Sildenafil, a selective inhibitor of phosphodiesterase type 5 (PDE5), is the first in a new class of orally effective treatments for erectile dysfunction. During sexual stimulation, the cavernous nerves release nitric oxide (NO), which induces cyclic guanosine monophosphate (cGMP) formation and smooth muscle relaxation in the corpus cavernosum. Sildenafil facilitates the erectile process during sexual stimulation by inhibiting PDE5 and thus blocking the breakdown of cGMP. Sildenafil alone can cause mean peak reductions in systolic/diastolic blood pressure of <em>1</em>0/<em>7</em> mm Hg that are not dose related, whereas the heart rate is unchanged. Sildenafil and nitrates both increase cGMP levels in the systemic circulation but at different points along the NO-cGMP pathway. The combination is contraindicated because they synergistically potentiate vasodilation and may cause excessive reductions in blood pressure. Erectile dysfunction is a significant medical condition that shares numerous risk factors with ischemic heart disease, and hence a substantial overlap exists between these patient groups. From extensive clinical trials, the most commonly reported cardiovascular adverse events in patients treated with sildenafil were headache (<em>1</em>6%), flushing (<em>1</em>0%), and dizziness (2%). The incidences of hypotension, orthostatic hypotension, and syncope and the rate of discontinuation of treatment due to adverse events were <2% and were the same in patients taking sildenafil and those taking placebo. Retrospective analysis of the concomitant use of antihypertensive medications (beta blockers, alpha blockers, diuretics, <em>angiotensin</em>-converting enzyme inhibitors, and calcium antagonists) in patients taking sildenafil did not indicate an increase in the reports of adverse events or significant episodes of hypotension compared with patients treated with sildenafil alone. In clinical trials, the incidence of serious cardiovascular adverse events, including stroke and myocardial infarction, was the same for patients treated with sildenafil or placebo. Concurrent disease states, such as renal or hepatic impairment, or concomitant use of inhibitors of the cytochrome P450 isozyme CYP3A4 could increase systemic exposure to sildenafil. Since the US market launch in April <em>1</em>998, monitoring of spontaneous adverse event reports in association with sildenafil has demonstrated a pattern that is generally consistent with the experience observed during clinical development, with the exception of infrequent reports of priapism. In conclusion, extensive clinical testing has shown that overall treatment with sildenafil for up to <em>1</em> year is well tolerated and is associated with a low incidence of adverse events that result in discontinuation of treatment in <3% of patients.

The renin-<em>angiotensin</em> system (RAS) conceived as a coordinated hormonal cascade plays an important role in controlling multiple functions in many organs and is much more complex than previously thought. The RAS has continued to expand, with the identification of new components, functions and subsystems. <em>Angiotensin</em>-converting enzyme (ACE) and its novel homolog <em>angiotensin</em> converting enzyme 2 (ACE2) are two key enzymes involved in the synthesis of bioactive components of the RAS. The main active peptides of the RAS include <em>angiotensin</em> II (Ang II), Ang III, Ang IV, and <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] among which Ang II and Ang-(<em>1</em>-<em>7</em>) are much more important in health and disease. The axis formed by ACE2 represents an endogenous counter-regulatory pathway within the RAS, and its actions are opposite to those of the ACE axis. Conventionally the RAS has been considered to be important in the cardiovascular system, metabolism, cell growth and homeostasis. In recent years, a key role of ACE and ACE2 and their peptides has been recognized in the inflammatory process in conditions such as cardiac hypertrophy, pulmonary hypertension, glomerulonephritis, lung injury, sepsis, and acute pancreatitis. Investigations are ongoing to better understand the role of the RAS in inflammation. A comprehensive understanding of the RAS components in inflammation can provide new possibilities for therapeutic approaches against inflammatory diseases. In this review, we discuss our current understanding of the subject, based on recent findings, on the role of ACE and ACE2 in inflammation.

BACKGROUND

The <em>angiotensin</em>-converting enzyme 2 and <em>angiotensin</em>-(<em>1</em>-<em>7</em>) (Ang <em>1</em>-<em>7</em>)/MasR (Mas receptor) axis are emerging as a key pathway that can modulate the development of diabetic cardiomyopathy. We studied the effects of Ang <em>1</em>-<em>7</em> on diabetic cardiomyopathy in db/db diabetic mice to elucidate the therapeutic effects and mechanism of action.

RESULTS

Ang <em>1</em>-<em>7</em> was administered to 5-month-old male db/db mice for 28 days via implanted micro-osmotic pumps. Ang <em>1</em>-<em>7</em> treatment ameliorated myocardial hypertrophy and fibrosis with normalization of diastolic dysfunction assessed by pressure-volume loop analysis and echocardiography. The functional improvement by Ang <em>1</em>-<em>7</em> was accompanied by a reduction in myocardial lipid accumulation and systemic fat mass and inflammation and increased insulin-stimulated myocardial glucose oxidation. Increased myocardial protein kinase C levels and loss of phosphorylation of extracellular signal-regulated kinase <em>1</em>/2 were prevented by Ang <em>1</em>-<em>7</em>. Furthermore, Ang <em>1</em>-<em>7</em> treatment decreased cardiac triacylglycerol and ceramide levels in db/db mice, concomitantly with an increase in myocardial adipose triglyceride lipase expression. Changes in adipose triglyceride lipase expression correlated with increased SIRT<em>1</em> (silent mating type information regulation 2 homolog <em>1</em>) levels and deacetylation of FOXO<em>1</em> (forkhead box O<em>1</em>).

CONCLUSIONS

We identified a novel beneficial effect of Ang <em>1</em>-<em>7</em> on diabetic cardiomyopathy that involved a reduction in cardiac hypertrophy and lipotoxicity, adipose inflammation, and an upregulation of adipose triglyceride lipase. Ang <em>1</em>-<em>7</em> completely rescued the diastolic dysfunction in the db/db model. Ang <em>1</em>-<em>7</em> represents a promising therapy for diabetic cardiomyopathy associated with type 2 diabetes mellitus.

BACKGROUND

Based on previous data, we hypothesized that an increase of <em>angiotensin</em> II (Ang II)-via the Ang II type <em>1</em> (AT-<em>1</em>) receptor-in the rostral ventrolateral medulla (RVLM) and the paraventricular nucleus (PVN) of the hypothalamus could activate NAD(P)H oxidase that will produce superoxides resulting in increased sympathetic activity and hypertension.

METHODS

The mRNA expression of AT-<em>1</em> receptors, NAD(P)H oxidase subunits (p47phox and gp9<em>1</em>phox), and CuZnSOD were analyzed in the RVLM and PVN of male Wistar rats (Goldblatt hypertension model, 2K-<em>1</em>C). In addition, we administered Tempol <em>1</em> and 5 nmol into the RVLM, PVN, or systemically. The mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) were analyzed.

RESULTS

The AT-<em>1</em> mRNA expression and NAD(P)H oxidase subunits was greater in the RVLM and PVN in 2K-<em>1</em>C compared to the control group. Furthermore, the CuZnSOD expression was similar in both groups. Tempol <em>1</em> nmol into the RVLM reduced MAP (<em>1</em>5 +/- <em>1</em>%) and RSNA (<em>1</em><em>1</em> +/- 2%) only in 2K-<em>1</em>C rats. Tempol (5 nmol) in the same region decreased the MAP (<em>1</em>2 +/- 4%) and RSNA (20 +/- 7%), respectively, only in 2K-<em>1</em>C. In the PVN, Tempol 5 nmol resulted in a significant fall in the MAP (24 +/- <em>1</em>%) and in the RSNA (7.9 +/- 2%) only in the 2K-<em>1</em>C. Acute intravenous (IV) infusion of Tempol decreased MAP and RSNA in the 2K-<em>1</em>C but not in the control rats.

CONCLUSIONS

The data suggest that the hypertension and sympathoexcitation in 2K-<em>1</em>C rats were associated with an increase in oxidative stress within the RVLM, the PVN and systemically.

Transgenic mice that overexpress <em>angiotensin</em>ogen, the sole precursor of <em>angiotensins</em>, in their renal proximal tubular cells develop hypertension, albuminuria, and tubular apoptosis. These pathological changes are due to enhanced generation of reactive oxygen species in the proximal tubule cells. Here, we determined whether overexpression of catalase to decrease oxidant injury in the proximal tubular cells could reverse these abnormalities. Double-transgenic mice specifically overexpressing <em>angiotensin</em>ogen and catalase in their renal proximal tubular cells were created by cross-breeding the single transgenics. Non-transgenic littermates served as controls. Overexpression of catalase prevented hypertension, albuminuria, tubulointerstitial fibrosis, and tubular apoptosis in the <em>angiotensin</em>ogen transgenic mice. Furthermore, the double transgenics had lower reactive oxygen species generation and reduced pro-fibrotic and apoptotic gene expression in the renal proximal tubular cells. Renal <em>angiotensin</em> converting enzyme-2 expression and urinary <em>angiotensin</em> <em>1</em>-<em>7</em> levels were downregulated in the single but normal in the double-transgenic mice. Thus, we suggest that the intrarenal renin-<em>angiotensin</em> system and reactive oxygen species generation have an important role in the development of hypertension and renal injury.

Aldosterone is implicated in cardiac hypertrophy and fibrosis. We tested the role of the mineralocorticoid receptor in a model of <em>angiotensin</em> II-induced cardiac injury. We administered spironolactone (SPIRO; 20 mg. kg(-<em>1</em>). d(-<em>1</em>)), valsartan (VAL; <em>1</em>0 mg. kg(-<em>1</em>). d(-<em>1</em>)), or vehicle to rats double transgenic for the human renin and <em>angiotensin</em>ogen genes (dTGR). We investigated basic fibroblast growth factor (bFGF), platelet-derived growth factor, transforming growth factor-beta(<em>1</em>), and the transcription factors AP-<em>1</em> and nuclear factor (NF)-kappaB. We used immunohistochemistry, electrophoretic mobility shift assays, and TaqMan RT-PCR. Untreated dTGR developed hypertension, cardiac hypertrophy, vasculopathy, and fibrosis with a 50% mortality rates at <em>7</em> weeks. SPIRO and VAL prevented death and reversed cardiac hypertrophy, while only VAL normalized blood pressure. Both drugs prevented vasculopathy. bFGF was markedly upregulated in dTGR, whereas platelet-derived growth factor-B and transforming growth factor-beta(<em>1</em>) were little changed. VAL and SPIRO suppressed this upregulation. Both AP-<em>1</em> and NF-kappaB were activated in dTGR compared with controls. VAL and SPIRO reduced both transcription factors and reduced bFGF, collagen I, fibronectin, and laminin in the interstitium. These findings show that aldosterone promotes hypertrophy, cardiac remodeling, and fibrosis, independent of blood pressure. The effects involve AP-<em>1</em>, NF-kappaB, and bFGF. Mineralocorticoid receptor blockade downregulates these effectors and reduces <em>angiotensin</em> II-induced cardiac damage.

<em>1</em>. The effects of <em>angiotensin</em> on the contractility of the transmurally stimulated rabbit portal vein and coeliac artery have been studied in conjunction with its effects on the release and uptake of (+/-)-(3)H-noradrenaline.2. <em>Angiotensin</em> contracted both vein and artery; these responses were enhanced by veratrine and reduced by tetradotoxin. At low (non-contractile) concentrations of <em>angiotensin</em>, contractions elicited by electrical stimulation (0.5-4 Hz) had a quicker onset and reached a higher maximal tension than control responses. Higher concentrations of <em>angiotensin</em> increased the degree of potentiation. Contractions to noradrenaline were not enhanced by <em>angiotensin</em>.3. Pretreatment of the coeliac artery, portal vein and perfused rat heart with <em>angiotensin</em> did not reduce the subsequent uptake of labelled noradrenaline in the presence of <em>angiotensin</em>. Simultaneous treatment with <em>angiotensin</em> and (3)H-noradrenaline caused a small, apparent inhibition of uptake into the portal vein.4. When the portal vein was incubated with (3)H-noradrenaline there was a marked accumulation of label within the tissue; over 90% of the radioactivity retained in the tissue was identified as intact noradrenaline. When the vein, or artery, was superfused with amine-free Krebs there was a steady basal release of label; the greater proportion of this label was identified as deaminated metabolites. Electrcal stimulation evoked a frequency dependent release of (3)H above basal levels. The greater proportion of this increased efflux was due to the release of intact (3)H-noradrenaline, with smaller increases in the amount of O-methylated and deaminated metabolites.5. <em>Angiotensin</em> increased the efflux of labelled noradrenaline + normetanephrine, or of total (3)H, during transmural stimulation (0.5-4 Hz) in both the vein and artery, but did not increase the efflux of deaminated products during electrical stimulation. The output of labelled noradrenaline + normetanephrine was usually doubled in the presence of <em>angiotensin</em> (200-500 ng/ml) during electrical stimulation of the portal vein.6. Cocaine (4 mug/ml) potentiated responses to noradrenaline and transmural stimulation, and doubled the output of (3)H or labelled noradrenaline + nor-metanephrine during electrical stimulation. Cocaine did not alter the potentiating effects of <em>angiotensin</em>.<em>7</em>. <em>Angiotensin</em> elicited transitory increases in the basal efflux of (3)H from both the portal vein and coeliac artery. However, this did not account for the marked increase in efflux seen during electrical stimulation. Vasopressin did not potentiate responses to transmural stimulation or significantly influence the efflux of (3)H from the vein or artery.8. Noradrenaline and 5-hydroxytryptamine, but not acetylcholine, markedly increased the efflux of (3)H from the portal vein. This increase in efflux of label is believed to result from an exchange of exogenous noradrenaline and 5-hydroxytryptamine with labelled tissue stores of noradrenaline.9. It is suggested that <em>angiotensin</em> is able to facilitate the release of the sympathetic transmitter and that this is the basis for its action in potentiating the responses to sympathetic stimulation. It is also suggested that the nor-adrenaline releasing action of <em>angiotensin</em> (by excitation of sympathetic nerve endings) has led to confusion in previous investigations of the effects of <em>angiotensin</em> on noradrenaline uptake.

Pressure-induced tone (myogenic, MT) and flow (shear stress)-induced dilation (FD) are potent modulators of resistance artery tone. We tested the hypothesis that locally produced <em>angiotensin</em> II interacts with MT and FD. Rat mesenteric resistance arteries were perfused in situ. Arterial diameter was measured by intravital microscopy after a bifurcation on 2 distal arterial branches equivalent in size (<em>1</em>50 microm, n=<em>7</em> per group). One was ligated distally and thus submitted to pressure only (MT, no FD). The second branch was submitted to flow and pressure (MT and FD). The difference in diameter between the 2 vessels was considered to be FD. Flow-diameter-pressure relationship was established in the absence and then in the presence of <em>1</em> of the following agents. In the nonligated segment (MT+FD), <em>angiotensin</em> II type <em>1</em> (AT(<em>1</em>)) receptor blockade (losartan) had no significant effect, whereas <em>angiotensin</em> II type 2 (AT(2)) receptor blockade (PD<em>1</em>233<em>1</em>9) or saralasin (AT(<em>1</em>)+AT(2) blocker) decreased the diameter significantly, by 9+/-<em>1</em> and <em>1</em>0+/-0.8 microm, respectively. <em>Angiotensin</em> II in the presence of losartan increased the diameter by <em>1</em>8+/-0.6 microm (inhibited by PD<em>1</em>233<em>1</em>9). PD<em>1</em>233<em>1</em>9 or saralasin had no effect after NO synthesis blockade or after endothelial disruption. In the arterial segment ligated distally (MT only), AT(<em>1</em>) or AT(2) receptor blockade had no significant effect. AT(2)-dependent dilation represented 20% to 39% of FD (shear stress from 22 to 3<em>7</em> dyn/cm(2)), and AT(2)-receptor mRNA was found in mesenteric resistance arteries. Thus, resistance arterial tone was modulated in situ by locally produced <em>angiotensin</em> II, which might participate in flow-induced dilation through endothelial AT(2) receptor activation of NO release.

BACKGROUND

The Global Registry of Acute Coronary Events (GRACE)-a prospective, multinational study of patients hospitalized with acute coronary syndromes (ACSs)-was designed to improve the quality of care for patients with an ACS. Expanded GRACE aims to test the feasibility of a simplified data collection tool and provision of quarterly feedback to index individual hospital management practices to an international reference cohort.

METHODS

We describe the objectives; study design; study and data management; and the characteristics, management, and hospital outcomes of patients>> or =<em>1</em>8 years old enrolled with a presumptive diagnosis of ACS.

RESULTS

From 200<em>1</em> to 2007, 3<em>1</em>,982 patients were enrolled at <em>1</em>84 hospitals in 25 countries; 30% were diagnosed with ST-segment elevation myocardial infarction, 3<em>1</em>% with non-ST-segment myocardial infarction, 26% with unstable angina, and <em>1</em>2% with another cardiac/noncardiac final diagnosis. The median age was 65 (interquartile range 55-75) years; 24% were >75 years old, and 33% were women. In general, increases were observed over time across the spectrum of ACS (<em>1</em>) in the use in the first 24 hours and at discharge of aspirin, clopidogrel, beta-blockers, and <em>angiotensin</em>-converting enzyme inhibitors/receptor blockers; (2) in the use at discharge of statins; (3) in the early use of glycoprotein IIb/IIIa inhibitors and low-molecular-weight heparin; and (4) in the use of cardiac catheterization and percutaneous coronary intervention. An increase in the use of primary percutaneous coronary intervention and a similar decrease in the use of fibrinolysis in ST-segment elevation myocardial infarction were also seen.

CONCLUSIONS

Over the course of 7 years, general increases in the use of evidence-based therapies for ACS patients were observed in the expanded GRACE.

A procedure for the isolation and long-term in vitro cultivation of endothelial cells from rat cerebral cortical microvasculature is described. Migrating cells emerged from collagenase-treated microvessel fragments as early as <em>1</em> to 2 days in culture. Migration continued and marked proliferation began 5 to <em>7</em> days after isolation and continued up to <em>1</em>2 to <em>1</em>4 days. Cell colonies developed and consisted of endothelial cells as determined by phase contrast microscopy and cell culture behavior. Proliferation of the endothelial cells was significantly enhanced (3- to 4-fold) with the addition of endothelial cell growth supplement at a concentration of <em>1</em>50 microgram. per ml. Cultures with endothelial cell growth supplement (ECGS) retained their characteristic endothelial morphology for 6 to 8 weeks, after which they exhibited a gradual deterioration and loss of their phenotypic appearance. The endothelial origin of these cells was demonstrated by positive immunofluorescent staining for factor VIII antigen and <em>angiotensin</em>-converting enzyme and lack of binding of rat smooth muscle myosin antibody. Ultrastructural examination of confluent endothelial cell cultures revealed intercellular junctional complexes consisting of gap junctions, punctate fusions, and tight junctions. Since long-term endothelial cell cultures derived from the cerebral microvasculature retain characteristic endothelial cell markers and in vivo markers and in vivo features of brain capillary endothelium, they can serve as a useful model system to characterize microvascular endothelium in a variety of disease states.

In this study we evaluated the cardiac effects of a pharmaceutical formulation developed by including <em>angiotensin</em> (Ang)-(<em>1</em>-<em>7</em>) in hydroxypropyl β-cyclodextrin (HPβCD), in normal, infarcted, and isoproterenol-treated rats. Myocardial infarction was produced by left coronary artery occlusion. Isoproterenol (2 mg/kg, IP) was administered daily for <em>7</em> days. Oral administration of HPβCD/Ang-(<em>1</em>-<em>7</em>) started immediately before infarction or associated with the first dose of isoproterenol. After <em>7</em> days of treatment, the rats were euthanized, and the Langendorff technique was used to analyze cardiac function. In addition, heart function was chronically (<em>1</em>5, 30, 50 days) analyzed by echocardiography. Cardiac sections were stained with hematoxylin/eosin and Masson trichrome to evaluate cardiac hypertrophy and damage, respectively. Pharmacokinetic studies showed that oral HPβCD/Ang-(<em>1</em>-<em>7</em>) administration significantly increased Ang-(<em>1</em>-<em>7</em>) on plasma whereas with the free peptide it was without effect. Oral administration of HPβCD/Ang-(<em>1</em>-<em>7</em>) (30 μg/kg) significantly reduced the deleterious effects induced by myocardial infarction on systolic and diastolic tension, ±dT/dt, perfusion pressure, and heart rate. Strikingly, a 50% reduction of the infarcted area was observed in HPβCD/Ang-(<em>1</em>-<em>7</em>)-treated rats. Furthermore, HPβCD/Ang-(<em>1</em>-<em>7</em>) attenuated the heart function impairment and cardiac remodeling induced by isoproterenol. In infarcted rats chronically treated with HPβCD/Ang-(<em>1</em>-<em>7</em>), the reduction of ejection fraction and fractional shorting and the increase in systolic and diastolic left ventricular volumes observed in infarcted rats were attenuated. Altogether, these findings further confirm the cardioprotective effects of Ang-(<em>1</em>-<em>7</em>). More importantly, our data indicate that the HPβCD/Ang-(<em>1</em>-<em>7</em>) is a feasible formulation for oral administration of Ang-(<em>1</em>-<em>7</em>), which can be used as a cardioprotective drug.

Collecting duct (CD) renin is stimulated by <em>angiotensin</em> (Ang) II, providing a pathway for Ang I generation and further conversion to Ang II. Ang II stimulates the epithelial sodium channel via the Ang II type <em>1</em> receptor and increases mineralocorticoid receptor activity attributed to increased aldosterone release. Our objective was to determine whether CD renin augmentation is mediated directly by Ang II type <em>1</em> receptor or via the epithelial sodium channel and mineralocorticoid receptor. In vivo studies examined the effects of epithelial sodium channel blockade (amiloride; 5 mg/kg per day) on CD renin expression and urinary renin content in Ang II-infused rats (80 ng/min, 2 weeks). Ang II infusion increased systolic blood pressure, medullary renin mRNA, urinary renin content, and intrarenal Ang II levels. Amiloride cotreatment did not alter these responses despite a reduction in the rate of progression of systolic blood pressure. In primary cultures of inner medullary CD cells, renin mRNA and (pro)renin protein levels increased with Ang II (<em>1</em>00 nmol/L), and candesartan (Ang II type <em>1</em> receptor antagonist) prevented this effect. Aldosterone (<em>1</em>0(-<em>1</em>0) to <em>1</em>0(-<em>7</em>) mol/L) with or without amiloride did not modify the upregulation of renin mRNA in Ang II-treated cells. However, inhibition of protein kinase C with calphostin C prevented the Ang II-mediated increases in renin mRNA and (pro)renin protein levels. Furthermore, protein kinase C activation with phorbol <em>1</em>2-myristate <em>1</em>3-acetate increased renin expression to the same extent as Ang II. These data indicate that an Ang II type <em>1</em> receptor-mediated increase in CD renin is induced directly by Ang II via the protein kinase C pathway and that this regulation is independent of mineralocorticoid receptor activation or epithelial sodium channel activity.

BACKGROUND

Heme oxygenase-<em>1</em> (HO-<em>1</em>) is a stress-response enzyme implicated in cardioprotection. To explore whether HO-<em>1</em> has a role in cardiac remodeling response, the effect of its overexpression on <em>angiotensin</em> II (Ang II)-induced cardiac hypertrophy was examined.

RESULTS

HO-<em>1</em> was induced in cultured rat neonatal cardiomyocytes by treatment with cobalt protoporphyrin IX (CoPPIX) or a recombinant adenovirus carrying the human HO-<em>1</em> gene. Ang II-induced myocyte hypertrophy assessed by increments in cell size, [3H]leucine uptake, and protein content was suppressed by HO-<em>1</em> overexpression. Cotreatment of cells with tin protoporphyrin IX, a HO inhibitor, significantly reversed the suppressive effect of HO-<em>1</em>. Bilirubin, one of the byproducts of heme degradation by HO-<em>1</em>, mediated the suppressive effect through the inhibition of Ang II-induced production of reactive oxygen species, as detected by a 2',7'-dichlorofluorescein probe. The antihypertrophic effect of HO-<em>1</em> was also demonstrated in rats receiving chronic Ang II infusions. Cotreatment of animals with CoPPIX significantly attenuated Ang II-induced left ventricular hypertrophy and hyperdynamic contractions, whereas concomitant treatment with tin protoporphyrin IX abolished CoPPIX-mediated cardioprotection in vivo.

CONCLUSIONS

HO-<em>1</em> attenuates Ang II-induced cardiac hypertrophy both in vitro and in vivo, and bilirubin mediates, at least in part, the antihypertrophic effect of HO-<em>1</em> via inhibition of reactive oxygen species production after Ang II stimulation.

OBJECTIVE

In in vitro studies, angiotensin (Ang) II has been demonstrated to promote podocyte apoptosis. The present study evaluates the effects of Ang II infusion in rats on podocyte nephrin expression and apoptosis and the molecular mechanisms involved in Ang II-induced proteinuria and mesangial expansion.

METHODS

Sprague-Dawley rats were randomly assigned to receive either normal saline or Ang II (400 ng x kg(-1) x min(-1)) by means of a mini-osmotic pump for variable time periods. Systolic blood pressure and urinary protein and albumin excretion rate measurements were carried out on days 7, 14, 21, and 28. The animals were sacrificed on days 14 and 28 and evaluated for serum creatinine, renal pathological changes, podocyte apoptosis, renal nephrin mRNA, and protein expression.

RESULTS

The Ang II-infused rats developed hypertension and proteinuria. On day 14, the Ang II-infused rats showed narrowing of the slit diaphragm, an increase in podocyte nephrin mRNA and protein expression, and alterations in its distribution along the foot processes. On day 28, the Ang II-infused rats demonstrated the presence of apoptotic podocytes and decreased nephrin mRNA and protein expression. There was a negative correlation between nephrin expression and the numbers of apoptotic podocytes (r = -0.63, p < 0.05).

CONCLUSIONS

These results suggest that changes in nephrin expression may play a role in the pathogenesis of Ang II-induced podocyte apoptosis.

We determined the levels of <em>angiotensin</em> I (ANG I), <em>angiotensin</em> II (ANG II), and the heptapeptide <em>angiotensin</em>(<em>1</em>-<em>7</em>) [ANG(<em>1</em>-<em>7</em>)] in the blood and brain of female Hannover Sprague-Dawley (SD) and transgenic hypertensive rats [mRen-2]2<em>7</em> by radioimmunoassay and high performance liquid chromatography. Hypertension was accompanied by higher plasma concentrations of ANG II, no statistical changes in ANG(<em>1</em>-<em>7</em>), and no differences in plasma ANG I levels. In the hypothalamus of transgenic rats, concentrations of ANG II and ANG(<em>1</em>-<em>7</em>) averaged 82<em>7</em>% and <em>1</em>68% above values in SD rats (p < 0.005) whereas both ANG I and ANG II increased in the medulla oblongata. The data showed that the established phase of hypertension in rats harboring the mouse Ren-2 gene is associated with overexpression of the renin-<em>angiotensin</em> system in brain regions participating in the endocrine regulation of blood pressure.

BACKGROUND

Arterial stiffness increases with age and contributes to the pathogenesis of systolic hypertension and cardiovascular disease in the elderly. Knowledge about the pathophysiological processes that determine arterial stiffness may help guide therapeutic approaches.

RESULTS

We related <em>7</em> circulating biomarkers representing distinct biological pathways (C-reactive protein, aldosterone-to-renin ratio, N-terminal proatrial natriuretic peptide and B-type natriuretic peptide, plasminogen activator inhibitor-<em>1</em>, fibrinogen, and homocysteine) to 5 vascular function measures (central pulse pressure, carotid-femoral pulse-wave velocity, mean arterial pressure, forward pressure wave amplitude [all measures of conduit artery stiffness], and augmented pressure, an indicator of wave reflection) in 2000 Framingham Offspring Study participants (mean age, 6<em>1</em> years; 55% women). Tonometry measures were obtained on average 3 years after the biomarkers were measured. In multivariable linear regression models adjusting for covariates, the biomarker panel was significantly associated with all 5 vascular measures (P<0.003 for all). On backward elimination, the aldosterone-to-renin ratio was positively associated with each stiffness measure (P< or =0.002 for all). In addition, C-reactive protein was positively related to augmented pressure (P=0.0003), whereas plasminogen activator inhibitor-<em>1</em> was positively associated with mean arterial pressure (P=0.003), central pulse pressure (P=0.00<em>1</em>), and forward pressure wave (P=0.0<em>1</em>).

CONCLUSIONS

Our cross-sectional data on a community-based sample suggest a distinctive pattern of positive associations of biomarkers of renin-angiotensin-aldosterone system activation with pan-arterial vascular stiffness, plasminogen activator inhibitor-<em>1</em> with central vascular stiffness indices, and C-reactive protein with wave reflection. These observations support the notion of differential influences of biological pathways on vascular stiffness measures.

ANG-(<em>1</em>-<em>7</em>) is associated with vasodilation and nitric oxide synthase stimulation. However, the role of ANG-(<em>1</em>-<em>7</em>) in type 2 diabetes mellitus is unknown. In this study, we examined the hypothesis that ANG-(<em>1</em>-<em>7</em>) attenuates ANG II-induced reactive oxygen species stress (ROS)-mediated injury in type 2 diabetic nephropathy of KK-A(y)/Ta mice. KK-A(y)/Ta mice were divided into four groups: <em>1</em>) a control group; 2) ANG II infusion group; 3) ANG II+ANG-(<em>1</em>-<em>7</em>) coinfusion group; and 4) ANG II+ANG-(<em>1</em>-<em>7</em>)+d-Ala(<em>7</em>)-ANG-(<em>1</em>-<em>7</em>) (A<em>7</em><em>7</em>9) coinfusion group. In addition, primary mesangial cells were cultured and then stimulated with 25 mM glucose with or without ANG II, ANG-(<em>1</em>-<em>7</em>), and A<em>7</em><em>7</em>9. The ANG II+ANG-(<em>1</em>-<em>7</em>) coinfusion group showed a lower urinary albumin/creatinine ratio increase than the ANG II group. ANG-(<em>1</em>-<em>7</em>) attenuated ANG II-mediated NAD(P)H oxidase activation and ROS production in diabetic glomeruli and mesangial cells. ANG II-induced NF-κB and MAPK signaling activation was also attenuated by ANG-(<em>1</em>-<em>7</em>) in the mesangial cells. These findings were related to improved mesangial expansion and to fibronectin and transforming growth factor-β<em>1</em> production in response to ANG II and suggest that ANG-(<em>1</em>-<em>7</em>) may attenuate ANG II-stimulated ROS-mediated injury in type 2 diabetic nephropathy. The ACE2-ANG-(<em>1</em>-<em>7</em>)-Mas receptor axis should be investigated as a novel target for treatment of type 2 diabetic nephropathy.

BACKGROUND

The recognized role of angiotensin II (Ang II) in the pathogenesis of the progression of renal disease cannot be solely attributed to Ang II's hemodynamic effects. Indeed, growth stimulating signals driven by Ang II promote mesangial cell (MC) hypertrophy and extracellular matrix production, prominent features of progressive glomerular injury. Superoxide anion (O2-) avidly interacts with nitric oxide, an endogenous vasodilator that inhibits growth factor stimulated MC growth and matrix production. In addition, O2- acting as an intracellular signal is linked to growth related responses such as activation of mitogen activated protein (MAP) kinases. The studies reported herein were designed to investigate: (a) whether Ang II induces MC O2-production and (b) if increased O2- production elicits growth responses in MC.

METHODS

MC were exposed to Ang II for 24 or 48 hours. In some experiments, in addition to Ang II, MC were exposed to: diphenylenieodonium (DPI), an inhibitor of the flavin containing NADH/NADPH oxidase; losartan (LOS), an Ang II type 1 (AT1) receptor blocker; PD 98059, a MAP kinases inhibitor; the protein kinase C inhibitors Calphostin C or H-7; and the tyrosine kinase inhibitors, herbymycin A or genistein.

RESULTS

Ang II (10(-5) M to 10(-8) M) dose dependently increased MC O2- production up to 125% above control (ED 50 5 x 10(-7) M). LOS as well as DPI, and the PKC inhibitors blocked Ang II stimulated MC O2- production. Ang II dose dependently increased MC 3H-leucine incorporation, and MC protein content, two markers of MC hypertrophy, as well as 3H-thymidine incorporation, a marker of MC hyperplasia. PD98059, a specific inhibitor of MAP kinases prevented Ang II induced MC hypertrophy. Moreover, LOS, DPI, and the PKC inhibitors each independently inhibited MC 3H-leucine incorporation, thereby establishing the specificity of Ang II induced O2- in driving MC hypertrophy.

CONCLUSIONS

The current studies demonstrate a previously unrecognized link between Ang II and MC O2- production that may participate in the pathophysiology of progressive renal disease by concomitantly affecting the hemodynamics of the glomerular microcirculation as well as growth related responses of MC to injury.

Vitamin D deficiency has been associated with cardiovascular dysfunction. We evaluated the role of the vitamin D receptor (VDR) in vascular endothelial function, a marker of cardiovascular health, at baseline and in the presence of <em>angiotensin</em> II, using an endothelial-specific knockout of the murine VDR gene. In the absence of endothelial VDR, acetylcholine-induced aortic relaxation was significantly impaired (maximal relaxation, endothelial-specific VDR knockout=58% versus control=<em>7</em>3%; P<0.05). This was accompanied by a reduction in endothelial NO synthase expression and phospho-vasodilator-stimulated phosphoprotein levels in aortae from the endothelial-specific VDR knockout versus control mice. Although blood pressure levels at baseline were comparable at <em>1</em>2 and 24 weeks of age, the endothelial VDR knockout mice demonstrated increased sensitivity to the hypertensive effects of <em>angiotensin</em> II compared with control mice (after <em>1</em>-week infusion: knockout=<em>1</em>55±<em>1</em>5 mm Hg versus control=<em>1</em>33±<em>7</em> mm Hg; P<0.0<em>1</em>; after 2-week infusion: knockout=<em>1</em>64±9 mm Hg versus control=<em>1</em>52±<em>1</em>3 mm Hg; P<0.05). By the end of 2 weeks, <em>angiotensin</em> II infusion-induced, hypertrophy-sensitive myocardial gene expression was higher in endothelial-specific VDR knockout mice (fold change compared with saline-infused control mice, type-A natriuretic peptide: knockout mice=3.<em>1</em>2 versus control=<em>1</em>.<em>7</em>; P<0.05; type-B natriuretic peptide: knockout mice=4.<em>7</em>2 versus control=2.68; P<0.05). These results suggest that endothelial VDR plays an important role in endothelial cell function and blood pressure control and imply a potential role for VDR agonists in the management of cardiovascular disease associated with endothelial dysfunction.

The renin-<em>angiotensin</em> system (RAS) plays a major role in liver fibrosis. Recently, a homolog of <em>angiotensin</em>-converting-enzyme <em>1</em> (ACE<em>1</em>), termed ACE2, has been identified that appears to be a negative regulator of the RAS by degrading Ang II to Ang(<em>1</em>-<em>7</em>). The aim of this study was to characterize the long-term effects of gene deletion of ACE2 in the liver, to define the role of ACE2 in acute and chronic liver disease, and to characterize the role of Ang(<em>1</em>-<em>7</em>) in hepatic stellate cell (HSC) activation. Ace2 knockout (KO) mice and wild-type (wt) littermates underwent different models of acute and chronic liver injury. Liver pathology was analyzed by histology, immunohistochemistry, alpha smooth muscle actin (alpha-SMA) immunoblotting, and quantitative polymerase chain reaction (qPCR). Murine HSCs were isolated by collagenase-pronase-perfusion, and density gradient centrifugation. One-year-old ace2 KO mice spontaneously developed an inflammatory cell infiltration and mild hepatic fibrosis that was prevented by treatment with irbesartan. Ace2 KO mice showed increased liver fibrosis following bile duct ligation for 2<em>1</em> days or chronic carbon tetrachloride (CCl(4)) treatment. In contrast, ace2 KO mice subjected to acute liver injury models did not differ from wt littermates. Treatment with recombinant ACE2 attenuated experimental fibrosis in the course of cholestatic and toxic liver injury. HSCs express the Ang(<em>1</em>-<em>7</em>) receptor Mas and Ang(<em>1</em>-<em>7</em>) inhibited Ang II-induced phosphorylation of extracellular signal-regulated kinase (ERK)-<em>1</em>/2 in cultured HSCs.

CONCLUSIONS

ACE2 is a key negative regulator of the RAS and functions to limit fibrosis through the degradation of Ang II and the formation of Ang(<em>1</em>-<em>7</em>). Whereas loss of ACE2 activity worsens liver fibrosis in chronic liver injury models, administration of recombinant ACE2 shows therapeutic potential.

A large body of evidence demonstrates that <em>angiotensin</em> II and <em>angiotensin</em> receptors are required for the pathogenesis of experimental lung fibrosis. <em>Angiotensin</em> has a number of profibrotic effects on lung parenchymal cells that include the induction of growth factors for mesenchymal cells, extracellular matrix molecules, cytokines and increased motility of lung fibroblasts. <em>Angiotensin</em> is also proapoptotic for lung epithelial cells, and is synthesized by a local system (i.e., entirely within the lung tissue) after lung injury by a variety of agents of both xenobiotic and endogenous origins. Recent evidence shows that the counterregulatory molecule <em>angiotensin</em> <em>1</em>-<em>7</em>, the product of the enzyme ACE-2, inhibits epithelial cell apoptosis and thus acts as an antifibrotic epithelial survival factor. This manuscript reviews the evidence supporting a role for <em>angiotensin</em> in lung fibrogenesis and discusses the signalling mechanisms underlying its action on lung parenchymal cells important in the pathogenesis of pulmonary fibrosis.

BACKGROUND

Peroxisome proliferator-activated receptor (PPAR)alpha is highly expressed in the heart. PPAR alpha may play a role in cardiac hypertrophy, but effects on cardiac function, inflammation, and fibrosis are unknown. We tested the hypothesis that the PPAR alpha activator fenofibrate prevents myocardial inflammation and fibrosis in angiotensin (Ang) II-infused rats.

RESULTS

Sprague Dawley rats received Ang II (120 ng/kg/min subcutaneously), fenofibrate (100 mg/kg/d p.o.), or Ang II + fenofibrate. After 7 d, systolic blood pressure (mmHg) was elevated (P < 0.01) in Ang II-infused rats (173 +/- 4) vs. controls (115 +/- 2) and reduced by fenofibrate (137 +/- 5). Electrophoretic mobility shift assay demonstrated that Ang II upregulated cardiac nuclear factor kappa B activity by 50%. Ang II significantly increased cardiac expression of vascular-cell adhesion molecule-1, platelet endothelial cell adhesion molecule, and intercellular adhesion molecule-1. Increases in expression of these inflammatory mediators were normalized by fenofibrate. Ang II-induced expression of transforming growth factor-beta 1, collagen deposition, and macrophage infiltration were partially prevented by fenofibrate.

CONCLUSIONS

The PPAR alpha activator fenofibrate prevented development of hypertension, and improved myocardial inflammation and collagen deposition in Ang II-infused rats. The hypolipidemic drug fenofibrate may be useful in prevention and treatment of myocardial disease associated with hypertension and hyperlipidemia.

Patients with essential hypertension are characterized by impaired basal and agonist-evoked nitric oxide release and increased endogenous endothelin (ET)-<em>1</em>-induced vasoconstriction. To assess whether candesartan, an <em>angiotensin</em> II type <em>1</em> receptor blocker, can improve endothelial function, we studied the changes in forearm blood flow (FBF) induced in <em>1</em>5 hypertensive patients and in <em>1</em>5 control subjects by the intrabrachial infusion of N(G)-monomethyl-L-arginine (L-NMMA), norepinephrine, the ET A/B receptor antagonist TAK 044, sodium nitroprusside, and acetylcholine. In hypertensive patients, the FBF study was repeated 2 and <em>1</em>2 months after the start of treatment with candesartan cilexetil (8 to <em>1</em>6 mg daily). Compared with controls (maximal FBF decrease, -46+/-<em>1</em><em>1</em>%), hypertensive patients showed a reduced (P<0.00<em>1</em>) vasoconstrictor response to L-NMMA (maximal FBF decrease, -28+/-<em>7</em>%); the response to norepinephrine was only slightly impaired, and the response to sodium nitroprusside was similar to that of controls. Finally, TAK-044 caused greater vasodilation in hypertensive patients (maximal FBF increase, <em>7</em><em>7</em>+/-9%) than in controls (maximal FBF increase, <em>1</em><em>7</em>+/-<em>1</em>0%). In hypertensive patients, candesartan cilexetil significantly enhanced vasoconstriction to L-NMMA after 2 and <em>1</em>2 months (maximal FBF decrease, 3<em>7</em>+/-2% [P<0.05] and 42+/-2% [P<0.00<em>1</em>], respectively). The responses to norepinephrine, acetylcholine, and sodium nitroprusside were not modified after 2 months. After <em>1</em>2 months, the responses to acetylcholine and sodium nitroprusside were significantly (P<0.05) enhanced at the highest rates. Vasodilation to TAK-044 was abolished after treatment with candesartan cilexetil; this effect is associated with a reduced plasma ET-<em>1</em> concentration. This study demonstrated that the <em>angiotensin</em> II receptor blocker candesartan improves tonic nitric oxide release and reduces vasoconstriction to endogenous ET-<em>1</em> in the forearm of hypertensive patients.

OBJECTIVE

Angiotensin-converting enzyme 2 (ACE2) has recently emerged as a key regulator of the renin-angiotensin system in both health and disease.

RESULTS

ACE2 deficiency is associated with elevated tissue and circulating levels of <em>angiotensin</em> II and reduced levels of <em>angiotensin</em> <em>1</em>-<em>7</em>. Phenotypically, this results in a modest elevation in systolic blood pressure and left ventricular hypertrophy. In atherosclerosis-prone apolipoprotein E knockout mice, ACE2 deficiency results in augmented vascular inflammation and an inflammatory response that contributes to increased atherosclerotic plaque formation. In the kidney, ACE2 deficiency is associated with progressive glomerulosclerosis. Interventions such as ACE2 replenishment or augmentation of its actions have proven successful in reducing hypertension, plaque accumulation, and renal and cardiac damage in a range of different models. Although promising, the balance of the renin-<em>angiotensin</em> system remains complicated, with some evidence that overexpression of ACE2 may have adverse cardiac effects, and ACE2 and its metabolic products may promote epithelial-to-mesenchymal transition.

CONCLUSIONS

Repletion of ACE2's activities offers a new strategy to complement current clinical interventions in treating hypertension, renal and cardiovascular disease. In particular conditions where ACE inhibition and angiotensin receptor blockade are partially effective, the adjunctive actions of ACE2 may not only reduce clinical escape but also augment the efficacy of interventions.