Severe acute respiratory syndrome coronavirus (SARS-CoV) can produce gastrointestinal symptoms. The intestinal tract is the only extrapulmonary site where viable viruses have been detected. This study examined seven established human intestinal cell lines, DLD-<em>1</em>, HCT-<em>1</em><em>1</em>6, HT-29, LoVo, LS-<em>1</em>80, SW-480 and SW-620, for their permissiveness to SARS-CoV infection. The results showed that only LoVo cells were permissive to SARS-CoV infection as evident by positive findings from indirect immunofluorescence staining for intracellular viral antigens, in situ hybridization for intracellular viral RNA, and electron microscopy for intracellular viral particles. In contrast to Vero cells, SARS-CoV did not produce cytopathic effects on LoVo cells. However, LoVo cells were found to be highly permissive for productive infection with a high viral titre (>3 x <em>1</em>0(<em>7</em>) viral copies/ml) produced in culture supernatant following a few days of incubation. SARS-CoV established a stable persistent chronic infection that could be maintained after multiple passages. Being a cell line of human origin, LoVo cells could be a useful in vitro model for studying the biology and persistent infection of SARS-CoV. Our results on the expression of <em>angiotensin</em>-converting enzyme 2 (ACE2), a recently identified cellular receptor for SARS-CoV, in these cell lines indicated that it might not be the sole determinant for cells to be susceptible to SARS-CoV infection.

The renin-<em>angiotensin</em> system (RAS) is a key component of cardiovascular physiology and homeostasis due to its influence on the regulation of electrolyte balance, blood pressure, vascular tone and cardiovascular remodeling. Deregulation of this system contributes significantly to the pathophysiology of cardiovascular and renal diseases. Numerous studies have generated new perspectives about a noncanonical and protective RAS pathway that counteracts the proliferative and hypertensive effects of the classical <em>angiotensin</em>-converting enzyme (ACE)/<em>angiotensin</em> (Ang) II/<em>angiotensin</em> type <em>1</em> receptor (AT<em>1</em>R) axis. The key components of this pathway are ACE2 and its products, Ang-(<em>1</em>-<em>7</em>) and Ang-(<em>1</em>-9). These two vasoactive peptides act through the Mas receptor (MasR) and AT2R, respectively. The ACE2/Ang-(<em>1</em>-<em>7</em>)/MasR and ACE2/Ang-(<em>1</em>-9)/AT2R axes have opposite effects to those of the ACE/Ang II/AT<em>1</em>R axis, such as decreased proliferation and cardiovascular remodeling, increased production of nitric oxide and vasodilation. A novel peptide from the noncanonical pathway, alamandine, was recently identified in rats, mice and humans. This heptapeptide is generated by catalytic action of ACE2 on Ang A or through a decarboxylation reaction on Ang-(<em>1</em>-<em>7</em>). Alamandine produces the same effects as Ang-(<em>1</em>-<em>7</em>), such as vasodilation and prevention of fibrosis, by interacting with Mas-related GPCR, member D (MrgD). In this article, we review the key roles of ACE2 and the vasoactive peptides Ang-(<em>1</em>-<em>7</em>), Ang-(<em>1</em>-9) and alamandine as counter-regulators of the ACE-Ang II axis as well as the biological properties that allow them to regulate blood pressure and cardiovascular and renal remodeling.

BACKGROUND

Diabetic nephropathy is characterized by glomerular and tubular hypertrophy, and angiotensin II receptor blockers (ARBs) are known to prevent renal hypertrophy in diabetic patients.

METHODS

To determine the effect of ARB on podocyte p27(Kip1) mRNA and protein expression, podocytes were exposed to 5.6 mmol/L normal glucose or 25 mmol/L high glucose with or without ARB, 10(-7) mol/L L-158,809. For animal studies, streptozotocin-induced diabetic rats were left untreated or were treated with 1 mg/kg/day L-158,809 for 3 months (diabetes mellitus + ARB). Competitive reverse transcription-polymerase chain reaction (RT-PCR), Western blot, immunohistochemistry, and morphometric analyses were performed.

RESULTS

p27(Kip1) mRNA and protein expression in podocytes exposed to high glucose and in 3-month diabetic glomeruli were significantly increased (P < 0.01). High glucose significantly increased angiotensin II levels both in cell lysates and in media compared with normal glucose (P < 0.05) and exogenous angiotensin II also increased p27(Kip1) mRNA and protein expression in podocytes. L-158,809 treatment in podocytes inhibited the increase in p27(Kip1) mRNA expression by 84%, and protein expression by 89% (P < 0.05). p27(Kip1) mRNA and protein expression in diabetic + ARB glomeruli were also significantly reduced by 78% and 85%, respectively, compared with diabetic glomeruli (P < 0.01). ARB treatment also significantly ameliorated increased glomerular p27(Kip1) expression in diabetes mellitus as assessed by immunohistochemistry (P < 0.01). The increase in glomerular volume in diabetes mellitus was also inhibited by 81% with ARB treatment (P < 0.05).

CONCLUSIONS

p27(Kip1) mRNA and protein expression were increased in diabetic glomeruli as well as in high glucose-stimulated podocytes, and this increment in p27(Kip1) expression was ameliorated by ARB treatment. These findings indicate that ARB treatment has an additional effect on preventing renal hypertrophy in diabetes mellitus.

BACKGROUND

Albuminuria with normal serum creatinine occurs frequently in patients with sickle cell disease (SCD), but the rate of progression to more advanced chronic renal disease is unknown. The purpose of this study was to investigate the rate of progression of children and young adults with SCD and albuminuria over time.

METHODS

Urine albumin/creatinine (A/C) ratios and serum creatinine were obtained serially. Serum cystatin C levels were determined in a subgroup of 20 patients.

RESULTS

Of 38 patients with SCD who had albuminuria (30 with microalbuminuria and 8 with proteinuria), <em>1</em>0.5% had progressive disease during follow-up of 20 +/- <em>1</em>2 months. Progressive disease was observed in 2 of 30 patients with MA because MA worsened to either intermittent proteinuria (<em>1</em> patient), or persistent proteinuria after <em>7</em> months follow-up (<em>1</em> patient). Two of eight patients with proteinuria worsened to nephrotic-range after 8 and <em>1</em><em>7</em> months with elevations of serum creatinine. All eight patients with proteinuria were treated with <em>angiotensin</em> blockade and/or hydroxyurea. Of those, six patients responded to treatment with decreased albuminuria and no changes in serum creatinine. Serum cystatin C level trended to increase before serum creatinine in patients with proteinuria.

CONCLUSIONS

Patients with rapid progression to nephrotic-range proteinuria showed decreased kidney function. Therefore, patients with albuminuria should be monitored closely for progression, and therapy with hydroxyurea and/or angiotensin blockade should be considered for patients who develop proteinuria. Serum cystatin C appears more sensitive than serum creatinine to detect early decrease in kidney function.

BACKGROUND

The aim of this study was to confirm the nature and number of genes contributing to stroke risk and qualify the genetic risk of each susceptibility gene in the Han Chinese population.

METHODS

After collecting all case-control studies related to DNA polymorphism of any candidate gene for ischemic stroke in Han Chinese, strict selection criteria and exclusion criteria were determined and different effect models were used according to the difference in heterogeneity. Meta-analyses were carried out by Revman 4.0 software and the publication bias was further evaluated through calculation of fail-safe numbers in the included gene polymorphisms.

RESULTS

Seventy-six studies were included in the meta-analyses which were all published in mainland China and referred to 6 candidate genes and <em>7</em> polymorphisms. Among the gene polymorphisms tested in the study, association of gene polymorphisms with increasing risk of ischemic stroke was confirmed in 6 polymorphisms including <em>angiotensin</em>-converting enzyme insertion/deletion (ACE I/D; OR = <em>1</em>.8<em>7</em>, 95% CI = <em>1</em>.45-2.42), methylenetetrahydrofolate reductase (MTHFR) C6<em>7</em><em>7</em>T (OR = <em>1</em>.55, 95% CI = <em>1</em>.26-<em>1</em>.90), plasminogen activator inhibitor <em>1</em> (PAI-<em>1</em>) 4G/5G (OR = <em>1</em>.<em>7</em>9, 95% CI = <em>1</em>.20-2.6<em>7</em>), beta-fibrinogen (beta-Fg) -455A/G (OR = <em>1</em>.48, 95% CI = <em>1</em>.<em>1</em>4-<em>1</em>.92), beta-Fg -<em>1</em>48T/C (OR = <em>1</em>.<em>7</em>2, 95% CI = <em>1</em>.42-2.0<em>7</em>), apolipoprotein E (ApoE) epsilon2-4 (OR = 2.39, 95% CI = <em>1</em>.94-2.95). Because of the obvious publication bias, the association between paraoxonase <em>1</em> (PON-<em>1</em>) polymorphisms and stroke risk was not established although the OR of the meta-analysis suggested a positive result (OR = <em>1</em>.<em>1</em>4, 95% CI = <em>1</em>.0<em>1</em>-<em>1</em>.35).

CONCLUSIONS

ACE D/I, MTHFR C6<em>7</em><em>7</em>T, beta-Fg -455A/G, beta-Fg -<em>1</em>48T/C, PAI-<em>1</em> 4G/5G, and ApoE epsilon2-4 were associated with risk of ischemic stroke in Han Chinese.

The activation of renin-<em>angiotensin</em>-aldosterine system(RAAS) is one of the main pathogenesis of hypertension. All the components of RAAS are present in the kidneys at higher concentrations compared to plasma levels, and intrarenal formation of <em>angiotensin</em> II (Ang II) is independent of the systemic RAAS. There are some unique features in intrarenal RAAS compared to systemic RAAS. Unlike JG cells where Ang II inhibits renin release via the AngII type <em>1</em> (AT<em>1</em>) receptor by negative feedback, in the collecting duct Ang II stimulates renin expression via the AT<em>1</em> receptor. Upregulated renin produced in the distal nephron may be able to support continued intrarenal Ang II formation leading to amplification or maintenance of the hypertensive state.The recently discovered <em>angiotensin</em>-converting enzyme-related carboxypeptidase 2 (ACE2)-<em>Angiotensin</em>-(<em>1</em>-<em>7</em>) Ang-(<em>1</em>-<em>7</em>)-Mas receptor axis has an opposing function to that of the ACE-Ang II-AT<em>1</em> receptor axis.The ACE2 deficiency was associated with an increase in blood pressure, and ACE2 knockout mice have highlighted hypertensive response to Ang II infusion associated with exaggerated accumulation of Ang II in the kidney. Recently, several numbers of patients have been evaluated as the activators of ACE2-Ang-(<em>1</em>-<em>7</em>)-Mas receptor axis, which can be divided into two main classes: aimed to increase the activity of ACE2, and directed to stimulate the Ang-(<em>1</em>-<em>7</em>) receptor Mas. In order to investigate new targets for hypertension and kidney disease, further research on the function of the ACE-Ang-(<em>1</em>-<em>7</em>)-Mas receptor axis is required.

<em>1</em>. The use of the rat colon as a blood-bathed organ is described for detecting changes in <em>angiotensin</em> concentration in the circulating blood of dogs.2. Partial occlusion of the aorta by a balloon inflated above the renal arteries leads to a contraction of the blood-bathed rat colon.3. From the experimental evidence, it is concluded that this contraction is due to an increased concentration of circulating <em>angiotensin</em>, brought about by the liberation of renin from the kidneys.4. The characteristics of renin release have been studied. It occurs within seconds of reducing the blood pressure to the kidneys and is proportional to the degree of reduction of blood pressure.5. With a prolonged reduction of renal blood pressure the concentration of <em>angiotensin</em> increases over the first <em>1</em>0-<em>1</em>2 min and then reaches a stable level.6. After a small haemorrhage <em>angiotensin</em> often appears in the circulation without a concomitant release of catechol amines.<em>7</em>. Greater haemorrhages induce the secretion of catechol amines as well as of renin. The catechol amine secretion is inhibited by ganglion block, but the renin secretion is not.8. It is concluded that the secretion of renin by the kidneys in response to a fall of renal blood pressure is a physiological response, probably of importance in homoeostasis.

OBJECTIVE

The circulating renin-<em>angiotensin</em> system (RAS) [plasma renin activity (PRA), <em>Angiotensin</em> (Ang) I, Ang II and Ang-(<em>1</em>-<em>7</em>)] was evaluated in a model of hepatic fibrosis in rats. To investigate the pathophysiological involvement of Ang-(<em>1</em>-<em>7</em>), animals were treated with the Ang-(<em>1</em>-<em>7</em>) Mas receptor antagonist, A-<em>7</em><em>7</em>9.

METHODS

RAS components, liver function and histology were examined in male Wistar rats (220-300 g). Animals were submitted to sham-surgery or ligature of the bile duct and evaluated <em>1</em>, 2, 4 and 6 weeks later. Blood samples were obtained to determine biochemical parameters and RAS components. A second group was treated with A-<em>7</em><em>7</em>9 or vehicle to measure liver hydroxyproline and total transforming growth factor beta-<em>1</em> (TGFbeta<em>1</em>).

RESULTS

PRA and Ang I were significantly elevated in rats at 4 and 6 weeks compared to sham-operated animals. Ang II and Ang-(<em>1</em>-<em>7</em>) progressively increased over the 6 weeks. Changes in RAS profile correlated with histological signs of fibrosis and deterioration in liver function. Pharmacological blockade of the Ang-(<em>1</em>-<em>7</em>) receptor aggravated liver fibrosis with a significant elevation in hydroxyproline and total TGFbeta(<em>1</em>).

CONCLUSIONS

Hepatic fibrosis was associated with RAS activation in our model. Our data also suggested that Ang-(<em>1</em>-<em>7</em>) played a protective role in hepatic fibrosis.

The renin-<em>angiotensin</em>-aldosterone-system appears to be involved in the development of cardiac fibrosis in rodents, characterized by nonepithelial cell proliferation and changes in the extracellular matrix. The aim of our study was to investigate the effect of high aldosterone concentrations on the proliferation of human cardiac interstitial cells in vitro. In addition, the effect of D-glucose as another risk factor for fibrosis, eg, in the diabetic heart, was investigated. Human cardiac myofibroblast cultures were established, and growth rates were measured by WST-<em>1</em> assay in fetal calf serum-free Dulbecco's modified Eagle's medium (DMEM). Cells in culture showed a significant increase in number between 24 to <em>7</em>2 hours of cultivation under basal conditions (DMEM, <em>1</em>0% fetal calf serum). Aldosterone at high concentrations (<em>1</em>0(-8) and <em>1</em>0(-<em>7</em>) mol/L) significantly (P<0.0<em>1</em>) increased the proliferation of cultured cardiac myofibroblasts. Comparable effects were observed after incubation of the cells with high D-glucose concentrations (<em>1</em>5 and 25 mmol/L, P<0.0<em>1</em>). No additive growth stimulation was evident when the cells were incubated in medium containing both aldosterone and D -glucose. These results suggest a role for aldosterone and glucose in mediating the cardiac fibrosis through stimulation of myofibroblast growth in patients with dysregulated renin-<em>angiotensin</em>-aldosterone-system (especially hyperaldosteronism) and impaired glucose homeostasis.

Women are less susceptible to the cerebrovascular complications of hypertension, such as a stroke and vascular dementia. The mechanism of such protection may be related to a reduced vulnerability of women to the cerebrovascular actions of hypertension. To test this hypothesis, we used a model of hypertension based on infusion of <em>angiotensin</em> II (ANG II), an octapeptide that plays a key role in hypertension and produces cerebrovascular dysregulation. Cerebral blood flow (CBF) was monitored by laser-Doppler flowmetry in anesthetized (urethane-chloralose) C5<em>7</em>BL/6J male and female mice equipped with a cranial window. ANG II administration (0.25 mug.kg(-<em>1</em>).min(-<em>1</em>) iv x 30-45 min) elevated arterial pressure equally in both sexes but attenuated the CBF increase induced by whisker stimulation or by the endothelium-dependent vasodilator acetylcholine (ACh) in male but not in female mice. The administration of ANG II for <em>7</em> days (2.<em>7</em>4 mg.kg(-<em>1</em>).day(-<em>1</em>)), using osmotic minipumps, also attenuated these cerebrovascular responses in male, but not female, mice. The reduced susceptibility to the effect of ANG II in female mice was abolished by ovariectomy and reinstated by estrogen administration to ovariectomized mice. Administration of estrogen to male mice abolished the ANG II-induced attenuation of CBF responses. We conclude that female mice are less susceptible to the cerebrovascular dysregulation induced by ANG II, an effect related to estrogen. Such protection from the deleterious cerebrovascular effects of hypertension may play a role in the reduced vulnerability to the cerebrovascular complications of hypertension observed in women.

<em>Angiotensin</em> II (Ang II) is implicated in fibrosis but the precise mechanism of this effect remains unclear. In a model of chronic cyclosporine (CsA) nephropathy, we previously showed that TGF-beta<em>1</em> plays a role in CsA-induced tubulointerstitial fibrosis and arteriolopathy by stimulating extracellular matrix (ECM) protein synthesis and inhibiting ECM degradation through increasing the synthesis of plasminogen activator inhibitor (PAI)-<em>1</em>. We hypothesized that Ang II contributes to fibrosis by inducing TGF-beta<em>1</em>. Salt-depleted rats were given placebo, CsA alone, CsA + nilvadipine, CsA + hydralazine/hydrochlorthiazide, CsA + losartan (AT<em>1</em> receptor antagonist) or CsA + enalapril (Ang converting enzyme inhibitor) and were sacrificed at <em>7</em> and 28 days. All treated groups achieved similar blood pressures and glomerular filtration rates. The lesion of chronic CsA nephropathy was ameliorated by concomitant therapy with losartan or enalapril at 28 days, a phenomenon not observed in the other treatment groups. Similarly, Ang II blockade resulted in decreased expression of TGF-beta<em>1</em> and PAI-<em>1</em> by Northern and ELISA. Similarly, the expression of ECM proteins directly influenced by TGF-beta decreased with Ang II blockade. These results suggest that CsA-induced fibrosis in this model is independent of renal hemodynamics and is mediated, at least partly, through Ang II induction of TGF-beta<em>1</em> expression.

The peptide hormone <em>angiotensin</em> II is well established to play an endocrine role in the regulation of blood pressure, fluid and electrolyte homeostasis. In addition to its hemodynamic function, recent studies have shown that numerous tissues and organs contain their own locally generated <em>angiotensin</em> products (<em>angiotensin</em> II, III, IV and Ang <em>1</em>-<em>7</em>) and they exhibit their respective activities. Such an intrinsic <em>angiotensin</em>-generating system renders to specific tissue function of our body, frequently via the regulatory mechanism of a paracrine, autocrine or intracrine manner. These tissues and organs include, to name but a few, the brain, bone marrow, adipose, epididymis, carotid body, liver, and pancreas. This local system has been shown to be responsive to various stimuli of physiological and pathophysiological importance. Moreover, the locally generated <em>angiotensin</em> peptides have multiple and novel actions including cell growth, anti-proliferation, apoptosis, reactive oxygen species generation, hormonal secretion, pro-inflammatory, and pro-fibrogenic actions, as well as vasoconstriction and vasodilatation. Notwithstanding the emerging roles of <em>angiotensin</em> II in various tissues and organs, the physiological significance and ultimately the clinical relevance remain largely undefined. Future target for these new functions by making use of specific renin-<em>angiotensin</em> system inhibitors, such as the <em>angiotensin</em>-converting enzyme and <em>angiotensin</em> II receptor blockers either in mono-therapy or in combination, could be of clinical importance. The current review is to focus on some of the new functions arising from the locally formed <em>angiotensin</em> II in tissues and organs, with particular attention to its emerging roles in the liver and the pancreas.

To study the metabolism and production of <em>angiotensin</em> I, highly purified monoiodinated [<em>1</em>25I] <em>angiotensin</em> I was given by constant systemic intravenous infusion, either alone (n = <em>7</em>) or combined with unlabeled <em>angiotensin</em> I (n = 5), to subjects with essential hypertension who were treated with the <em>angiotensin</em> converting enzyme inhibitor captopril (50 mg b.i.d.). Blood samples were taken from the aorta and the renal, antecubital, femoral, and hepatic veins. [<em>1</em>25I]<em>Angiotensin</em> I and <em>angiotensin</em> I were extracted from plasma, separated by high-performance liquid chromatography, and quantitated by gamma counting and radioimmunoassay. Plasma renin activity was measured at pH <em>7</em>.4. The plasma decay curves after discontinuation of the infusions of [<em>1</em>25I]<em>angiotensin</em> I and unlabeled <em>angiotensin</em> I were similar for the two peptides. The regional extraction ratio of [<em>1</em>25I]<em>angiotensin</em> I was 4<em>7</em> +/- 4% (mean +/- SEM) across the forearm, 59 +/- 3% across the leg, 8<em>1</em> +/- <em>1</em>% across the kidneys, and 96 +/- <em>1</em>% across the hepatomesenteric vascular bed. These results were not different from those obtained for infused unlabeled <em>angiotensin</em> I. Despite the rapid removal of arterially delivered <em>angiotensin</em> I, no difference was found between the venous and arterial levels of endogenous <em>angiotensin</em> I across the various vascular beds, with the exception of the liver where <em>angiotensin</em> I in the vein was 50% lower than in the aorta. Thus, 50-90% of endogenous <em>angiotensin</em> I in the veins appeared to be derived from regional de novo production. The blood transit time is 0.<em>1</em>-0.2 minute in the limbs and in the kidneys and 0.3-0.5 minute in the hepatomesenteric vascular bed. This is too short for plasma renin activity to account for the measured de novo <em>angiotensin</em> I production. It was calculated that less than 20-30% in the limbs and in the kidneys and approximately 60% in the hepatomesenteric region of de novo-produced <em>angiotensin</em> I could be accounted for by circulating renin. These results indicate that a high percentage of plasma <em>angiotensin</em> I may be produced locally (i.e., not in circulating plasma).

Impaired cardiac sympathetic activity can be evaluated by <em>1</em>23I-metaiodobenzylguanidine (MIBG) imaging.

METHODS

We studied the significance of MIBG imaging for 24 patients (age 58+/-<em>1</em>2 y) with dilated cardiomyopathy (DCM). We compared <em>1</em>2 patients (group A) treated with metoprolol (dose from 30-60 mg/d) with <em>1</em>2 patients treated with <em>angiotensin</em>-converting enzyme (ACE) inhibitors. Patients were studied before treatment, after 5 mo of treatment (only in group A) and after <em>1</em> y of treatment. Cardiac MIBG uptake was assessed as the heart-to-mediastinum activity ratio (H/M) and total defect score (TDS) from anterior planar and SPECT MIBG images, which were acquired in 4 h after tracer injection. New York Heart Association (NYHA) class and left ventricular ejection fraction (LVEF) calculated by echocardiography were also assessed.

RESULTS

TDS decreased in both groups (in group A, from 30+/-7 through 23+/-9 to <em>1</em>8+/-<em>1</em>0; P < 0.0<em>1</em>, in group B, from 30+/-6 to 24+/-8; P < 0.0<em>1</em>) and H/M was increased in both groups (in group A, from <em>1</em>.87+/-0.3<em>1</em> through 2.03+/-0.28 to 2.<em>1</em>4+/-0.29; P < 0.0<em>1</em>, in group B, from <em>1</em>.82+/-0.28 to <em>1</em>.94+/-0.26; P < 0.05). But TDS and H/M were more improved in group A than in group B (P < 0.05). LVEF was significantly increased in only group A (from 38+/-6 through 43+/-8 to 49%+/-9%; P < 0.0<em>1</em>). NYHA improved in both groups (in group A, from mean 2.5 through 2.<em>1</em> to <em>1</em>.8; P < 0.0<em>1</em>, in group B, from mean 2.6 to 2.<em>1</em>; P < 0.05) but was more improved in group A than in group B (P < 0.05).

CONCLUSIONS

Cardiac function, symptom and cardiac sympathetic activity evaluated by MIBG images improved after the beta-blocker therapy more than with the treatment that used ACE inhibitors.

The pulmonary endothelium is susceptible to oxidative insults. Catalase conjugated with monoclonal antibodies (MAbs) against endothelial surface antigens, <em>angiotensin</em>-converting enzyme (MAb 9B9) or intercellular adhesion molecule-<em>1</em> (MAb <em>1</em>A29), accumulates in the lungs after systemic injection in rats (V. Muzykantov, E. Atochina, H. Ischiropoulos, S. Danilov, and A. Fisher. Proc. Natl. Acad. Sci. USA 93: 52<em>1</em>3-52<em>1</em>8, <em>1</em>996). The present study characterizes the augmentation of antioxidant defense by these antibody-catalase conjugates in isolated rat lungs perfused for <em>1</em> h with catalase conjugated with either MAb 9B9, MAb <em>1</em>A29, or control mouse IgG. Approximately 20% of the injected dose of Ab-<em>1</em>25I-catalase accumulated in the perfused rat lungs (vs. <5% for IgG-<em>1</em>25I-catalase). After elimination of nonbound material, the lungs were perfused further for <em>1</em> h with 5 mM hydrogen peroxide (H2O2). H2O2 induced an elevation in tracheal and pulmonary arterial pressures (<em>1</em>26 +/- <em>7</em> and <em>1</em>32 +/- 5%, respectively, of the control level), lung wet-to-dry weight ratio (<em>7</em>.<em>1</em> +/- 0.4 vs. 6.0 +/- 0.0<em>1</em> in the control lungs), and ACE release into the perfusate (436 +/- 20 vs. <em>7</em>5 +/- <em>7</em> mU in the control perfusates). Both MAb 9B9-catalase and MAb <em>1</em>A29-catalase significantly attenuated the H2O2-induced elevation in <em>1</em>) <em>angiotensin</em>-converting enzyme release to the perfusate (2<em>1</em>5 +/- <em>1</em>4 and 2<em>1</em><em>7</em> +/- 38 mU, respectively), 2) lung wet-to-dry ratio (6.25 +/- 0.<em>1</em> and 6.3 +/- 0.3, respectively), 3) tracheal pressure (94 +/- 4 and <em>1</em>0<em>1</em> +/- 4%, respectively, of the control level), and 4) pulmonary arterial pressure (<em>1</em>03 +/- 3 and <em>1</em>04 +/- <em>7</em>%, respectively, of the control level). Nonconjugated catalase, nonconjugated antibodies, nonspecific IgG, and IgG-catalase conjugate had no protective effect, thus confirming the specificity of the effect of MAb-catalase. These results support a strategy of catalase immunotargeting for protection against pulmonary oxidative injury.

OBJECTIVE

Epoxygenase metabolites produced by the kidney affect renal blood flow and tubular transport function and 11,12-epoxyeicosatrienoic acid (11,12-EET) has been putatively identified as an endothelium-derived hyperpolarizing factor. The current studies were performed to determine the influence of 11,12-EET on the regulation of afferent arteriolar diameter in angiotensin II-infused hypertensive rats.

METHODS

Male Sprague-Dawley rats received angiotensin II (60 ng/min) or vehicle via an osmotic minipump. Angiotensin II-infused hypertensive and vehicle-infused normotensive rats were studied for 2 weeks following implantation of the minipump. Renal microvascular responses to the sulfonimide analog of 11,12-EET (11,12-EET-SI) and angiotensin II were observed utilizing the in-vitro juxtamedullary nephron preparation. Renal cortical epoxygenase enzyme protein levels were quantified by Western blot analysis. Renal microvessels were also isolated and epoxygenase metabolite levels measured by negative ion chemical ionization (NICI)/gas chromatography-mass spectroscopy.

RESULTS

Systolic blood pressure averaged 118 +/- 2 mmHg prior to pump implantation and increased to 185 +/- 7 mmHg in rats infused with angiotensin II for 2 weeks. Afferent arteriolar diameters of 2-week normotensive animals averaged 22 +/- 1 microm. Diameters of the afferent arterioles were 17% smaller in hypertensive rats (P< 0.05); however, arterioles from both groups responded to 11,12-EET-SI (100 nmol) with similar 15-17% increases in diameter. As we previously demonstrated, the afferent arteriolar reactivity to angiotensin II was enhanced in angiotensin II-infused animals. Interestingly, elevation of 11,12-EET-SI levels to 100 nmol reversed the enhanced vascular reactivity to angiotensin II associated with angiotensin II hypertension. Renal microvascular EET levels were not different between groups and averaged 81 +/- 9 and 87 +/- 13 pg/mg per 30 min in normotensive and hypertensive animals, respectively. Renal cortical microsomal levels of the epoxygenase CYP2C23 and CYP2C11 proteins were also similar in normotensive and angiotensin II hypertensive rats.

CONCLUSIONS

Taken together, these data support the concept that renal microvascular 11,12-EET activity and levels may not properly offset the enhanced angiotensin II renal vasoconstriction during angiotensin II hypertension.

Activation of G protein-coupled receptors by agonists involves significant movement of transmembrane domains (TM) following binding of agonist. The underlying structural mechanism by which receptor activation takes place is largely unknown but can be inferred by detecting variability within the environment of the ligand-binding pocket, which constitutes a water-accessible crevice surrounded by the seven TM helices. Using the substituted cysteine accessibility method, we initially identified those residues within the seventh transmembrane domain (TM7) of wild type <em>angiotensin</em> II type <em>1</em> (AT<em>1</em>) receptor that contribute to forming the binding site pocket. We have substituted successively TM7 residues ranging from Ile276 to Tyr302 to cysteine. Treatment of A277C, V280C, T282C, A283C, I286C, A29<em>1</em>C, and F30<em>1</em>C mutant receptors with the charged sulfhydryl-specific alkylating agent MTSEA significantly inhibited ligand binding, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT<em>1</em> receptor. Interestingly, this pattern of acquired MTSEA sensitivity was greatly reduced for TM7 reporter cysteines engineered in a constitutively active mutant of the AT<em>1</em> receptor. Our data suggest that upon activation, TM7 of the AT<em>1</em> receptor goes through a pattern of helical movements that results in its distancing from the binding pocket per se. These studies support accumulating evidence whereby elements of TM7 of class A GPCRs promote activation of the receptor through structural rearrangements.

This review examines the recent progress in the field of <em>angiotensin</em> receptors. Multiplicity of these receptors was demonstrated initially on the basis of pharmacologic differences and then confirmed by expression cloning. AT<em>1</em> receptors are predominant in the adult. They are widely distributed and mediate all of the known biologic effects of <em>angiotensin</em> II (AngII) through a variety of signal transduction systems, including activation of phospholipases C and A2, inhibition of adenylate cyclase, opening of calcium channels, and activation of tyrosine kinases. AT2 receptors are predominant in the fetus, but also present in adult tissues such as the adrenals, ovaries, uterus, and brain. AngII via these receptors exerts effects often opposed to those mediated by the AT<em>1</em> receptors. Signal transduction implicates protein tyrosine phosphatase stimulation. AT<em>1</em> and AT2 receptor expressions are regulated differently, and regulation is also tissue-specific. AT<em>1</em> and AT2 receptors have been demonstrated in endothelial cells. Activation of AT<em>1</em> receptors results in production of vasodilatory agents, nitric oxide, and prostacyclin (PGI2), which counteract the direct vasoconstrictor effects of Ang II on the adjacent smooth muscle cells. AT<em>1</em> receptors on mesangial cells, smooth muscle cells, and fibroblasts are involved in cell growth and fibrosis, the latter being due both to an increase in the synthesis and a decrease in the degradation of the main components of the extracellular matrix. These AT<em>1</em> receptor-dependent effects are for the most part indirect and mediated by growth factors, cytokines, and other peptides, including endothelin, transforming growth factor-beta<em>1</em>, and platelet-derived growth factor. AngII is metabolized into active fragments by deletion of the terminal amino acids on both ends. AngIII and AngIV are formed by successive deletions of aspartic acid and arginine at the N terminus. AngII (<em>1</em>-<em>7</em>) is obtained by deletion of phenylalanine at the C terminus. AngIII shares the same receptors and exerts the same effects as AngII. AngIV and AngII (<em>1</em>-<em>7</em>) recognize the AT<em>1</em> and AT2 receptors with a lesser affinity than AngII and, in addition, possess their own receptors that mediate effects often opposed to those of AngII.

Six weeks after the onset of insulin-treated streptozotocin diabetes (STZ) in Munich-Wistar rats, the effect of a low-sodium (LNa) and a low-salt (LNaCl) diet on renal function and on plasma and kidney tissue <em>angiotensin</em> II (AIIp, AIIk) was tested. Clearance experiments were performed in anesthetized rats <em>7</em> days after starting on LNa or LNaCl. On a control diet, STZ exhibited an increase in GFR, RBF, and kidney weight (KW) and a reduction in renal vascular resistance (RVR) and AIIk, but no change in AIIp, compared with nondiabetic normal rats (CON). Although sodium restriction reduced and salt restriction increased AIIk in CON, both diets increased AIIp without affecting renal hemodynamics or KW. In diabetic rats, both salt and sodium restriction further increased GFR and RBF by reducing RVR, increased KW, and changed AIIk and AIIp in a similar pattern, but at significantly lower values compared with CON. Daily treatment of STZ-LNa with the AII-receptor blocker losartan (20 mg/L, in drinking water) did not affect the reduction in RVR and the increase in KW but slightly reduced RBF because of a decrease in mean arterial blood pressure and further increased GFR. It was concluded that (<em>1</em>) AIIk but not AIIp is affected differently by LNa compared with LNaCl in both CON and STZ; (2) LNaCl and LNa change AIIp and AIIk in a similar pattern but at significant lower values in STZ compared with CON; and (3) with regard to renal hemodynamics and KW, the response to LNa and LNaCl is different in CON compared with rats 6 wk after the onset of diabetes mellitus, the latter exhibiting a further increase in renal hyperfiltration and KW by a mechanism that is not directly AII receptor dependent.

Controversy exists regarding whether all patients with acute myocardial infarction (AMI) benefit from <em>angiotensin</em>-converting enzyme inhibitors (ACEIs). We examined the association between ACEI treatment and mortality in a large, unselected population of patients with AMI. The present study included <em>1</em>05,224 patients with AMI who were not treated with ACEIs on admission. A logistic regression analysis, including 33 variables, calculated a propensity score for each patient to estimate the probability of receiving ACEIs at discharge, given the background. The association between ACEI treatment at discharge and the <em>1</em>-year outcome was evaluated in prespecified subgroups using the Cox regression analyses, adjusting for the propensity score and medications at discharge. A total of 38,395 patients (36.5%) received ACEIs at discharge. After adjustment, ACEI treatment was associated with a 24% reduction in mortality (relative risk 0.<em>7</em>6, 95% confidence interval 0.<em>7</em>3 to 0.80). The benefit was largest in patients with a history or present signs of heart failure. In patients without heart failure, a significant benefit of ACEI treatment was seen only in patients with renal dysfunction (relative risk 0.69, 95% confidence interval 0.54 to 0.88). In the whole group, the risk of AMI decreased by <em>7</em>% (relative risk 0.93, 95% confidence interval 0.90 to 0.96), with a larger effect seen in patients with ST-segment elevation AMI or systolic left ventricular dysfunction. In conclusion, in unselected patients with AMI, ACEI treatment was associated with a reduction in <em>1</em>-year mortality, mainly in patients with heart failure or renal dysfunction, and a small reduction in the risk of reinfarction, mainly in patients with ST-segment elevation AMI or systolic left ventricular dysfunction.

<em>Angiotensin</em> II (Ang II) type <em>1</em> receptor (AT(<em>1</em>)) antagonists such as losartan (LOS) are widely used for the treatment of hypertension and elicit antiinflammatory and antiaggregatory in vitro and in patients, although the underlying mechanism are unclear. Following computer-based molecule similarity, we proposed that on cytochrome-P450 degradation, the LOS metabolite EXP3<em>1</em><em>7</em>9 is generated, which shows molecule homology to indomethacin, a cyclooxygenase inhibitor with antiinflammatory and antiaggregatory properties. Subsequently, serum-levels of EXP3<em>1</em><em>7</em>9 were determined for 8 hours in patients receiving a single oral dose of <em>1</em>00 mg LOS. High-performance liquid chromatography followed by liquid chromatography-mass spectrometry (GC-MS) [corrected] from serum samples revealed a maximum of <em>1</em>0(-<em>7</em>) mol/L for EXP3<em>1</em><em>7</em>9 peaking between 3 to 4 hours. The increase in serum-EXP3<em>1</em><em>7</em>9 levels was associated with a significant reduction in platelet aggregation in vivo (-35+/-4%, P<0.00<em>1</em> versus control). EXP3<em>1</em><em>7</em>9 generation was investigated in a chemical reaction mimicking the liver cytochrome-P450-dependent LOS-degradation and human endothelial cells were exposed to Ang II or lipopolysaccharides (LPS) in the presence of EXP3<em>1</em><em>7</em>9 (<em>1</em>0(-<em>7</em>) mol/L). LPS- and Ang II-induced COX-2 transcription was abolished by EXP3<em>1</em><em>7</em>9. Moreover, EXP3<em>1</em><em>7</em>9 significantly reduced Ang II- and LPS-induced formation of prostaglandin F2alpha as determined by GC-MS [corrected]. Thus, antiinflammatory properties of LOS are mediated via its EXP3<em>1</em><em>7</em>9 metabolite by abolishing COX-2 mRNA upregulation and COX-dependent TXA2 and PGF2alpha generation. Serum levels of EXP3<em>1</em><em>7</em>9 are detectable in patients in concentrations that exhibit antiinflammatory and antiaggregatory properties in vitro.

-Losartan was the first available orally administered selective antagonist of the <em>angiotensin</em> II type <em>1</em> receptor developed for the treatment of hypertension. The Losartan Intervention For Endpoint (LIFE) Reduction in Hypertension Study is a double-blind, prospective, parallel group study designed to compare the effects of losartan with those of the beta-blocker atenolol on the reduction of cardiovascular morbidity and mortality. Patients with essential hypertension, aged between 55 and 80 years, and ECG-documented left ventricular hypertrophy (LVH) were included. Altogether, 9223 patients in Scandinavia, the United Kingdom, and the United States were randomized from June <em>1</em>995 through April <em>1</em>99<em>7</em>, and 9<em>1</em>94 remain after exclusion of a study center at which irregularities were discovered. This population of hypertensives (mean systolic/diastolic blood pressure, <em>1</em><em>7</em>4.4/9<em>7</em>.8 mm Hg) with LVH comprises women (54.<em>1</em>%) and men, mostly retired from active work (mean age, 66.9 years), with a high prevalence of overweight (mean body mass index, 28.0 kg/m2), diabetes mellitus (<em>1</em>2.3%), lipid disorders (<em>1</em>8.0%), and symptoms or signs of coronary heart disease (<em>1</em>5.<em>1</em>%). There were fewer current smokers ((<em>1</em><em>7</em>%) than in the general population, and approximately <em>7</em>% were nonwhite. Almost 30% of participants had been untreated for at least 6 months when screened for the study. Only <em>1</em>55<em>7</em> persons who entered the placebo run-in period of <em>1</em>4 days were excluded, predominantly because of sitting blood pressures above or below the predetermined range of <em>1</em>60-200/95-<em>1</em><em>1</em>5 mm Hg and ECG-LVH criteria not met. By application of simple <em>1</em>2-lead ECG criteria for LVH (Cornell voltage QRS duration product formula plus Sokolow-Lyon voltage read by a core laboratory), hypertensive patients with LVH with an average 5-year coronary heart disease risk of 22.3% according to the Framingham score were identified. This population is now being treated (goal, (<em>1</em>40/90 mm Hg) in adherence with the protocol for at least 4 years after final enrollment (ie, through April 200<em>1</em>) and until at least <em>1</em>040 patients suffer myocardial infarction, stroke, or cardiovascular death.

BACKGROUND

Genetic studies may help us to understand the mechanisms underlying the involvement of various neuro-humoral factors in the regulation of the mechanical properties of large arteries. We have shown previously that the <em>angiotensin</em> II type <em>1</em> receptor gene polymorphism was a strong determinant of aortic stiffness in hypertensives.

OBJECTIVE

To assess the contribution of two polymorphisms of the endothelial nitric oxide synthase gene to aortic stiffness in normotensive and hypertensive subjects in the same cohort.

METHODS

We studied 309 untreated hypertensive and <em>1</em>23 normotensive subjects. Aortic stiffness was evaluated by measuring the carotid-femoral pulse-wave velocity non-invasively. The endothelial nitric oxide synthase gene polymorphisms G<em>1</em>0-T at intron 23 (GIN23T) and G298-T at exon 7 (Glu298Asp) were determined in each subject.

RESULTS

The distributions of genotypes and allele prevalences of the endothelial nitric-oxide synthase G<em>1</em>0-T polymorphisms among hypertensive and normotensive subjects were similar. In contrast, the prevalence of the nitric oxide synthase 298G allele was higher in the hypertensive group than it was among normotensive subjects. We found no association of the endothelial nitric oxide synthase genotypes with blood pressure levels or pulse-wave velocity for either population.

CONCLUSIONS

The present results do not suggest that two common polymorphisms of the endothelial nitric oxide synthase gene are involved in the regulation of aortic stiffness in hypertensive and normotensive individuals. The higher prevalence of endothelial nitric oxide synthase 298G allele among hypertensives suggests that this gene is involved in essential hypertension but this observation needs further confirmation.

<em>Angiotensin</em> (Ang) II is not the only active peptide of the renin-<em>angiotensin</em> system. Several of its degradation products including Ang III (obtained by deletion of the N terminal amino acid), Ang IV (obtained by deletion of the two N terminal amino acids) and Ang II(<em>1</em>-<em>7</em>) (obtained by deletion of the C terminal amino acid) also possess biological functions. These peptides are formed via the activity of several enzymes, aminopeptidase A for Ang III, aminopeptidases A and N for Ang IV, prolylendopeptidase and carboxypeptidases for Ang II(<em>1</em>-<em>7</em>). Ang III possesses most of the properties of Ang II and shares the same receptors. This peptide is particularly important in brain and pituitary physiology and plays a major role in the secretion of arginine vasopressin. Ang IV possesses its own receptors distinct from AT<em>1</em> and AT2. Some of its effects (for example, stimulation of the synthesis of the type <em>1</em> inhibitor of plasminogen activator by endothelial cells) were previously attributed to Ang II. Others are opposed to Ang II effects (renal and cerebral vasodilation). Its role in vascular, renal and cerebral physiology remains to be determined. Ang II(<em>1</em>-<em>7</em>) exhibits direct and indirect effects, the latter resulting from Ang II(<em>1</em>-<em>7</em>)-dependent formation of nitric oxide and vasodilatory prostaglandins. Ang II(<em>1</em>-<em>7</em>) recognizes both specific receptors and AT<em>1</em> receptors as shown by the partial antagonistic properties of losartan. Ang II(<em>1</em>-<em>7</em>) plays essentially a role in the control of the hydroelectrolytic balance by increasing glomerular filtration rate, urinary output and sodium excretion rate.