ml(-<em>1</em>) x min x <em>1</em>00 g). Blood flow to the spleen, muscle, lungs, and adrenals, as well as the blood pressure and heart rate, were not altered by the ANG-(<em>1</em>-<em>7</em>) infusion. The selective ANG-(<em>1</em>-<em>7</em>) antagonist A-<em>7</em><em>7</em>9 reduced the blood flow in renal, cerebral, mesenteric, and cutaneous beds and blocked the ANG-(<em>1</em>-<em>7</em>)-induced vasodilatation in the kidney, mesentery, and skin, suggesting a significant role of endogenous ANG-(<em>1</em>-<em>7</em>) in these territories. The effects of ANG-(<em>1</em>-<em>7</em>) on the cerebral blood flow, cardiac index, systolic volume, and total peripheral resistance were partially attenuated by A-<em>7</em><em>7</em>9. A high dose of ANG-(<em>1</em>-<em>7</em>) (<em>1</em><em>1</em> pmol x min(-<em>1</em>) x <em>1</em>0 min(-<em>1</em>)) caused an opposite effect of that produced by the low dose. Our results show for the first time that ANG-(<em>1</em>-<em>7</em>) has a previously unsuspected potent effect in the blood flow distribution and systemic hemodynamics.

Although <em>angiotensin</em> II (Ang II) and the heptapeptide Ang-(<em>1</em>-<em>7</em>) differ by only one amino acid, the two peptides produce different responses in vascular smooth muscle cells. We previously showed that Ang II stimulated phosphoinositide hydrolysis, whereas Ang II and Ang-(<em>1</em>-<em>7</em>) released prostaglandins. We now report that Ang II and Ang-(<em>1</em>-<em>7</em>) differentially modulate rat aortic vascular smooth muscle cell growth. Ang-(<em>1</em>-<em>7</em>) inhibited [3H]thymidine incorporation in response to stimulation by fetal bovine serum, platelet-derived growth factor, or Ang II. The reduction in serum-stimulated thymidine incorporation by Ang-(<em>1</em>-<em>7</em>) depended on the concentration of the heptapeptide over the range of <em>1</em> nmol/L to <em>1</em> mumol/L, with a maximal inhibition of 60% by <em>1</em> mumol/L Ang-(<em>1</em>-<em>7</em>). Ang-(<em>1</em>-<em>7</em>) also inhibited the serum-stimulated increase in cell number to a maximum of <em>7</em><em>7</em>% by <em>1</em> mumol/L Ang-(<em>1</em>-<em>7</em>). The attenuation of serum-stimulated thymidine incorporation by Ang-(<em>1</em>-<em>7</em>) was unaffected by antagonists selective for <em>angiotensin</em> type <em>1</em> (AT<em>1</em>) or type 2 (AT2) receptors; however, [Sar<em>1</em>,Ile<em>1</em>]Ang II and [Sar<em>1</em>,Thr2]Ang II were effective antagonists, indicating that growth inhibition by Ang-(<em>1</em>-<em>7</em>) was a result of <em>angiotensin</em> receptor activation. In contrast, Ang II stimulated [3H]thymidine incorporation in cultured vascular smooth muscle cells over the same concentration range, with a maximal stimulation of 3<em>1</em>4% at <em>1</em> mumol/L Ang II. Ang II also increased the total number of cells (to <em>1</em>45% of control), suggesting that enhanced thymidine incorporation was associated with vascular smooth muscle cell proliferation. The AT<em>1</em> antagonist losartan or L-<em>1</em>58,809 but not AT2 antagonists blocked [3H]thymidine incorporation by Ang II. These results suggest that Ang-(<em>1</em>-<em>7</em>) and Ang II exhibit opposite effects on the regulation of vascular smooth muscle cell growth. The inhibition of proliferation by Ang-(<em>1</em>-<em>7</em>) appears to be mediated by a novel <em>angiotensin</em> receptor that is not inhibited by AT<em>1</em> or AT2 receptor antagonists.

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND<em>1</em>0. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-<em>1</em>,2, thromboxane, <em>angiotensin</em> II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF<em>1</em>2<em>1</em>, VEGF<em>1</em>65) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-<em>1</em> and PIGF-2 effectively stimulated cell proliferation on PLEC (<em>1</em>42 +/- <em>7</em>% and <em>1</em><em>7</em>3 +/- <em>1</em>0%) and MIEC (<em>1</em>60 +/- 20% and <em>1</em>43 +/- 28%) in contrast to HUVEC (9 +/- 8% and <em>1</em>5 +/- 20%) and HPEC-A2 (<em>1</em>5 +/- <em>7</em>% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (<em>1</em>) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.

Homogeneous porcine calpain (Ca2+-dependent cysteine proteinase) was found to hydrolyze a variety of peptides and synthetic substrates. Leu-Trp-Met-Arg-Phe-Ala, eledoisin-related peptide, alpha-neoendorphin, <em>angiotensin</em> I, luteinizing hormone-releasing hormone, neurotensin, dynorphin, glucagon, and oxidized insulin B chain were cleaved with a general preference for a Tyr, Met, or Arg residue in the P<em>1</em> position preceded by a Leu or Val residue in the P2 position. No great difference in specificity was found between low-Ca2+-requiring calpain I and high-Ca2+-requiring calpain II. 4-Methylcoumaryl-<em>7</em>-amide (MCA) derivatives having a Leu(or Val)-Met(or Tyr)-MCA or a Leu-Lys-MCA sequence were also cleaved by either calpain I or calpain II with preference for Leu over Val by a factor of 9 to <em>1</em>6. Calpains I and II showed similar but not identical kinetic behavior for individual substrates. The Km and kcat values ranged from 0.23 to <em>7</em>.08 mM and 0.062 to 0.805 s-<em>1</em> for the calpains, while kcat/Km values for the calpains were only <em>1</em>/433 to <em>1</em>/5 of those for papain with a given substrate. With succinyl-Leu-Met(or Tyr)-MCA, calpains I and II were half-maximally activated at <em>1</em>2 and 260 microM Ca2+, respectively, and competitively inhibited by leupeptin (Ki = 0.32 microM for I and 0.43 microM for II) or antipain (Ki = <em>1</em>.4<em>1</em> microM for I and <em>1</em>.45 microM for II). Thus, this is the first report describing the specificity and kinetics of calpains I and II.

<em>1</em>. Recent studies in animals have linked fetal exposure to excess maternal glucocorticoids with the later occurrence of cardiovascular disorders, particularly hypertension. 2. To test the hypothesis that prenatal treatment could impact on adult blood pressure two groups of pregnant ewes were transported from the farm to the Institute at either 22-29 days of pregnancy (pretreatment group <em>1</em>) or 59-66 days of pregnancy (pretreatment group 2), subjected to 48 h treatment with dexamethasone (0.28 mg day-<em>1</em> kg-<em>1</em> for 2 days) and then returned to the farm. The control group remained at the farm for the entire pregnancy. Lambs were then studied at approximately 4, <em>1</em>0 and <em>1</em>9 months after birth. 3. The basal mean arterial pressure in pretreatment group <em>1</em> (80 +/- <em>1</em> mmHg at <em>1</em>24 days; 83 +/- <em>1</em> mmHg at 309 days and 89 +/- <em>1</em> mmHg at 558 days; n = 6) was significantly different (P < 0.05 in all groups) from that in the control group of lambs (<em>7</em>4 +/- 2 mmHg at <em>1</em><em>1</em>0 days; <em>7</em>6 +/- <em>1</em> mmHg at 323 days and 8<em>1</em> +/- <em>1</em> mmHg at 568 days; n = <em>7</em>). However, prenatal glucocorticoid exposure did not alter vascular sensitivity to noradrenaline, <em>angiotensin</em> II and adrenocorticotropic hormone in these sheep at any of the ages studied, nor did it affect basal or adrenocorticotropic hormone-induced concentrations of cortisol or basal plasma renin concentrations in the lambs at any age. 4. These data support the hypothesis that excess glucocorticoid exposure in early pregnancy, during a critical developmental stage or 'window', programmes higher blood pressure that persists in later life.

Monocyte chemoattractant protein-<em>1</em> (MCP-<em>1</em>), a potent monocyte chemoattractant synthesized by vascular cells and monocytes, has been proposed to be an important mediator of inflammatory responses in the arterial vasculature. It was recently demonstrated that hypertension is associated with an inflammatory response in the arterial wall. To determine the effect of hypertension on arterial MCP-<em>1</em> expression, we induced hypertension in Sprague-Dawley rats by infusing <em>angiotensin</em> II (0.<em>7</em>5 mg x kg[-<em>1</em>] x d[-<em>1</em>] SC) for <em>7</em> days. Using Northern blot analysis, we detected a 3.6-fold increase in MCP-<em>1</em> mRNA in the aortas of hypertensive rats. When we normalized blood pressure in <em>angiotensin</em> II-treated rats through oral administration of the nonspecific vasodilator hydralazine (<em>1</em>5 mg x kg[-<em>1</em>] x d[-<em>1</em>]), aortic MCP-<em>1</em> mRNA expression was significantly reduced. Similar results were obtained with a norepinephrine model of hypertension. Taken together, these data suggest that mechanical factors may be responsible in part for the upregulation of expression. Consistent with this interpretation, we found that cultured rat aortic vascular smooth muscle cells exposed to mechanical strain (20% peak deformation at <em>1</em> Hz) exhibited a marked increase in MCP-<em>1</em> expression, suggesting the hemodynamic strain imparted onto arterial cells in hypertension is an important stimulus underlying this phenomenon. These results provide important insights into the in vivo regulation of MCP-<em>1</em> and have potential implications for understanding the influence of hypertension on atherosclerosis.

Interleukin (IL)-6 acts via a receptor complex consisting of the cognate IL-6 receptor (IL-6R) or the soluble IL-6 receptor (sIL-6R) and glycoprotein <em>1</em>30 (gp<em>1</em>30). Here, we investigated the role of these IL-6R components in hypertension and vascular hypertrophy in mice. <em>Angiotensin</em> (Ang) II (<em>1</em>.<em>1</em> mg/kg/day) caused hypertension and cardiac/aortic hypertrophy in wild-type, but not IL-6(-/-), mice throughout <em>7</em> days. A recombinant dimeric soluble gp<em>1</em>30 (sgp<em>1</em>30Fc; 50 to <em>1</em>00 microg, i.p.) blocked Ang II hypertension but not hypertrophy in wild-type mice. Cognate IL-6R was detected in aortic smooth muscle, but its levels and those of plasma sIL-6R were approximately 50% decreased in IL-6(-/-) mice. Ang II infusion activated signal transducer and activator of transcription-3 in heart of WT and decreased Ang II receptor <em>1</em> (ATR<em>1</em>) expression in aorta. Both responses were unaffected by sgp<em>1</em>30Fc and absent in IL-6(-/-) mice. In summary, we show that IL-6 trans-signaling is required for Ang II-dependent hypertension, but that hypertrophy, down-regulation of AT<em>1</em>R, and cardiac signal transducer and activator of transcription-3 activation are mediated via cognate IL-6R. These data show that IL-6 responses in a single disease context are governed by both modes of IL-6 signaling, with each pathway eliciting different outcomes. Inhibition of IL-6 signaling is suggested as a potential therapy for hypertension and cardiac hypertrophy.

BACKGROUND

Aldosterone has been implicated in the effects of angiotensin II in the vasculature. We hypothesized that there is local expression of the mineralocorticoid receptor (MR) in the vasculature and that the use of a selective aldosterone receptor antagonist (SARA) improves endothelial function in early atherosclerosis.

RESULTS

New Zealand rabbits were placed on normal chow or 1% cholesterol diets, randomized to placebo or SARA (eplerenone, 50 mg/kg twice daily), and killed at the end of 6 weeks for various studies. In the hyperlipidemic (HL) chow group, there was a 2.3-fold increase in superoxide (O2*-)) generation. SARA normalized O2*- generation in intact aortas and reduced NADH and NADPH oxidase activity to basal levels (0.31+/-0.04 and 0.27+/-0.02 in HL versus 0.16+/-0.05 and 0.07+/-0.02 in HL-SARA, respectively; P<0.01 by ANOVA). This was associated with improvements in peak relaxations to the endothelial-dependent agonist acetylcholine (82+/-6% in HL-SARA versus 61+/-4 in HL; P<0.01 by ANOVA; ED(50) 6.8x10(-8) mol/L in HL-SARA and 1.2x10(-7) mol/L in HL; P=NS) to near-normal levels. Vessels from the HL group demonstrated hyperreactivity to angiotensin II that could not be corrected with SARA. Plasma aldosterone levels by radioimmunoassay demonstrated a 4- to 5-fold increase in response to SARA but no differences with lipid feeding. Real-time reverse transcriptase-polymerase chain reaction studies revealed expression of MR in the aorta of HL rabbits and those of controls.

CONCLUSIONS

MR antagonism improves endothelial function and reduces O2*- generation in diet-induced atherosclerosis. Targeting aldosterone by blocking its receptor has potential antiatherosclerotic effects.

We report the existence of two structurally distinct forms of the <em>angiotensin</em> receptor AT-<em>1</em> in the mouse. A Balb/c mouse genomic library was screened by homology screening with a polymerase chain reaction (PCR) amplified probe. Restriction mapping and sequencing of the isolated genes revealed the presence of two receptor isoforms, here named the mouse AT-<em>1</em>a and AT-<em>1</em>b receptors, containing 22 different amino acids. Receptor binding studies performed on COS-<em>7</em> cells transfected with the two receptors revealed that they had similar binding profiles for <em>angiotensin</em> II, <em>angiotensin</em> III and AT-<em>1</em> or AT-2 specific antagonists. Because many of the structural differences were in the carboxy terminal putative intracellular domain, we speculate that these isoforms may differ in their regulation, signal transduction, or desensitization mechanisms.

BACKGROUND

In a double-transgenic human renin and human angiotensinogen rat model, we found that mineralocorticoid receptor (MR) blockade ameliorated angiotensin II (Ang II)-induced renal and cardiac damage. How Ang II and aldosterone (Ald) might interact is ill defined.

RESULTS

We investigated the effects of Ang II (10(-7) mol/L) and Ald (10(-7) mol/L) on extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling in vascular smooth muscle cells (VSMCs) with Western blotting and confocal microscopy. Ang II induced ERK 1/2 and JNK phosphorylation by 2 minutes. Ald achieved the same at 10 minutes. Ang II+Ald had a potentiating effect by 2 minutes. Two oxygen radical scavengers and the epidermal growth factor receptor (EGFR) antagonist AG1478 reduced Ang II-, Ald-, and combination-induced ERK1/2 phosphorylation. Preincubating the cells with the MR blocker spironolactone (10(-6) mol/L) abolished Ang II-induced ROS generation, EGFR transactivation, and ERK1/2 phosphorylation.

CONCLUSIONS

Ald potentiates Ang II-induced ERK-1/2 and JNK phosphorylation. Oxygen radicals, the MR, and the EGFR play a role in early signaling induced by Ang II and Ald in VSMCs. These in vitro data may help explain the effects of MR blockade on Ang II-induced end-organ damage in vivo.

Given that <em>angiotensin</em>-(<em>1</em>-<em>7</em>) (Ang-[<em>1</em>-<em>7</em>]) has been frequently reported to exert direct in vitro vascular effects but less often in vivo, we investigated whether a vasodepressor effect of Ang-(<em>1</em>-<em>7</em>) could be unmasked acutely in conscious spontaneously hypertensive rats (SHR) against a background of <em>angiotensin</em> II type <em>1</em> (AT<em>1</em>) receptor blockade. Mean arterial pressure (MAP) and heart rate were measured over a 5-day protocol in various groups of rats randomized to receive the following drug combinations: saline, AT<em>1</em> receptor (AT<em>1</em>R) antagonist candesartan (0.0<em>1</em> or 0.<em>1</em> mg/kg IV) alone, Ang-(<em>1</em>-<em>7</em>) (5 pmol/min) alone, candesartan plus Ang-(<em>1</em>-<em>7</em>), and candesartan plus Ang-(<em>1</em>-<em>7</em>) and <em>angiotensin</em> II type 2 (AT2) receptor (AT2R) antagonist PD<em>1</em>233<em>1</em>9 (50 microg/kg per minute). In Wistar-Kyoto (WKY) rats, saline, Ang-(<em>1</em>-<em>7</em>), or candesartan alone caused no significant alteration in MAP, whereas Ang-(<em>1</em>-<em>7</em>) coadministered with candesartan caused a marked, sustained reduction in MAP. A similar unmasking of a vasodepressor response to Ang-(<em>1</em>-<em>7</em>) during AT<em>1</em>R blockade was observed in SHR. Moreover, the AT(2)R antagonist PD<em>1</em>233<em>1</em>9 markedly attenuated the enhanced depressor response evoked by the Ang-(<em>1</em>-<em>7</em>)/candesartan combination in SHR and WKY rats, whereas in other experiments, the putative Ang-(<em>1</em>-<em>7</em>) antagonist A-<em>7</em><em>7</em>9 (5 and 50 pmol/min) did not attenuate this vasodepressor effect. In separate experiments, the bradykinin type 2 receptor antagonist HOE <em>1</em>40 (<em>1</em>00 microg/kg IV) or the NO synthase inhibitor Nomega-nitro-L-arginine methyl ester (<em>1</em> mg/kg IV) abolished the depressor effect of Ang-(<em>1</em>-<em>7</em>) in the presence of candesartan. Collectively, these results suggest that Ang-(<em>1</em>-<em>7</em>) evoked a depressor response during AT<em>1</em>R blockade via activation of AT2R, which involves the bradykinin-NO cascade.

Wnt/β-catenin signaling is an evolutionarily conserved, highly complex, key developmental pathway that regulates cell fate, organ development, tissue homeostasis, as well as injury and repair. Although relatively silent in normal adult kidney, Wnt/β-catenin signaling is re-activated after renal injury in a wide variety of animal models and in human kidney disorders. Whereas some data point to a protective role of this signaling in healing and repair after acute kidney injury, increasing evidence suggests that sustained activation of Wnt/β-catenin is associated with the development and progression of renal fibrotic lesions. In kidney cells, Wnt/β-catenin promotes the expression of numerous fibrosis-related genes such as Snail<em>1</em>, plasminogen activator inhibitor-<em>1</em>, and matrix metalloproteinase-<em>7</em>. Recent studies also indicate that multiple components of the renin-<em>angiotensin</em> system are the direct downstream targets of Wnt/β-catenin. Consistently, inhibition of Wnt/β-catenin signaling by an assortment of strategies ameliorates kidney injury and mitigates renal fibrotic lesions in various models of chronic kidney disease, suggesting that targeting this signaling could be a plausible strategy for therapeutic intervention. In this mini review, we will briefly discuss the regulation, downstream targets, and mechanisms of Wnt/β-catenin signaling in the pathogenesis of kidney fibrosis.