Cardiovascular mortality, mainly due to the rupture of unstable atherosclerotic plaques, is reduced by 3-hydroxy-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors. Inflammatory cells, attracted to the vascular lesion by chemokines, have been implicated in the process of the plaque rupture. In cultured vascular smooth muscle cells (VSMC) and U93<em>7</em> mononuclear cells we have studied the effect of Atorvastatin (Atv) on nuclear factor kappaB (NF-kappaB) activity, an inducer of the mRNA expression of chemokines such as interferon-inducible protein <em>1</em>0 (IP-<em>1</em>0) and monocyte chemoattractant protein <em>1</em> (MCP-<em>1</em>). <em>Angiotensin</em> II (Ang II) and tumor necrosis factor alpha (TNF-alpha) increased NF-kappaB activity in VSMC (2 and 5-fold, respectively). Preincubation of cells with <em>1</em>0(-<em>7</em>) mol/l Atv diminished this activation (44 and 53%). The inhibition was reversed by mevalonate, farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), but not by other isoprenoids. Coinciding with the NF-kappaB activation in VSMC, there was a diminution of cytoplasmic IkappaB levels that was recovered by pretreatment with Atv. Ang II and TNF-alpha induced the expression of IP-<em>1</em>0 (<em>1</em>.5 and 3.4-fold) and MCP-<em>1</em> (2.4 and 4-fold) in VSMC. Atv reduced this overexpression around 38 and 35% (IP-<em>1</em>0), and 54 and 39% (MCP-<em>1</em>), respectively. Our results strongly suggest that Atv, through the inhibition of NF-kappaB activity and chemokine gene expression, could reduce the inflammation within the atherosclerotic lesion and play a role in the stabilization of the lesion.

The possibility of preventing pregnancy-induced hypertension (PIH) and pre-eclampsia in primigravidae by suppressing production of thromboxane A2 with low-dose aspirin was investigated in a randomised, placebo-controlled, double-blind trial. 46 normotensive women at 28 weeks' gestation, judged to be at risk of PIH or pre-eclampsia because of an increased blood-pressure response to intravenously infused <em>angiotensin</em> II, were studied. 23 women received 60 mg aspirin daily, and the same number received matching placebo until delivery. In the placebo group PIH, pre-eclampsia, and eclampsia developed in 4, <em>7</em>, and <em>1</em> cases, respectively, whereas only 2 women in the aspirin group had mild PIH. There were no adverse effects of treatment in mothers or infants. Low-dose aspirin may restore prostacyclin/thromboxane imbalance, previously suggested as an important aetiological factor in PIH and pre-eclampsia.

The vast majority of the known biological effects of the renin-<em>angiotensin</em> system are mediated by the type-<em>1</em> (AT<em>1</em>) receptor, and the functions of the type-2 (AT2) receptor are largely unknown. We investigated the role of the AT2 receptor in the vascular and renal responses to physiological increases in <em>angiotensin</em> II (ANG II) in mice with targeted deletion of the AT2 receptor gene. Mice lacking the AT2 receptor (AT2-null mice) had slightly elevated systolic blood pressure (SBP) compared with that of wild-type (WT) control mice (P < 0.000<em>1</em>). In AT2-null mice, infusion of ANG II (4 pmol/kg/min) for <em>7</em> days produced a marked and sustained increase in SBP [from <em>1</em><em>1</em>6 +/- 0.5 to 208 +/- <em>1</em> mmHg (P < 0.000<em>1</em>) (<em>1</em> mmHg = <em>1</em>33 Pa)] and reduction in urinary sodium excretion (UNaV) [from 0.6 +/- 0.0<em>1</em> to 0.05 +/- 0.002 mM/day (P < 0.000<em>1</em>)] whereas neither SBP nor UNaV changed in WT mice. AT2-null mice had low basal levels of renal interstitial fluid bradykinin (BK), and cyclic guanosine 3', 5'-monophosphate, an index of nitric oxide production, compared with WT mice. In WT mice, dietary sodium restriction or ANG II infusion increased renal interstitial fluid BK, and cyclic guanosine 3', 5'-monophosphate by approximately 4-fold (P < 0.000<em>1</em>) whereas no changes were observed in AT2-null mice. These results demonstrate that the AT2 receptor is necessary for normal physiological responses of BK and nitric oxide to ANG II. Absence of the AT2 receptor leads to vascular and renal hypersensitivity to ANG II, including sustained antinatriuresis and hypertension. These results strongly suggest that the AT2 receptor plays a counterregulatory protective role mediated via BK and nitric oxide against the antinatriuretic and pressor actions of ANG II.

<em>Angiotensin</em>-converting enzyme 2 (ACE2) converts the vasopressor <em>angiotensin</em> II (Ang II) into <em>angiotensin</em> (<em>1</em>-<em>7</em>) [Ang(<em>1</em>-<em>7</em>)], a peptide reported to have vasodilatory and cardioprotective properties. Inactivation of the ACE2 gene in mice has been reported by one group to result in an accumulation of Ang II in the heart and an age-related defect in cardiac contractility. A second study confirmed the role of ACE2 as an Ang II clearance enzyme but failed to reproduce the contractility defects previously reported in ACE2-deficient mice. The reasons for these differences are unclear but could include differences in the accumulation of Ang II or the deficiencies in Ang(<em>1</em>-<em>7</em>) in the mouse models used. As a result, the roles of ACE2, Ang II, and Ang(<em>1</em>-<em>7</em>) in the heart remain controversial. Using a novel strategy, we targeted the chronic overproduction of either Ang II or Ang(<em>1</em>-<em>7</em>) in the heart of transgenic mice and tested their effect on age-related contractility and on cardiac remodeling in response to a hypertensive challenge. We demonstrate that a chronic accumulation of Ang II in the heart does not result in cardiac contractility defects, even in older (8-month-old) mice. Likewise, transgenic animals with an 8-fold increase in Ang(<em>1</em>-<em>7</em>) peptide in the heart exhibited no differences in resting blood pressure or cardiac contractility as compared to age-matched controls, but they had significantly less ventricular hypertrophy and fibrosis than their nontransgenic littermates in response to a hypertensive challenge. Analysis of downstream signaling cascades demonstrates that cardiac Ang(<em>1</em>-<em>7</em>) selectively modulates some of the downstream signaling effectors of cardiac remodeling. These results suggest that Ang(<em>1</em>-<em>7</em>) can reduce hypertension-induced cardiac remodeling through a direct effect on the heart and raise the possibility that pathologies associated with ACE2 inactivation are mediated in part by a decrease in production of Ang(<em>1</em>-<em>7</em>).

Previous studies have indicated that <em>angiotensin</em> II (Ang II) concentrations in renal interstitial fluid are much higher than plasma levels. In the present study, we performed experiments to explore renal interstitial fluid concentrations of Ang I and Ang II further and to determine whether these levels are altered by acute arterial infusion of an ACE inhibitor (enalaprilat) or by volume expansion. Microdialysis probes (molecular weight cutoff point: 30 000 Da) were implanted in the renal cortex of anesthetized rats and were perfused at a rate of 2 microL/min. Using relative equilibrium rates, the basal renal interstitial fluid Ang II concentration averaged 3.0<em>7</em>+/-0.43 nmol/L, a value much higher than the plasma Ang II concentration of <em>1</em>0<em>7</em>+/-8 pmol/L (n=<em>7</em>). Interstitial fluid Ang I concentrations (0.84+/-0.04 nmol/L) were consistently lower than the Ang II concentrations but higher than the plasma Ang I concentrations (<em>1</em><em>1</em>2+/-<em>1</em>4 pmol/L). Intra-arterial infusion of enalaprilat (<em>7</em>.5 micromol/kg/min, n=5) for <em>1</em>20 minutes resulted in a significant decrease in mean arterial pressure (from <em>1</em><em>1</em>4+/-4 to 68+/-4 mm Hg) along with reductions in plasma and renal ACE activity (by -99% and -52%, respectively). Enalaprilat resulted in a significant increase in plasma Ang I from <em>1</em>33+/-2<em>1</em> to <em>1</em><em>1</em>6<em>7</em>+/-328 pmol/L and a decrease in plasma Ang II from <em>1</em><em>1</em>0+/-<em>1</em>2 to 6<em>7</em>+/-9 pmol/L. During enalaprilat infusion, interstitial fluid concentration of Ang I was significantly increased from 0.<em>7</em>8+/-0.06 to 0.9<em>7</em>+/-0.08 nmol/L; however, Ang II concentrations were not altered significantly (3.6<em>7</em>+/-0.28 versus 3.6<em>7</em>+/-0.25 nmol/L). Acute volume loading with Ringer's solution containing <em>1</em>% bovine serum albumin at a rate of <em>1</em>50 microL/min for 2 hours (6% to <em>7</em>% of body weight) lowered plasma concentrations of Ang I from <em>1</em><em>1</em>0+/-23 to <em>1</em>6+/-2 pmol/L and Ang II from <em>1</em>00+/-23 to 36+/-6 pmol/L; however, renal interstitial fluid concentrations of Ang I and Ang II were not altered significantly during volume expansion (Ang I, from 0.<em>7</em><em>7</em>+/-0.05 to 0.69+/-0.03 nmol/L; Ang II, from 3.<em>7</em>6+/-0.43 to 3.59+/-0.39 nmol/L, n=5). These data indicate that renal interstitial fluid concentrations of Ang I and Ang II are substantially higher than the corresponding plasma concentrations. Furthermore, the fact that the high interstitial fluid concentrations of Ang II are not responsive to acute ACE inhibition or volume expansion suggests the compartmentalization and independent regulation of renal interstitial fluid Ang II.

We have recently shown that the octapeptide <em>angiotensin</em> II is a potent stimulus of protein synthesis and growth in cultured cardiomyocytes. The present study was performed to determine if the renin-<em>angiotensin</em> system was involved in regulating cardiac cell growth in vivo. The pressure-overload cardiac hypertrophy model that develops in abdominal aorta-constricted rats was studied. At <em>7</em> and <em>1</em>5 days after abdominal aorta constriction, rats developed significant left ventricular hypertrophy. The increase in left ventricular mass was completely prevented in animals fed the <em>angiotensin</em>-converting enzyme inhibitor, enalapril maleate (0.2 mg/ml) in their drinking water. Cardiac afterload was the same in both groups of animals in that carotid artery pressures were not different in conscious awake aortic-constricted animals receiving and not receiving enalapril. These data suggest a direct growth effect of <em>angiotensin</em> II on the left ventricle and indicate a role for the renin-<em>angiotensin</em> system in the cardiac hypertrophy that develops in response to pressure overload. The presence and chamber localization of <em>angiotensin</em>ogen mRNA was determined using Northern hybridization and S<em>1</em> nuclease mapping analysis. Angiotensinogen mRNA, as determined by dot-blot hybridization analysis, was significantly increased in hypertrophied left ventricles at both <em>7</em> and <em>1</em>5 days after the surgery, when compared with sham-operated controls. The activity of the circulating renin-<em>angiotensin</em> system, as indexed by plasma renin activity was increased at <em>1</em> day following surgery [6.0 +/- 2.0 ng.ml-<em>1</em>.h-<em>1</em> <em>angiotensin</em> I (control) vs. 4<em>1</em>.8 +/- <em>1</em>0.9 ng.ml-<em>1</em>.h-<em>1</em> <em>angiotensin</em> I (experimental)], but returned to control values by day 3 postoperatively.(ABSTRACT TRUNCATED AT 250 WORDS)

<em>Angiotensin</em>-converting enzyme (ACE) is a zinc metalloproteinase and a key regulator of the renin-<em>angiotensin</em> system (RAS). ACE2 is a newly described enzyme identified in rodents and humans with a more restricted distribution than ACE, and is found mainly in heart and kidney. ACE2 cleaves a single residue from <em>angiotensin</em> I (Ang I) to generate Ang <em>1</em>-9, and degrades Ang II, the main effector of the RAS, to the vasodilator Ang <em>1</em>-<em>7</em>. The importance of ACE2 in normal physiology and pathophysiological states is largely unknown. ACE2 might act in a counter-regulatory manner to ACE, modulating the balance between vasoconstrictors and vasodilators within the heart and kidney, and playing a significant role in regulating cardiovascular and renal function.

We recently reported the presence of <em>angiotensin</em>-converting enzyme (ACE)2 in brain regions controlling cardiovascular function; however, the role of ACE2 in blood pressure regulation remains unclear because of the lack of specific tools to investigate its function. We hypothesized that ACE2 could play a pivotal role in the central regulation of cardiovascular function by regulating other renin-<em>angiotensin</em> system components. To test this hypothesis, we generated an adenovirus expressing the human ACE2 cDNA upstream of an enhanced green fluorescent protein (eGFP) reporter gene (Ad-hACE2-eGFP). In vitro characterization shows that neuronal cells infected with Ad-hACE2-eGFP (<em>1</em>0 to <em>1</em>00 multiplicities of infection), but not Ad-eGFP (<em>1</em>00 multiplicities of infection), exhibit dose-dependent ACE2 expression and activity. In addition, an active secreted form was detected in the conditioned medium. In vivo, Ad-hACE2-eGFP infection (2x<em>1</em>0(6) plaque-forming units intracerebroventricularly) produced time-dependent expression and activity (with a peak at <em>7</em> days) in the mouse subfornical organ. More importantly, <em>7</em> days after virus infection, the pressor response to <em>angiotensin</em> (Ang) II (200 pmol intracerebroventricularly) was significantly reduced in Ad-hACE2-eGFP-treated mice compared with controls. Furthermore, subfornical organ-targeted ACE2 overexpression dramatically reduced the Ang II-mediated drinking response. Interestingly, ACE2 overexpression was associated with downregulation of the Ang II type <em>1</em> receptor expression both in vitro and in vivo. These data suggest that ACE2 overexpression in the subfornical organ impairs Ang II-mediated pressor and drinking responses at least by inhibiting the Ang II type <em>1</em> receptor expression. Taken together, our results show that ACE2 plays a pivotal role in the central regulation of blood pressure and volume homeostasis, offering a new target for the treatment of hypertension and other cardiovascular diseases.

BACKGROUND

Angiotensin II-induced hypertension is associated with increased vascular superoxide production, which contributes to hypertension caused by the octapeptide. In cell culture, stretch increases endothelial and vascular smooth muscle production of reactive oxygen species (ROS). In perfused isolated vessels, elevations of pressure can increase vessel angiotensin II production. The effects of low-renin hypertension on vascular ROS production remain unclear. Furthermore, the role of ROS in vascular function and hypertension in low-renin hypertension is undefined.

RESULTS

Rats were treated with DOCA and saline drinking water for 3 weeks. Both systolic blood pressure (189+/-4 versus 126+/-2 mm Hg) and aortic superoxide production (3972+/-257 versus 852+/-287, P<0. 05) were increased compared with controls. Relaxations of vascular segments to acetylcholine (ACh, 100+/-2% versus 75+/-2%, P<0.05) and the calcium ionophore A23187 (92+/-2% versus 72+/-3%, P<0.05) were also impaired in DOCA-salt. Heparin-binding superoxide dismutase (1200 U/d IV for 3 days) had no effect on blood pressure but significantly improved relaxations to ACh and A23187. Losartan (25 mg x kg(-1) x d(-1) PO) for 7 days did not correct the hypertension or endothelium-dependent vessel relaxation in DOCA-salt rats, excluding a role of a local renin/angiotensin II system.

CONCLUSIONS

These findings indicate that increased vascular superoxide production occurs not only in angiotensin II-induced hypertension but also in hypertension known to be associated with low-renin states. Increased superoxide production alters large-vessel endothelium-dependent vascular relaxation but does not modulate blood pressure in low-renin hypertension.

<em>Angiotensin</em> converting enzyme (ACE) generates <em>angiotensin</em> II from <em>angiotensin</em> I, which plays a critical role in the pathophysiology of diabetic nephropathy. However, ACE2 generates <em>angiotensin</em> <em>1</em>-<em>7</em>, which may protect the kidney by attenuating the effects of <em>angiotensin</em> II, since deletion of the Ace2 gene leads to glomerulosclerosis in mice, and pharmacologic inhibition of ACE2 exacerbates experimental diabetic nephropathy. We measured ACE2 and ACE expression in renal biopsies of patients with kidney disease due to type 2 diabetes to determine if the expression pattern is specific to diabetic nephropathy. ACE2 and ACE mRNA levels were measured by real-time PCR in laser microdissected renal biopsies from <em>1</em>3 diabetic and 8 control patients. ACE2 mRNA was significantly reduced by more than half in both the glomeruli and proximal tubules of the diabetic patients compared to controls, but ACE mRNA was increased in both compartments. There was a significant parallel decrease in ACE2 protein expression, determined by immunohistochemistry, in proximal tubules, a pattern not found in <em>1</em>2 patients with focal glomerulosclerosis or <em>1</em>0 patients with chronic allograft nephropathy. Our results suggest that the kidney disease of patients with type 2 diabetes is associated with a reduction in ACE2 gene and protein expression and this may contribute to the progression of renal injury.

BACKGROUND

Angiotensin (Ang) II-induced apoptosis was reported to be mediated by different signaling molecules. Whether these molecules are either interconnected in a single pathway or constitute different and alternative cascades by which Ang II exerts its apoptotic action, is not known.

OBJECTIVE

To investigate in cultured myocytes from adult cat and rat, 2 species in which Ang II has opposite inotropic effects, the signaling cascade involved in Ang II-induced apoptosis.

RESULTS

Ang II (1 micromol/L) reduced cat/rat myocytes viability by approximately 40%, in part, because of apoptosis (TUNEL/caspase-3 activity). In both species, apoptosis was associated with reactive oxygen species (ROS) production, Ca(2+)/calmodulin-dependent protein kinase (CaMK)II, and p38 mitogen-activated protein kinase (p38MAPK) activation and was prevented by the ROS scavenger MPG (2-mercaptopropionylglycine) or the NADPH oxidase inhibitor DPI (diphenyleneiodonium) by CaMKII inhibitors (KN-93 and AIP [autocamtide 2-related inhibitory peptide]) or in transgenic mice expressing a CaMKII inhibitory peptide and by the p38MAPK inhibitor, SB202190. Furthermore, p38MAPK overexpression exacerbated Ang II-induced cell mortality. Moreover, although KN-93 did not affect Ang II-induced ROS production, it prevented p38MAPK activation. Results further show that CaMKII can be activated by Ang II or H(2)O(2), even in the presence of the Ca(2+) chelator BAPTA-AM, in myocytes and in EGTA-Ca(2+)-free solutions in the presence of the calmodulin inhibitor W-7 in in vitro experiments.

CONCLUSIONS

(1) The Ang II-induced apoptotic cascade converges in both species, in a common pathway mediated by ROS-dependent CaMKII activation which results in p38MAPK activation and apoptosis. (2) In the presence of Ang II or ROS, CaMKII may be activated at subdiastolic Ca(2+) concentrations, suggesting a new mechanism by which ROS reset the Ca(2+) dependence of CaMKII to extremely low Ca(2+) levels.

At maximally effective concentrations, vasopressin (<em>1</em>0(-<em>7</em>) M) increased myo-inositol trisphosphate (IP3) in isolated rat hepatocytes by <em>1</em>00% at 3 s and <em>1</em>50% at 6 s, while adrenaline (epinephrine) (<em>1</em>0(-5) M) produced a <em>1</em><em>7</em>% increase at 3 s and a 30% increase at 6 s. These increases were maintained for at least <em>1</em>0 min. Both agents increased cytosolic free Ca2+ [( Ca2+]i) maximally by 5 s. Increases in IP3 were also observed with <em>angiotensin</em> II and ATP, but not with glucagon or platelet-activating factor. The dose-responses of vasopressin and adrenaline on phosphorylase and [Ca2+]i showed a close correspondence, whereas IP3 accumulation was 20-30-fold less sensitive. However, significant (20%) increases in IP3 could be observed with <em>1</em>0(-9) M-vasopressin and <em>1</em>0(-<em>7</em>) M-adrenaline, which induce near-maximal phosphorylase activation. Vasopressin-induced accumulation of IP3 was potentiated by <em>1</em>0mM-Li+, after a lag of approx. <em>1</em> min. However the rise in [Ca2+]i and phosphorylase activation were not potentiated at any time examined. Similar data were obtained with adrenaline as agonist. Lowering the extracellular Ca2+ to 30 microM or 250 microM did not affect the initial rise in [Ca2+]i with vasopressin but resulted in a rapid decline in [Ca2+]i. Brief chelation of extracellular Ca2+ for times up to 4 min also did not impair the rate or magnitude of the increase in [Ca2+]i or phosphorylase a induced by vasopressin. The following conclusions are drawn from these studies. IP3 is increased in rat hepatocytes by vasopressin, adrenaline, <em>angiotensin</em> II and ATP. The temporal relationships of its accumulation to the increases in [Ca2+]i and phosphorylase a are consistent with it playing a second message role. Influx of extracellular Ca2+ is not required for the initial rise in [Ca2+]i induced by these agonists, but is required for the maintenance of the elevated [Ca2+]i.

We studied the role of aldosterone (aldo) in myocardial injury in a model of <em>angiotensin</em> (Ang) II-hypertension. Wistar rats were given <em>1</em>% NaCl (salt) to drink and randomized into one of the following groups (n = <em>1</em>0; treatment, 2<em>1</em> d): <em>1</em>) vehicle control (VEH); 2) Ang II infusion (25 ng/min, sc); 3) Ang II infusion plus the selective aldo blocker, eplerenone (epl, <em>1</em>00 mg/kg.d, orally); 4) Ang II infusion in adrenalectomized (ADX) rats; and 5) Ang II infusion in ADX rats with aldo treatment (20 micro g/kg.d, sc). ADX rats received also dexamethasone (<em>1</em>2 micro g/kg.d, sc). Systolic blood pressure increased with time in all treatment groups except the VEH group (VEH, <em>1</em>36 +/- 6; Ang II/NaCl, 203 +/- <em>1</em>2; Ang II/NaCl/epl, <em>1</em>96 +/- <em>1</em>0; Ang II/NaCl/ADX, <em>1</em>8<em>1</em> +/- <em>7</em>; Ang II/NaCl/ADX/aldo, 236 +/- 8 mm Hg). Despite similar levels of hypertension, epl and ADX attenuated the increase in heart weight/body weight induced by Ang II. Histological examination of the hearts evidenced myocardial and vascular injury in the Ang II/salt (<em>7</em> of <em>1</em>0 hearts with damage, P < 0.05 vs. VEH) and Ang II/salt/ADX/aldo groups (<em>1</em>0 of <em>1</em>0 hearts with damage, P < 0.05). Injury included arterial fibrinoid necrosis, perivascular inflammation (primarily macrophages), and focal infarctions. Vascular lesions were associated with expression of the inflammatory mediators cyclooxygenase 2 (COX-2) and osteopontin in the media of coronary arteries. Myocardial injury, COX-2, and osteopontin expression were markedly attenuated by epl treatment (<em>1</em> of <em>1</em>0 hearts with damage, P < 0.05 vs. Ang II/salt) and adrenalectomy (2 of <em>1</em>0 hearts with damage, P < 0.05 vs. Ang II/salt). Our data indicate that aldo plays a major role in Ang II-induced vascular inflammation in the heart and implicate COX-2 and osteopontin as potential mediators of the damage.

<em>Angiotensin</em>-converting enzyme 2 (ACE2) preferentially forms <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [ANG-(<em>1</em>-<em>7</em>)] from ANG II. We showed that cardiac ACE2 is elevated following treatment of coronary artery-ligated rats with AT<em>1</em> receptor blockers (ARBs). Cardiac myocytes and fibroblasts were isolated from neonatal rats to determine the molecular mechanisms for the ACE2 upregulation by ARB treatment. ANG II significantly reduced ACE2 activity and downregulated ACE2 mRNA in cardiac myocytes, effects blocked by the ARB losartan, indicating that ANG II regulates ACE2. ANG II also reduced ACE2 mRNA in cardiac fibroblasts; however, no enzyme activity was detected, reflecting the limited expression of ACE2 in these cells. Endothelin-<em>1</em> (ET-<em>1</em>) also significantly reduced myocyte ACE2 mRNA. The reduction in ACE2 mRNA by ANG II or ET-<em>1</em> was blocked by inhibitors of mitogen-activated protein kinase kinase <em>1</em>, suggesting that ANG II or ET-<em>1</em> activates extracellular signal-regulated kinase (ERK) <em>1</em>/ERK2 to reduce ACE2. Although ACE2 mRNA was not affected by ANG-(<em>1</em>-<em>7</em>), both the ANG II- and ET-<em>1</em>-mediated reductions in ACE2 mRNA were blocked by the heptapeptide. The ANG-(<em>1</em>-<em>7</em>) modulatory effect was prevented by the ANG-(<em>1</em>-<em>7</em>) receptor antagonist [D-Ala<em>7</em>]-ANG-(<em>1</em>-<em>7</em>), indicating that the ANG-(<em>1</em>-<em>7</em>) response was mediated by a specific AT(<em>1</em>-<em>7</em>) receptor. Myocyte treatment with atrial natriuretic peptide (ANP) also reversed the ACE2 mRNA downregulation by ANG II or ET-<em>1</em>, whereas treatment with ANP alone was ineffective. These results indicate that multiple hypertrophic and anti-hypertropic peptides regulate ACE2 production in myocytes, suggesting that ACE2 expression in the heart is dependent upon the compliment and concentration of regulatory molecules.

BACKGROUND

Postprandial hypertriglyceridemia and hyperglycemia are considered risk factors for cardiovascular disease. Evidence suggests that postprandial hypertriglyceridemia and hyperglycemia induce endothelial dysfunction and inflammation through oxidative stress. Statins and <em>angiotensin</em> type <em>1</em> receptor blockers have been shown to reduce oxidative stress and inflammation, improving endothelial function.

RESULTS

Twenty type 2 diabetic patients ate 3 different test meals: a high-fat meal, 75 g glucose alone, and a high-fat meal plus glucose. Glycemia, triglyceridemia, endothelial function, nitrotyrosine, C-reactive protein, intercellular adhesion molecule-<em>1</em>, and interleukin-6 were assayed during the tests. Subsequently, diabetics took atorvastatin 40 mg/d, irbesartan 300 mg/d, both, or placebo for <em>1</em> week. The 3 tests were performed again between 5 and 7 days after the start of each treatment. High-fat load and glucose alone produced a decrease in endothelial function and increases in nitrotyrosine, C-reactive protein, intercellular adhesion molecule-<em>1</em>, and interleukin-6. These effects were more pronounced when high-fat load and glucose were combined. Short-term atorvastatin and irbesartan treatments significantly counterbalanced these phenomena, and their combination was more effective than either therapy alone.

CONCLUSIONS

This study confirms an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on endothelial function and inflammation, suggesting oxidative stress as a common mediator of such an effect. Short-term treatment with atorvastatin and irbesartan may counterbalance this phenomenon; the combination of the 2 compounds is most effective.

The peroxisome proliferator activated receptor (PPARgamma) agonist rosiglitazone has been reported to yield cardiovascular benefits in patients by a mechanism that is not completely understood. We tested whether oral rosiglitazone (25 mg/kg per day, 2<em>1</em> days) treatment improves blood pressure and vascular function in a transgenic mouse expressing both human renin and human <em>angiotensin</em>ogen transgenes (R(+)A(+)). Rosiglitazone decreased systolic (<em>1</em>38+/-5 versus <em>1</em>28+/-5 mm Hg) and mean blood pressure (<em>1</em>45+/-5 versus <em>1</em>26+/-<em>7</em> mm Hg) of R(+)A(+) mice as measured by tail-cuff and indwelling carotid catheters, respectively. Relaxation of carotid arteries to acetylcholine and authentic nitric oxide, but not papaverine, was impaired in R(+)A(+) mice when compared with littermate controls (RA(-)). There were no effects of rosiglitazone on RA(-) mice; however, relaxation to acetylcholine (49+/-<em>1</em>0 versus 82+/-9% at <em>1</em>00 micromol/L) and nitric oxide (5<em>1</em>+/-<em>1</em><em>1</em> versus <em>7</em>2+/-6% at <em>1</em>0 micromol/L) was significantly improved in treated R(+)A(+) mice. Rosiglitazone treatment of R(+)A(+) mice did not alter the expression of genes, including endothelial nitric oxide synthase (eNOS), <em>angiotensin</em> <em>1</em> receptors, and preproendothelin-<em>1</em>, nor did it alter the levels of eNOS or soluble guanylyl cyclase protein. In separate studies, carotid arteries from R(+)A(+) and RA(-) mice relaxed in a concentration-dependent manner to rosiglitazone, suggesting possible PPARgamma-independent effects in the vasculature. This response was not inhibited with the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester (200 micromol/L) or the PPARgamma antagonist bisphenol A diglycidyl ether; 4,4'-isopropylidenediphenol diglycidyl ether (<em>1</em>00 micromol/L). These data suggest that in addition to potential genomic regulation caused by PPARgamma activation, the direct effect of rosiglitazone in blood vessels may contribute to the improved blood pressure and vessel function.

<em>Angiotensin</em> II (AII) is a potent stimulus for HCO3- reabsorption in the rat proximal tubule in vivo. To determine the ionic mechanism of increased HCO3- reabsorption, we have examined the effect of AII on luminal Na(+)-H+ exchange and basolateral Na+/HCO3- cotransport in perfused S<em>1</em> proximal tubules isolated from superficial nephrons of the rabbit kidney. Transporter activity was assessed by removing Na+ from both luminal and basolateral (i.e., bath) solutions and determining the rate at which intracellular pH (pHi) increased after Na+ was returned to only the lumen or only the bath. pHi was measured with the pH-sensitive fluorescent dye 2', <em>7</em>'-bis(2-carboxyethyl)-5(and 6)-carboxyfluorescein. We found that basolateral administration of <em>1</em> nM AII not only increased the rate of luminal Na(+)-H+ exchange approximately 3.5-fold but also increased the rate of basolateral Na+/HCO3- cotransport approximately 2.5-fold. 5-(N-Ethyl-N-isopropyl)amiloride (50 microM) blocked luminal Na(+)-H+ exchange before and after stimulation by AII but had no effect on basolateral Na+/HCO3- cotransport. Conversely, 4,4'-diisothiocyanato-2,2'-stilbenedisulfonate (50 microM) blocked basolateral Na+/HCO3- cotransport before and after AII but had no effect on luminal Na(+)-H+ exchange. Our data thus indicate that, at least under the conditions of our assay, AII independently stimulates the transporters responsible for both the luminal and basolateral steps of transepithelial HCO3- reabsorption.

A rapid method for isolating highly purified rat liver plasma membrane vesicles using isotonic medium and Percoll self-forming gradient centrifugation is described. The vesicles were characterized by enzyme markers and electron microscopy. The method also yielded a fraction rich in nuclei. The vesicles transported Ca2+ in an ATP-dependent manner and this was enhanced by oxalate. The Vmax for Ca2+ uptake was 0.65 +/- 0.08 nmol/mg X min, which was approximately <em>1</em>8-fold higher than for other liver plasma membrane preparations, and the Km for Ca2+ was 5.2 +/- 0.4 nM. Calcium uptake was inhibited by 40-50% in vesicles isolated from rat livers perfused for 3 min with <em>1</em>0(-<em>7</em>)M vasopressin. The half-maximally effective concentration of vasopressin was 5 X <em>1</em>0(-<em>1</em>0)M which correlates with that for raising cytosolic Ca2+ and phosphorylase a. Inhibition was not significant in vesicles from livers perfused with vasopressin for only <em>1</em> min, indicating that inhibition of the Ca2+ pump may not be involved in the rise in cytosolic Ca2+ observed at <em>1</em>-2 s with this hormone. Epinephrine (<em>1</em>0(-5)M) and <em>angiotensin</em> II (<em>1</em>0(-<em>7</em>)M) inhibited Ca2+ uptake by 3<em>1</em> +/- <em>1</em>0 and 26 +/- 5%, respectively, at 3 min. Glucagon (<em>1</em>0(-<em>7</em>)M) had no effect. It is proposed that the inhibitory action of the Ca2+-dependent hormones on the plasma membrane Ca2+ pump plays an important role in the actions of these hormones by prolonging the elevation in cytosolic Ca2+.

Our recent investigations have postulated a human umbilical vein endothelial cell (HUVEC)-associated prekallikrein activator (PKA). When prekallikrein (PK) assembles on high molecular weight kininogen on HUVEC, PK is activated to kallikrein. PKA was found in the <em>1</em>5,800 x g pellet of HUVEC lysates using an assay that measures PK activation only when bound to high molecular weight kininogen linked to microtiter plates. Sequential DEAE, wheat germ lectin affinity, and hydroxyapatite chromatography resulted in four protein bands on SDS-PAGE. One protein in the <em>7</em>3-kDa band was identified by amino acid sequencing as prolylcarboxypeptidase (PRCP). On gel filtration, PKA activity was a single homogenous peak identical in migration to the <em>7</em>3-kDa immunoblot of PRCP. Anti-PRCP inhibits PKA activity and PK activation on HUVEC. Purified PKA was blocked by diisopropyl fluorophosphate (<em>1</em> mm), phenylmethylsulfonyl fluoride (3 mm), leupeptin (<em>1</em>00 microm), antipain (IC(50) = 2 microm), HgCl(2) (IC(50) = 500 microm), Z-Pro-Pro-aldehyde-dimethyl acetate (IC(50) = <em>1</em> microm), and corn trypsin inhibitor (IC(50) = 40 nm). PKA did not correct the coagulant defect in factor XII deficient plasma, was purified from HUVEC cultured in factor XII-deficient serum, was not detected by antibody to factor XII, did not activate FXI, and was not inhibited by a neutralizing antibody to FXII. <em>Angiotensin</em> II (IC(50) = 2 microm) or bradykinin (IC(50) = <em>1</em>00 microm), but not <em>angiotensin</em> II-(<em>1</em>-<em>7</em>) or bradykinin(<em>1</em>-5), and the prolyl oligopeptidase inhibitor Fmoc-Ala-Pyr-CN (IC(50) = 50 nm) also blocked purified PKA activation of PK. The K(m) of PK activation by PRCP is 6.<em>7</em> nm. PRCP antigen is present on the membrane of fixed but not permeabilized HUVEC. PRCP appears to be a HUVEC-associated PK activator.

We hypothesized that the downregulation of Cyp2c by tumor necrosis factor (TNF) alpha contributes to hypertension and renal injury in salt-sensitive <em>angiotensin</em> hypertension. Male Sprague-Dawley rats were fed a high-salt diet (8% NaCl), and osmotic minipumps were implanted to deliver <em>angiotensin</em> II for <em>1</em>4 days. Rats were divided into 3 groups: high salt, <em>angiotensin</em> high salt, and <em>angiotensin</em> high salt administered the TNF-alpha blocker, etanercept. Arterial pressure increased from 94+/-5 to <em>1</em>48+/-<em>7</em> mm Hg during week <em>1</em> in the <em>angiotensin</em> high-salt group, whereas etanercept slowed blood pressure elevation during the first week in the treated group (90+/-2 to <em>1</em>09+/-6 mm Hg). After 2 weeks, arterial pressure increased to <em>1</em>56+/-<em>1</em><em>1</em> mm Hg in the <em>angiotensin</em> high-salt group and <em>1</em>4<em>1</em>+/-6 mm Hg in the etanercept-treated group. Albuminuria and proteinuria were significantly elevated in <em>angiotensin</em> high-salt rats and were reduced in the etanercept-treated rats. Urinary monocyte chemoattractant protein-<em>1</em> excretion significantly increased in the <em>angiotensin</em> high-salt group (2<em>7</em>5+/-4<em>7</em> versus 8<em>1</em>+/-<em>1</em>9 ng/day) and was decreased in the etanercept-treated group (<em>1</em>53+/-3<em>1</em> ng/day). <em>Angiotensin</em> high-salt rats also had a significant increase in renal monocyte/macrophage infiltration, and this was again attenuated by etanercept treatment. Renal expression of Cyp2c23 decreased, whereas renal epoxide hydrolase expression increased in <em>angiotensin</em> high-salt rats. Etanercept treatment increased Cyp2c23 expression and lowered epoxide hydrolase expression. These data suggest that TNF-alpha contributes to downregulation of Cyp2c23, blood pressure regulation, and renal injury in <em>angiotensin</em> high-salt hypertension.

The temporal and spatial expression of transforming growth factor (TGF)-beta(<em>1</em>) and connective tissue growth factor (CTGF) was assessed in the left ventricle of a myocardial infarction (MI) model of injury with and without <em>angiotensin</em>-converting enzyme (ACE) inhibition. Coronary artery ligated rats were killed <em>1</em>, 3, <em>7</em>, 28, and <em>1</em>80 days after MI. TGF-beta(<em>1</em>), CTGF, and procollagen alpha<em>1</em>(I) mRNA were localized by in situ hybridization, and TGF-beta(<em>1</em>) and CTGF protein levels by immunohistochemistry. Collagen protein was measured using picrosirius red staining. In a separate group, rats were treated for 6 months with an ACE inhibitor. There were temporal and regional differences in the expression of TGF-beta(<em>1</em>), CTGF, and collagen after MI. Procollagen alpha<em>1</em>(I) mRNA expression increased in the border zone and scar peaking <em>1</em> week after MI, whereas collagen protein increased in all areas of the heart over the <em>1</em>80 days. Expression of TGF-beta(<em>1</em>) mRNA and protein showed major increases in the border zone and scar peaking <em>1</em> week after MI. The major increases in CTGF mRNA and protein occurred in the viable myocardium at <em>1</em>80 days after MI. Long-term ACE inhibition reduced left ventricular mass and decreased fibrosis in the viable myocardium, but had no effect on cardiac TGF-beta(<em>1</em>) or CTGF. TGF-beta(<em>1</em>) is involved in the initial, acute phase of inflammation and repair after MI, whereas CTGF is involved in the ongoing fibrosis of the heart. The antifibrotic benefits of captopril are not mediated through a reduction in CTGF.

<em>Angiotensin</em>-converting enzyme 2 (ACE2) is a recently discovered homologue of <em>angiotensin</em>-converting enzyme (ACE) that is thought to counterbalance ACE. ACE2 cleaves <em>angiotensin</em> I and <em>angiotensin</em> II into the inactive <em>angiotensin</em> <em>1</em>-9, and the vasodilator and anti-proliferative <em>angiotensin</em> <em>1</em>-<em>7</em>, respectively. ACE2 is known to be present in human kidney, but no data on renal disease are available to date. Renal biopsies from 58 patients with diverse primary and secondary renal diseases were studied (hypertensive nephropathy n = 5, IgA glomerulopathy n = 8, minimal change nephropathy n = <em>7</em>, diabetic nephropathy n = 8, focal glomerulosclerosis n = 5, vasculitis n = <em>7</em>, and membranous glomerulopathy n = <em>1</em>8) in addition to <em>1</em><em>7</em> renal transplants and <em>1</em>8 samples from normal renal tissue. Immunohistochemical staining for ACE2 was scored semi-quantitatively. In control kidneys, ACE2 was present in tubular and glomerular epithelium and in vascular smooth muscle cells and the endothelium of interlobular arteries. In all primary and secondary renal diseases, and renal transplants, neo-expression of ACE2 was found in glomerular and peritubular capillary endothelium. There were no differences between the various renal disorders, or between acute and chronic rejection and control transplants. ACE inhibitor treatment did not alter ACE2 expression. In primary and secondary renal disease, and in transplanted kidneys, neo-expression of ACE2 occurs in glomerular and peritubular capillary endothelium. Further studies should elucidate the possible protective mechanisms involved in the de novo expression of ACE2 in renal disease.

Despite the evidence that <em>angiotensin</em>-converting enzyme (ACE)2 is a component of the renin-<em>angiotensin</em> system (RAS), the influence of ACE2 on <em>angiotensin</em> metabolism within the kidney is not well known, particularly in experimental models other than rats or mice. Therefore, we investigated the metabolism of the <em>angiotensins</em> in isolated proximal tubules, urine, and serum from sheep. Radiolabeled [(<em>1</em>25)I]ANG I was hydrolyzed primarily to ANG II and ANG-(<em>1</em>-<em>7</em>) by ACE and neprilysin, respectively, in sheep proximal tubules. The ACE2 product ANG-(<em>1</em>-9) from ANG I was not detected in the absence or presence of ACE and neprilysin inhibition. In contrast, the proximal tubules contained robust ACE2 activity that converted ANG II to ANG-(<em>1</em>-<em>7</em>). Immunoblots utilizing an NH(2) terminal-directed ACE2 antibody revealed a single <em>1</em>20-kDa band in proximal tubule membranes. ANG-(<em>1</em>-<em>7</em>) was not a stable product in the tubule preparation and was rapidly hydrolyzed to ANG-(<em>1</em>-5) and ANG-(<em>1</em>-4) by ACE and neprilysin, respectively. Comparison of activities in the proximal tubules with nonsaturating concentrations of substrate revealed equivalent activities for ACE (ANG I to ANG II: 248 +/- <em>1</em><em>7</em> fmol x mg(-<em>1</em>) x min(-<em>1</em>)) and ACE2 [ANG II to ANG-(<em>1</em>-<em>7</em>): 253 +/- <em>1</em><em>1</em> fmol x mg(-<em>1</em>) x min(-<em>1</em>)], but lower neprilysin activity [ANG II to ANG-(<em>1</em>-4): <em>1</em><em>1</em>9 +/- 24 fmol x mg(-<em>1</em>) x min(-<em>1</em>); P < 0.05 vs. ACE or ACE2]. Urinary metabolism of ANG I and ANG II was similar to the proximal tubules; soluble ACE2 activity was also detectable in sheep serum. In conclusion, sheep tissues contain abundant ACE2 activity that converts ANG II to ANG-(<em>1</em>-<em>7</em>) but does not participate in the processing of ANG I into ANG-(<em>1</em>-9).

Tissue accumulation of circulating prorenin results in <em>angiotensin</em> generation, but could also, through binding to the recently cloned (pro)renin receptor, lead to <em>angiotensin</em>-independent effects, like p42/p44 mitogen-activated protein kinase (MAPK) activation and plasminogen-activator inhibitor (PAI)-<em>1</em> release. Here we investigated whether prorenin exerts <em>angiotensin</em>-independent effects in neonatal rat cardiomyocytes. Polyclonal antibodies detected the (pro)renin receptor in these cells. Prorenin affected neither p42/p44 MAPK nor PAI-<em>1</em>. PAI-<em>1</em> release did occur during coincubation with <em>angiotensin</em>ogen, suggesting that this effect is <em>angiotensin</em> mediated. Prorenin concentration-dependently activated p38 MAPK and simultaneously phosphorylated HSP2<em>7</em>. The latter phosphorylation was blocked by the p38 MAPK inhibitor SB203580. Rat microarray gene (n=4800) transcription profiling of myocytes stimulated with prorenin detected 260 regulated genes (P<0.00<em>1</em> versus control), among which genes downstream of p38 MAPK and HSP2<em>7</em> involved in actin filament dynamics and (cis-)regulated genes confined in blood pressure and diabetes QTL regions, like Syntaxin-<em>7</em>, were overrepresented. Quantitative real-time RT-PCR of <em>7</em> selected genes (Opg, Timp<em>1</em>, Best5, Hsp2<em>7</em>, pro-Anp, Col3a<em>1</em>, and Hk2) revealed temporal regulation, with peak levels occurring after 4 hours of prorenin exposure. This regulation was not altered in the presence of the renin inhibitor aliskiren or the <em>angiotensin</em> II type <em>1</em> receptor antagonist eprosartan. Finally, pilot 2D proteomic differential display experiments revealed actin cytoskeleton changes in cardiomyocytes after 48 hours of prorenin stimulation. In conclusion, prorenin exerts <em>angiotensin</em>-independent effects in cardiomyocytes. Prorenin-induced stimulation of the p38 MAPK/HSP2<em>7</em> pathway, resulting in alterations in actin filament dynamics, may underlie the severe cardiac hypertrophy that has been described previously in rats with hepatic prorenin overexpression.