BACKGROUND

Endothelial dysfunction is an initial step in the pathogenesis of cardiovascular diseases. Since we previously identified the G protein-coupled receptor Mas as a receptor for <em>angiotensin</em> (Ang)-(<em>1</em>-<em>7</em>), a heptapeptide with endothelium-dependent vasorelaxant properties, we investigated whether alterations on the Ang-(<em>1</em>-<em>7</em>)/Mas axis alter endothelial function.

RESULTS

Ang-(<em>1</em>-<em>7</em>)-mediated relaxation of murine wild-type mesenteric arteries was equally impaired in both wild-type arteries pretreated with the Ang-(<em>1</em>-<em>7</em>) receptor blocker, A<em>7</em><em>7</em>9, and arteries isolated from Mas-deficient mice. Importantly, the response to the endothelium-dependent vasorelaxant, bradykinin (BK), and acetylcholine (ACh) effects were comparably inhibited, while endothelium-independent vessel relaxation by sodium nitroprusside was unaltered in these vessels. Hypothesizing endothelial dysfunction, we proved the in-vivo relevance of the ex-vivo findings investigating mesenteric properties after <em>1</em> week of minipump infusion of A<em>7</em><em>7</em>9 in wild-type mice. Both BK- and ACh-induced relaxation were significantly impaired in wild-type vessels of pretreated animals. A<em>7</em><em>7</em>9-induced impairment of endothelial function was confirmed in vitro, since BK-mediated nitric oxide (NO) release was increased by Ang-(<em>1</em>-<em>7</em>) and blunted by A<em>7</em><em>7</em>9 pretreatment in primary human endothelial cell cultures.

CONCLUSIONS

Our data highlight a pivotal role for the receptor Mas in preserving normal vascular relaxation. Consequently, Mas agonists arise as a promising tool in the treatment of cardiovascular diseases characterized by endothelial dysfunction.

Following cerebral ischemia, i.v. infusion of <em>angiotensin</em> II increases cerebral edema and mortality. <em>Angiotensin</em> type <em>1</em> receptor blockage should therefore improve acute cerebral ischemia. Left middle cerebral artery occlusion (<em>1</em>20 min) followed by reperfusion was performed with the thread method under halothane anesthesia in Sprague-Dawley rats. Olmesartan (<em>angiotensin</em> type <em>1</em> receptor blocker; 0.0<em>1</em> or 0.<em>1</em>mumol/kg/h) was infused i.p. for <em>7</em> days following middle cerebral artery occlusion followed by reperfusion. Stroke index score, infarct volume, specific gravity, and brain <em>angiotensin</em> II and matrix metalloproteinases were quantified in the ischemic and non-ischemic hemispheres. Olmesartan treatment improved stroke index score, infarct volume, and cerebral edema in our cerebral ischemia model. In particular, stroke index score, infarct volume, and cerebral edema were reduced even with a low dose of olmesartan that did not decrease blood pressure. Paralleling these effects on cerebral ischemia, olmesartan treatment also reduced the reactive upregulation in brain <em>angiotensin</em> II, matrix metalloproteinase-2, matrix metalloproteinase-9, and membrane type <em>1</em>-matrix metalloproteinase in the ischemic area. <em>Angiotensin</em> type <em>1</em> receptor stimulation may be one of the important factors that cause cerebral edema following cerebral ischemia, and that its inhibition may be of therapeutic advantage in cerebral ischemia.

Bradykinin (BK) liberates nitric oxide, prostacyclin, and tissue plasminogen activator from endothelial cells. We hypothesized that BK B2 receptor knockout (KO) mice (BKB2R(-/-)) have increased thrombosis risk. Paradoxically, the BKB2R(-/-) mice have long bleeding times and delayed carotid artery thrombosis, <em>7</em>8 +/- 6.<em>7</em> minutes, versus 3<em>1</em> +/- 2.<em>7</em> minutes in controls. The mechanism(s) for thrombosis protection was sought. In BKB2R(-/-) plasma coagulation, fibrinolysis and anticoagulant proteins are normal except for an increased prekallikrein and decreased factor XI. BKB2R(-/-) mice have elevated BK <em>1</em>-5 (<em>1</em>60 +/- <em>7</em>5 fmol/mL, vs 44 +/- 29 fmol/mL in controls) and <em>angiotensin</em> II (<em>1</em>82 +/- 4<em>1</em> pg/mL, vs 49 +/- <em>7</em> pg/mL in controls). Ramipril treatment shortens vessel occlusion time. BKB2R(-/-) mice have elevated plasma 6-keto-PGF<em>1</em>alpha (666 +/- 232 ng/mL, vs 23 +/- 5.3 ng/mL in controls) and serum nitrate (6<em>1</em> +/- 5.3 microM, vs 24 +/- <em>1</em>.8 microM in controls). Treatment with L-NAME (NG-mono-methyl-L-arginine ester) or nimesulide shortens the thrombosis time. BKB2R(-/-) mice have increased <em>angiotensin</em> receptor 2 (AT2R) mRNA and protein expression. Treatment with an AT2R antagonist, PD<em>1</em>23 3<em>1</em>9, normalizes the thrombosis time and nitrate and 6-keto-PGF<em>1</em>alpha. The long bleeding times in BKB2R(-/-) mice also correct with L-NAME and nimesulide therapy. In BKB2R(-/-) mice, <em>angiotensin</em> II binding to an overexpressed AT2R promotes thromboprotection by elevating nitric oxide and prostacyclin. These investigations indicate a pathway for thrombosis risk reduction via the plasma kallikrein/kinin and renin <em>angiotensin</em> systems.

To identify genes influencing blood pressure response to an <em>angiotensin</em> II receptor blocker, single nucleotide polymorphisms identified by genome-wide association analysis of the response to candesartan were validated by opposite direction associations with the response to a thiazide diuretic, hydrochlorothiazide. We sampled <em>1</em>98 white and <em>1</em>93 blacks with primary hypertension from opposite tertiles of the race-sex-specific distributions of age-adjusted diastolic blood pressure response to candesartan. There were 285 polymorphisms associated with the response to candesartan at P(<em>1</em>0(-4) in whites. A total of 2<em>7</em>3 of the 285 polymorphisms, which were available for analysis in a separate sample of <em>1</em>96 whites, validated for opposite direction associations with the response to hydrochlorothiazide (Fisher χ(2) <em>1</em>-sided P=0.02). Among the 2<em>7</em>3 polymorphisms, those in the chromosome <em>1</em><em>1</em>q2<em>1</em> region were the most significantly associated with response to candesartan in whites (eg, rs<em>1</em><em>1</em>02082<em>1</em> near FUT4, P=8.98 × <em>1</em>0(-<em>7</em>)), had the strongest opposite direction associations with response to hydrochlorothiazide (eg, rs3<em>7</em>58<em>7</em>85 in GPR83, P=<em>7</em>.<em>1</em>0 × <em>1</em>0(-3)), and had the same direction associations with response to candesartan in the <em>1</em>93 blacks (eg, rs<em>1</em>6924603 near FUT4, P=<em>1</em>.52 × <em>1</em>0(-2)). Also notable among the 2<em>7</em>3 polymorphisms was rs<em>1</em><em>1</em>649420 on chromosome <em>1</em>6 in the amiloride-sensitive sodium channel subunit SCNN<em>1</em>G involved in mediating renal sodium reabsorption and maintaining blood pressure when the renin-<em>angiotensin</em> system is inhibited by candesartan. These results support the use of genomewide association analyses to identify novel genes predictive of opposite direction associations with blood pressure responses to inhibitors of the renin-<em>angiotensin</em> and renal sodium transport systems.

Release of endothelin-<em>1</em>, a novel potent vasoconstrictor peptide originally isolated from endothelial cells, from cultured bovine endothelial cells has been shown to be stimulated by arginine vasopressin and <em>angiotensin</em> II. To elucidate the cellular mechanism by which endothelin-<em>1</em> is released by these vasoconstrictors, we tested the effects of several compounds on the agonist-induced endothelin-<em>1</em> release and studied the changes of cytosolic free Ca2+ concentrations and phosphoinositide breakdown by these agonists in cultured bovine endothelial cells. Protein kinase C inhibitors (H-<em>7</em>, staurosporine), an intracellular Ca2+ chelator, and an inhibitor of phospholipase C (neomycin), all abolished the agonist-induced endothelin-<em>1</em> release, whereas the Ca2+ channel blocker nicardipine was ineffective. Although synthetic <em>1</em>,2-diglyceride (diolein) dose dependently stimulated endothelin-<em>1</em> release, downregulation of protein kinase C after pretreatment with phorbol ester resulted in decreased effects to increase endothelin-<em>1</em> release by the agonists. Both arginine vasopressin and <em>angiotensin</em> II induced immediate and transient increases in intracellular Ca2+ levels of fura-2-loaded endothelial cells as well as formation of inositol trisphosphate; the agonist-induced intracellular Ca2+ increases were not affected either by nicardipine or by chelating extracellular Ca2+. The arginine vasopressin- and <em>angiotensin</em> II-induced intracellular Ca2+ increases, inositol trisphosphate formation, and endothelin-<em>1</em> release were completely abolished by V<em>1</em>-receptor antagonist and saralasin, respectively. It is concluded that arginine vasopressin and <em>angiotensin</em> II stimulate the release of endothelin-<em>1</em> by a common mechanism, involving receptor-mediated mobilization of intracellular Ca2+ and activation of protein kinase C in endothelial cells.

Young-onset hypertension has a stronger genetic component than late-onset counterpart; thus, the identification of genes related to its susceptibility is a critical issue for the prevention and management of this disease. We carried out a two-stage association scan to map young-onset hypertension susceptibility genes. The first-stage analysis, a genome-wide association study, analyzed <em>1</em><em>7</em>5 matched case-control pairs; the second-stage analysis, a confirmatory association study, verified the results at the first stage based on a total of <em>1</em>,008 patients and <em>1</em>,008 controls. Single-locus association tests, multilocus association tests and pair-wise gene-gene interaction tests were performed to identify young-onset hypertension susceptibility genes. After considering stringent adjustments of multiple testing, gene annotation and single-nucleotide polymorphism (SNP) quality, four SNPs from two SNP triplets with strong association signals (-log(<em>1</em>0)(p>><em>7</em>) and <em>1</em>3 SNPs from 8 interactive SNP pairs with strong interactive signals (-log(<em>1</em>0)(p)>8) were carefully re-examined. The confirmatory study verified the association for a SNP quartet 2<em>1</em>9 kb and 495 kb downstream of LOC3443<em>7</em><em>1</em> (a hypothetical gene) and RASGRP3 on chromosome 2p22.3, respectively. The latter has been implicated in the abnormal vascular responsiveness to endothelin-<em>1</em> and <em>angiotensin</em> II in diabetic-hypertensive rats. Intrinsic synergy involving IMPG<em>1</em> on chromosome 6q<em>1</em>4.2-q<em>1</em>5 was also verified. IMPG<em>1</em> encodes interphotoreceptor matrix proteoglycan <em>1</em> which has cation binding capacity. The genes are novel hypertension targets identified in this first genome-wide hypertension association study of the Han Chinese population.

The renin-<em>angiotensin</em> system (RAS) plays key roles throughout the cardiovascular continuum, and blockade of this system--either through <em>angiotensin</em>-converting enzyme (ACE) inhibition or through <em>angiotensin</em> II type <em>1</em> (AT(<em>1</em>)) receptor antagonism--now occupies a central place in the management of cardiovascular disease (CVD). Understanding of the RAS has expanded in recent years with the identification of new pathways for formation of <em>angiotensin</em> II and novel effector peptides, such as <em>angiotensin</em>-(<em>1</em>-<em>7</em>), which may constitute new therapeutic targets. A substantial proportion of the benefits of ACE inhibitors, including vasodilation, improvements in endothelial function, and inhibition of cell proliferation, appear to be attributable to decreases in <em>angiotensin</em> II and increases in bradykinin. In addition, however, there is evidence that other mechanisms, such as modulation of ACE signaling, may also contribute. <em>Angiotensin</em> receptor blockers (ARBs) selectively block AT(<em>1</em>) receptors and allow unopposed stimulation of AT(2) receptors, with potentially beneficial vasodilatory, anti-inflammatory, and antiproliferative effects. As a result, these agents share many of the clinical benefits of ACE inhibitors. Both ACE inhibitors and ARBs have been shown to exert multiple antiatherogenic actions, and to reduce clinical events in high-risk participants; their use is recommended in current guidelines for the secondary prevention of CVD.

<em>1</em>. Sodium nitroprusside is a potent relaxant of smooth muscles with a predominantly tonic response, e.g. rat aorta contracted by noradrenaline, <em>angiotensin</em> II, Phe2-Lys8-vasopressin, BaC<em>1</em>(2), or KC<em>1</em>, and guinea-pig tracheal smooth muscle contracted by carbachol. 2. Smooth muscle preparations from the splanchnic region and with varying degrees of phasic contractility are less sensitive and develop tachyphylaxis (portal vein, duodenum of the rat) or are unresponsive to sodium nitroprusside (vas deferens, uterus of the rat). 3. Cardiac auricles of the guinea pig are not affected by sodium nitroprusside in either frequency or amplitude or spontaneous contractions. 4. Sdium nitroprusside causes a parallel shift of the dose-response curve of rat aorta to noradrenaline to the right and reduces the maximum response. 5. The drug has no blocking or stimulant effect on alpha- or beta-adrenoceptors, respectively. 6. Sodium nitroprusside inhibits the contractile response of calcium-depleted depolarized rat aorta to extra-cellular calcium. Like verapamil, it inhibits the increment in 45calcium uptake of rabbit aorta elicited by K+. Sodium nitroprusside significantly reduced 45calcium binding by microsomes prepared from rabbit aorta. <em>7</em>. Rabbit aorta was incubated with lanthanum chloride to prevent calcium influx; sodium nitroprusside reduced the maintained rapid contraction phase in response to noradrenaline which is believed to be based on the intracellular activation of calcium. 8. In rat aorta, cellular cAMP and ATP levels were not found to be affected by the drug. 9. Rabbit aorta, "skinned" by glycerination is unresponsive to sdoium nitroprusside. <em>1</em>0. It is concluded that sodium nitropruside acts on exictation-contraction coupling predominantly in tonic smooth muscle by interfering with both the influx and the intracellular activation of calcium.

BACKGROUND

We wished to determine the role of NO in exercise-induced metabolic forearm vasodilation.

RESULTS

Young healthy volunteers (n = <em>1</em><em>1</em>) underwent static handgrip exercise (4 to 5 kg, 3 minutes). Forearm blood flow (FBF) measured by strain plethysmography increased from 4.<em>1</em> +/- 0.<em>7</em> mL.min-<em>1</em>.<em>1</em>00 mL-<em>1</em> at rest to 9.8 +/- <em>1</em>.2 mL.min-<em>1</em>.<em>1</em>00 mL-<em>1</em> immediately after exercise and gradually decreased thereafter. Exercise was repeated after intrabrachial artery infusion of NG-monomethyl-L-arginine (L-NMMA) at 4.0 mumol/min for 5 minutes. L-NMMA did not alter blood pressure and heart rate. L-NMMA decreased FBF at rest to 2.9 +/- 0.4 mL.min-<em>1</em>.<em>1</em>00 mL-<em>1</em> (P < .0<em>1</em>), peak FBF immediately after exercise to <em>7</em>.2 +/- 0.<em>7</em> mL.min-<em>1</em>.<em>1</em>00 mL-<em>1</em> (P < .0<em>1</em>), and FBF during the mid to late phase of metabolic vasodilation (P < .0<em>1</em>). Calculated oxygen consumption during peak exercise was comparable before and after L-NMMA. Intra-arterially infused L-arginine (<em>1</em>0 mg/min, 5 minutes) reversed the inhibitory effect of L-NMMA. To determine the effect of the decrease in resting FBF on exercise-induced hyperemia, we normalized FBF after exercise by resting FBF. The percent increases in FBF after exercise from resting FBF were similar before and after L-NMMA. Furthermore, we examined the effect of intra-arterially infused <em>angiotensin</em> II on FBF at rest and after exercise (n = <em>7</em>). <em>Angiotensin</em> II decreased FBF at rest from 3.<em>1</em> +/- 0.3 to <em>1</em>.8 +/- 0.3 mL.min-<em>1</em>.<em>1</em>00 mL-<em>1</em> (P < .0<em>1</em>), peak FBF after exercise from 8.<em>1</em> +/- 0.5 to 5.6 +/- 0.5 mL.min-<em>1</em>.<em>1</em>00 mL-<em>1</em> (P < .0<em>1</em>), and FBF during the mid to late phase of metabolic vasodilation. The effects of L-NMMA and <em>angiotensin</em> II on FBF at rest and exercise were similar.

CONCLUSIONS

Our results suggest that L-NMMA decreased FBF after exercise largely by decreasing resting FBF. These results suggest that NO may not play a significant role in exercise-induced metabolic arteriolar vasodilation in the forearm of healthy humans.

<em>1</em>. Recently it has been shown that injection of <em>angiotensin</em> II into the anterior diencephalon causes the rat to drink water. In the present experiments the dipsogenic action of a number of other substances including substances related to <em>angiotensin</em> was tested.2. Injection of 0.00<em>1</em> Goldblatt u. renin into the <em>angiotensin</em>-sensitive region causes the water-replete rat to drink. Drinking is slower in onset and continues for longer than after injection of <em>angiotensin</em> II.3. Synthetic tetradecapeptide renin substrate and <em>angiotensin</em> I were as effective as <em>angiotensin</em> II at causing water-replete rats to drink.4. beta-aspartic acid(<em>1</em>)-valine(5)-<em>angiotensin</em> II was also fully effective; but the D-arginine substituted octapeptide was much less effective.5. The (2-8) heptapeptide retained about 50% of the dipsogenic activity of the octapeptide, whereas the absence of phenylalanine at the other end of the peptide chain in the (<em>1</em>-<em>7</em>) heptapeptide results in an inactive compound.6. The (3-8) hexapeptide and the (4-8) pentapeptide, both of which have phenylalanine at the end of the chain, and the (<em>1</em>-4) and (5-8) tetrapeptide fragments of <em>angiotensin</em> II showed only a slight action on intake of water.<em>7</em>. Kallikrein, bradykinin, adenosine-3'5-cyclic phosphate, vasopressin and oxytocin caused no drinking when injected into the <em>angiotensin</em>-sensitive region.8. It is concluded that the requirements for the dipsogenic activity of <em>angiotensin</em> are the same as those for its other biological actions with the qualification that the precursor peptides are also active, presumably because they give rise to <em>angiotensin</em> II locally.

We studied the structural and the functional alterations of perivascular adipose tissue (PVAT) in hypertension with spontaneously hypertensive rats (SHR). Measured with dual energy X-ray absorptiometry, a smaller body fat mass and a greater lean mass were found in SHR than in Wistar-Kyoto (WKY) rats, while body weight was comparable between them. In the thoracic PVAT, the density and the total number of brown adipocytes were greater in SHR than in WKY rats, while the cross section area of PVAT was similar between them. In functional assessment, four types of vessel preparations (with either intact PVAT or intact endothelium, or with both, or without both) were employed. Vessels with intact PVAT from SHR contracted more to phenylephrine than that from WKY rats, while vessels without PVAT exhibited comparable contractile response to phenylephrine between SHR and WKY rats. Both endothelium-dependent and -independent components of PVAT-associated attenuation of phenylephrine-induced contraction were reduced in SHR as compared with that of WKY rats. Bioassay experiments were carried out to assess the transferable relaxation factor from the PVAT. Transfer of bathing solution incubated with PVAT-intact vessel caused less relaxation in SHR recipients than in WKY rats, and the relaxation response was abolished by D-Ala(<em>7</em>)-<em>angiotensin</em>-(<em>1</em>-<em>7</em>), an <em>angiotensin</em>-(<em>1</em>-<em>7</em>) receptor antagonist. In summary, PVAT-associated inhibition of vessel contractile response to agonist was impaired in SHR, and the impairment involved both endothelium-dependent and -independent mechanisms. The functional impairment observed in SHR PVAT may be related to changes in adipocyte composition but not to reduced PVAT mass in SHR.

BACKGROUND

Growth arrest-specific gene 6 (Gas6) and its binding partner, the receptor tyrosine kinase Axl, are important mediators in experimental nephritis. The authors tested whether the Gas6/Axl signaling pathway participates in human renal diseases.

METHODS

The authors compared 26 human renal specimens from patients with IgA nephritis, acute diffuse immune complex glomerulonephritis, acute lupus nephritis, antineutrophil cytoplasmic antibody--associated glomerulonephritis, acute transplant rejection, and normal renal tissue. Because reactive oxygen species are pivotal in inflammation, the authors tested whether the Axl/Gas6 expression is influenced by NADPH oxidase in vitro.

RESULTS

Gas6 and Axl immunofluorescence was barely detectable in normal kidney. However, in disease Axl was copiously expressed in the small vessel media, glomeruli, distal tubules, and collecting ducts. Similarly, Gas6 was upregulated in the small vessel intima and media, all segments of the renal tubules, the brush border, and glomeruli. Gas6 and Axl upregulation was a prominent but nonspecific finding in these renal diseases. Cultured rat vascular smooth muscle cells and immortalized human mesangial cells were stimulated with <em>angiotensin</em> (Ang) II (<em>1</em> x <em>1</em>0(-<em>7</em>) mol/L) for 6 or <em>1</em>8 hours. Confocal microscopy and Western blot showed Ang II-dependent Gas6 and Axl expression. An antisense probe against the p22 phox unit of NADPH-oxidase suppressed Ang II-induced Gas6 and Axl expression. In addition, in p4<em>7</em> phox knockout cells Ang II-induced Gas6 and Axl expression were blocked.

CONCLUSIONS

GAS6/Axl signaling is involved in human renal disease. The Ang II-induced Gas6 and Axl expression may be dependent on NADPH-oxidase. Gas6 and Axl are important signaling molecules in human renal disease and may be potential therapeutic targets.

Halothane is a well known cerebral vasodilator that can produce dangerous increases in intracranial pressure (ICP) in certain neurosurgical patients. It has been suggested that isoflurane may be a less potent cerebral vasodilator. The authors therefore undertook a direct comparison of the effects of halothane and isoflurane on cerebral blood flow (CBF), cerebral vascular resistance (CVR), intracranial pressure, and cerebral metabolic rate for oxygen (CMRO2). Studies were carried out in normocarbic mechanically ventilated cats, using the intracarotid <em>1</em>33Xe injection technique to measure CBF. The effects of three doses were examined: 0.5, <em>1</em>.0, and <em>1</em>.5 MAC, studied in the continued presence of <em>7</em>5% N2O. Autoregulation also was tested at <em>1</em>.0 MAC (plus <em>7</em>5% N2O) by recording CBF and CVR before and after elevation of blood pressure with <em>angiotensin</em>. Both agents had similar effects on blood pressure and ICP. However, while halothane produced significant increases in CBF at all doses, with values of 6<em>1</em> +/- 5 ml X <em>1</em>00 g-<em>1</em> X min-<em>1</em> (<em>1</em>23 +/- 8% of control, mean +/- SE) at <em>1</em>.0 MAC, isoflurane anesthesia caused no significant changes in CBF at any level, (e.g., 48 +/- 8 ml X <em>1</em>00 g-<em>1</em> X min-<em>1</em> or 94 +/- <em>1</em>2% of control at <em>1</em>.0 MAC). Both drugs produced dose-related decreases in CVR, but the changes were greater with halothane, e.g., CVR at <em>1</em>.0 MAC halothane = <em>1</em>.46 +/- 0.20 mmHg X ml-<em>1</em> X <em>1</em>00 g X min (4<em>7</em> +/- <em>7</em>% of control) compared with 2.23 +/- 0.40 mmHg X ml-<em>1</em> X <em>1</em>00 g X min (<em>7</em>2 +/- 9% of control).(ABSTRACT TRUNCATED AT 250 WORDS)

Ang-(<em>1</em>-<em>7</em>) (<em>angiotensin</em>-<em>1</em>-<em>7</em>), a peptide product of the recently described ACE (<em>angiotensin</em>-converting enzyme) homologue ACE2, opposes the harmful actions of AngII (<em>angiotensin</em> II) in cardiovascular tissues, but its role in liver disease is unknown. The aim of the present study was to assess plasma levels of Ang-(<em>1</em>-<em>7</em>) in human liver disease and determine its effects in experimental liver fibrosis. <em>Angiotensin</em> peptide levels were measured in cirrhotic and non-cirrhotic patients with hepatitis C. The effects of Ang-(<em>1</em>-<em>7</em>) on experimental fibrosis were determined using the rat BDL (bile-duct ligation) model. Liver histology, hydroxyproline quantification and expression of fibrosis-related genes were assessed. Expression of RAS (renin-<em>angiotensin</em> system) components and the effects of Ang-(<em>1</em>-<em>7</em>) were examined in rat HSCs (hepatic stellate cells). In human patients with cirrhosis, both plasma Ang-(<em>1</em>-<em>7</em>) and AngII concentrations were markedly elevated (P<0.00<em>1</em>). Non-cirrhotic patients with hepatitis C had elevated Ang-(<em>1</em>-<em>7</em>) levels compared with controls (P<0.05), but AngII concentrations were not increased. In BDL rats, Ang-(<em>1</em>-<em>7</em>) improved fibrosis stage and collagen Picrosirius Red staining, and reduced hydroxyproline content, together with decreased gene expression of collagen <em>1</em>A<em>1</em>, alpha-SMA (smooth muscle actin), VEGF (vascular endothelial growth factor), CTGF (connective tissue growth factor), ACE and mas [the Ang-(<em>1</em>-<em>7</em>) receptor]. Cultured HSCs expressed AT<em>1</em>Rs (AngII type <em>1</em> receptors) and mas receptors and, when treated with Ang-(<em>1</em>-<em>7</em>) or the mas receptor agonist AVE 099<em>1</em>, produced less alpha-SMA and hydroxyproline, an effect reversed by the mas receptor antagonist A<em>7</em><em>7</em>9. In conclusion, Ang-(<em>1</em>-<em>7</em>) is up-regulated in human liver disease and has antifibrotic actions in a rat model of cirrhosis. The ACE2/Ang-(<em>1</em>-<em>7</em>)/mas receptor axis represents a potential target for antifibrotic therapy in humans.

Superoxide radical (O2-) is ubiquitously critical to the bioactivity of endothelial nitric oxide. In <em>angiotensin</em>-dependent hypertension, vascular O2- levels rise and impede endothelium/nitric oxide-dependent vascular relaxation. We have reported that the major O2- source in the rabbit aorta is adventitial fibroblast phagocyte-like NADPH oxidase and shown that <em>angiotensin</em> (Ang) II treatment of adventitial fibroblasts causes a concentration-dependent increase in particulate NADPH-dependent O2-. From cultured rabbit aortic adventitial fibroblasts treated or not treated with Ang II, we prepared particulate fractions and measured lucigenin-enhanced chemiluminescence. Because [Sar<em>1</em>,Thr8]-Ang II, a generalized antagonist of Ang II and plausible inhibitor of the conversion of Ang II, reversed Ang II (<em>1</em>0 nmol/L)-induced NADH- and NADPH-dependent O2- to basal levels, we tested the effect of the inhibitor of aminopeptidase N, amastatin (<em>1</em>0 micromol/L), and found no effect on Ang II-stimulated O2-. Ang(<em>1</em>-<em>7</em>), Ang III, and Ang IV also were not effective in stimulating O2- levels at concentrations similar to those of Ang II. Kinetic analysis showed a rise in NADPH oxidase O2- production in response to Ang II, which peaks at 3 hours and returns to basal levels by <em>1</em>6 hours. p6<em>7</em>phox, a cytosolic factor, appears to be affected at both the level of transcription and protein synthesis because actinomycin and cycloheximide individually inhibited the observed effect. A partial sequence of p6<em>7</em>phox was recovered by reverse transcriptase from mRNA harvested from cultured rabbit aortic adventitial fibroblasts. Furthermore, the p6<em>7</em>phox mRNA transcript in aortic fibroblasts is induced by Ang II before the peak of NADPH oxidase by Northern analysis and ribonuclease protection assays. These data suggest that Ang II stimulates NAD(P)H oxidase O2- generation in fibroblasts of aortic adventitia via transcriptional activation of p6<em>7</em>phox. These data also provide preliminary evidence for the regulation of factors of the NADPH oxidase and potentially provide a novel means by which to abrogate the development of O2(-)-dependent hypertension.

<em>Angiotensin</em>-(<em>1</em>-<em>1</em>2) [ANG-(<em>1</em>-<em>1</em>2)] is a newly identified peptide detected in a variety of rat tissues, including the brain. To determine whether brain ANG-(<em>1</em>-<em>1</em>2) participates in blood pressure regulation, we treated male adult (mRen2)2<em>7</em> hypertensive rats (24-28 wk of age) with Anti-ANG-(<em>1</em>-<em>1</em>2) IgG or Preimmune IgG via an intracerebroventricular cannula for <em>1</em>4 days. Immunoneutralization of brain ANG-(<em>1</em>-<em>1</em>2) lowered systolic blood pressure (-43 +/- 8 mmHg on day 3 and -26 +/- <em>7</em> mmHg on day <em>1</em>0 from baseline, P < 0.05). Water intake was lower on intracereroventricular day 6 in the Anti-ANG-(<em>1</em>-<em>1</em>2) IgG group, accompanied by higher plasma osmolality on day <em>1</em>3, but there were no differences in urine volume, food intake, or body weight during the 2-wk treatment. In Preimmune IgG-treated animals, there were no significant changes in these variables over the 2-wk period. The antihypertensive effects produced by endogenous neutralization of brain ANG-(<em>1</em>-<em>1</em>2) suggest that ANG-(<em>1</em>-<em>1</em>2) is functionally active in brain pathways regulating blood pressure.

The purpose of this study was to identify changes in venomotor tone in the chronic low-dose <em>angiotensin</em> II (Ang II) model of hypertension and to establish the contribution of sympathetic nerve activation to these venomotor tone changes. Male Sprague-Dawley rats were acclimatized to a 0.4% or 2.0% NaCl diet for <em>7</em> days and then catheterized to allow chronic and repeated measures of arterial pressure, central venous pressure, and mean circulatory filling pressure (MCFP), an index of venous smooth muscle tone, in conscious undisturbed rats. After 4 days of recovery and a 3-day control period, an Ang II or physiological saline-filled osmotic minipump was implanted subcutaneously to deliver Ang II (<em>1</em>50 ng/kg per minute) or vehicle control for <em>1</em>4 days. MCFP was measured in duplicate before and after acute ganglionic blockade with hexamethonium (30 mg/kg i.v.) on control day 2 and Ang II infusion on days <em>1</em>, 3, <em>7</em>, and <em>1</em>4. Blood volume was also measured on these days and was unchanged for the duration of the study in all of the groups. Arterial pressure was increased for the duration of Ang II infusion in rats on both 0.4% and 2% NaCl diets, but the increase was significantly greater in the 2% NaCl group and completely abolished by hexamethonium. MCFP was significantly increased for the entire Ang II infusion period only in rats fed 2% NaCl, and this increase was completely abolished by hexamethonium. We conclude that the combination of chronic low-dose Ang II infusion and high dietary salt intake engages the sympathetic nervous system to increase venomotor tone.

Bird fancier's lung (BFL) is one of the most common types of hypersensitivity pneumonitis. Nevertheless, the criteria for diagnosing this condition are not standardized. The current study is an in-depth investigation into the clinical characteristics of BFL in the largest series examined for this purpose by a single group, to our knowledge, taking into account the acute, subacute, or chronic clinical presentation. From <em>1</em>9<em>7</em><em>7</em> to 2003, BFL was diagnosed in 86 patients using a homogeneous protocol. Data from the clinical history and physical examination were analyzed, as well as the results from the following complementary examinations: laboratory analyses, specific serum IgG antibodies determination, chest X-ray, chest computed tomography (CT), pulmonary function testing, immediate hypersensitivity skin testing, delayed cutaneous hypersensitivity testing, bronchofibroscopy with bronchoalveolar lavage (BAL) and/or transbronchial biopsy, bronchial challenge testing, and surgical lung biopsy. In addition, clinical and epidemiologic characteristics were determined in a control group of 60 pigeon breeders who did not meet the diagnostic criteria of BFL. Eighty-six patients (2<em>1</em> men and 65 women) with a mean age of 4<em>7</em> years were studied. Seven (8%) patients were younger than <em>1</em>5 years of age at the time of the diagnosis. In 3 cases, the disease was caused by exposure to feather-filled bedding. Nearly <em>1</em> in 5 patients was diagnosed in the chronic phase of the disease. The mean diagnostic delay was <em>1</em>.6 years overall, and 3.2 years in patients diagnosed in the chronic phase of the disease. Among the <em>1</em><em>7</em>% of patients with chronic disease, the mean interval from initiation of exposure to diagnosis was <em>1</em>6 years, a higher value than in the acute or subacute presentation forms. Dyspnea and cough were the most common clinical symptoms (98% and 82%, respectively), and nearly 25% had grade III or IV dyspnea at diagnosis. Only <em>1</em>8% of patients experienced chest tightness, a symptom classically considered to be frequent in this condition. Erythrocyte sedimentation rate was elevated (>30 mm/h) in 44% of patients. Urinary calcium was elevated in 20% of patients. <em>Angiotensin</em>-converting enzyme was not elevated in any of the patients in which it was measured. Lactate dehydrogenase increases were found in 5<em>1</em>% of patients. Specific IgG antibodies to avian antigens were documented in 92% of BFL patients, but also in 8<em>7</em>% of pigeon breeder controls. The most frequent radiologic finding was an interstitial pattern in <em>7</em>9% of patients. Common chest CT features were ground glass areas (68%) and a mosaic pattern (6<em>1</em>%); areas of emphysema were found in <em>7</em>/4<em>1</em> (<em>1</em><em>7</em>%) patients, 5 of whom had never smoked. Two patients had a CT pattern of pulmonary fibrosis indistinguishable from idiopathic pulmonary fibrosis. Immediate hypersensitivity skin testing with bird sera and pigeon bloom was positive in <em>7</em>8% and <em>1</em>00% of BFL patients, respectively, and in 64% and 88% of control pigeon breeders, respectively. Almost one-third of the patients (29%) presented an anergic response on delayed cutaneous hypersensitivity testing. Restrictive ventilatory impairment was the most frequent functional pattern (<em>7</em><em>7</em>%), although 9% and 4% showed a pure obstructive and mixed pattern, respectively. The carbon monoxide diffusing capacity was decreased (<80% of the predicted value) in 85% of cases. Forty-one percent of patients had PaO2 <60 mm Hg at diagnosis when blood gas analysis was performed. Lymphocytosis (>20% lymphocytes) was documented in 83% of patients who underwent BAL, with a similar frequency in the 3 presentation forms: <em>7</em>0% acute, 89% subacute, and 85% chronic. In addition, inversion of the CD4/CD8 ratio ((<em>1</em>) was observed in 62% of the patients, but 38% of cases showed a CD4 predominance. The characteristic triad of histopathologic findings in hypersensitivity pneumonitis was found in only 9% of patients undergoing transbronchial biopsy, but at least <em>1</em> of these findings was seen in 69%. Surgical lung biopsy was undertaken in <em>1</em>4/86 (<em>1</em>6%) patients; the complete triad was observed in 50% and at least <em>1</em> finding in <em>1</em>00%. In 54/86 (63%) patients, the diagnosis was confirmed by bronchial challenge testing, a test with a sensitivity of 92% and specificity of <em>1</em>00%. BFL is a potentially severe disease that can progress to respiratory failure secondary to pulmonary fibrosis or chronic obstructive pulmonary disease, as a form of chronic occupational respiratory disease. Respiratory symptoms in exposed patients, including children and adults who have only <em>1</em> pet bird at home, should raise the suspicion of BFL. Diagnosis in the chronic phase is frequent, and the delay to diagnosis was greatest in these cases. Elevated urinary calcium, lactate dehydrogenase, and erythrocyte sedimentation rate in a bird fancier may constitute a combined marker for suspected BFL. Chest CT frequently discloses emphysema and a pattern of idiopathic pulmonary fibrosis in some patients. An anergic response on delayed cutaneous hypersensitivity testing is not infrequent. The presentation with respiratory failure and the predominance of CD4 T lymphocytes in some patients' BAL are both remarkable. Lymphocytosis on BAL also persists in the chronic phase of the disease. Bronchial challenge testing has a high diagnostic yield, and surgical lung biopsy is not needed to reach the final diagnosis in the vast majority of cases.

Using B(2) kinin receptor gene knockout mice (B(2)(-/-)), we tested the hypothesis that (l) lack of B(2) receptors may affect blood pressure and cardiac function and aggravate cardiac remodeling after myocardial infarction (MI), and (2) kinins partially mediate the cardiac beneficial effect of <em>angiotensin</em>-converting enzyme inhibitors (ACEi) or <em>angiotensin</em> II type <em>1</em> receptor antagonists (AT(<em>1</em>)-ant), whereas lack of B(2) receptors may diminish this cardioprotective effect. Chronic heart failure (HF) was induced by MI, which was caused by coronary artery ligation in both B(2)(-/-) and <em>1</em>29/SvEvTac mice (wild-type control, B(2)(+/+)). An ACEi (ramipril, 2.5 mg/kg/d) or AT(<em>1</em>)-ant (L-<em>1</em>58809, 3 mg/kg/d) was given <em>1</em> week after MI and was continued for <em>1</em>2 weeks. Left ventricular (LV) ejection fraction, cardiac output (CO), diastolic LV dimension (LVDd), and LV mass were evaluated by echocardiography. Myocyte cross-sectional area and interstitial collagen fraction were studied histopathologically. We found that basal blood pressure and cardiac function were similar in B(2)(+/+) and B(2)(-/-) mice. After MI, development of HF and remodeling were also similar between the 2 strains. The ACEi improved cardiac function and remodeling in both strains; however, its effects were attenuated in B(2)(-/-) mice (respective values for B(2)(+/+) versus B(2)(-/-) mice: overall increase in ejection fraction, 64+/-<em>1</em>0% versus 2<em>1</em>+/-5% [P<0.0<em>1</em>]; increase in CO, 69+/-<em>1</em><em>7</em>% versus 23+/-9% [P<0.0<em>1</em>]; overall decrease in LVDd, -24+/-3% versus -<em>7</em>+/-4% [P<0.0<em>1</em>]; and decrease in LV mass, -38+/-3% versus -6+/-6% [P<0.0<em>1</em>]). AT(<em>1</em>)-ant had a beneficial cardiac effect similar to that produced by ACEi, and this effect was also diminished in B(2)(-/-) mice (respective values for B(2)(+/+) versus B(2)(-/-) mice: overall increase in ejection fraction, 46+/-<em>1</em>0% versus 25+/-9% [P<0.0<em>1</em>]; increase in CO, 44+/-<em>1</em>4% versus <em>1</em>5+/-5% [P<0.0<em>1</em>]; overall decrease in LVDd, -<em>1</em>4+/-4% versus -6+/-3% [P<0.0<em>1</em>]; and decrease in LV mass, -33+/-4 versus -<em>1</em>6+/-<em>7</em>% [P<0.0<em>1</em>]). The effect of ACEi or AT(<em>1</em>)-ant on myocyte cross-sectional area was similar between strains; however, their effect on the interstitial collagen fraction was diminished in B(2)(-/-) mice. We concluded that (<em>1</em>) lack of B(2) kinin receptors does not affect cardiac phenotype or function, either under normal physiological conditions or during the development of HF; and (2) kinins acting via the B(2) receptor play an important role in the cardioprotective effect of ACEi and AT(<em>1</em>)-ant.

The nonpeptide AVE-099<em>1</em>, which has been reported as a selective ligand for the <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [ANG-(<em>1</em>-<em>7</em>)] receptor Mas, has actions similar to those attributed to the cardioprotective product of the renin-<em>angiotensin</em> system, ANG-(<em>1</em>-<em>7</em>). In this study, we evaluated the cardiac effects of AVE-099<em>1</em> in normal and infarcted male Wistar rats. Myocardial infarction was induced by left coronary artery ligation. At the end of the treatment, the Langendorff technique was used to analyze cardiac function. Left ventricle serial sections were dyed with Gomori trichrome stain to quantify the infarcted area. In normal hearts, AVE-099<em>1</em> produced a significant decrease in perfusion pressure and an increase in systolic tension, rate of tension rise and fall (+/-dT/dt), and heart rate. These effects were completely blocked by the perfusion of the hearts with a solution containing the selective ANG-(<em>1</em>-<em>7</em>) antagonist A-<em>7</em><em>7</em>9. N(G)-nitro-l-arginine methyl ester treatment abolished the AVE-099<em>1</em>-induced vasodilation in isolated hearts. AVE-099<em>1</em> significantly attenuated the decrease in systolic tension (sham operated, <em>1</em>3.00 +/- <em>1</em>.02 g; infarction, <em>7</em>.<em>1</em>8 +/- 0.66 g; AVE treated, 9.23 +/- <em>1</em>.05 g, n = 5), +dT/dt, -dT/dt, and heart rate induced by myocardial infarction. Infarction-induced vasoconstriction was completely prevented by AVE-099<em>1</em> treatment. Furthermore, AVE-099<em>1</em> significantly decreased the infarcted area (6.98 +/- <em>1</em>.0<em>1</em> vs. 3.94 +/- <em>1</em>.04 mm(2) in AVE-treated rats). These data indicate that the compound AVE-099<em>1</em> produces beneficial effects in isolated perfused rat hearts involving the ANG-(<em>1</em>-<em>7</em>) receptor Mas and the release of nitric oxide. In addition, our results indicate that AVE-099<em>1</em> attenuates postischemic heart failure.

The renin-<em>angiotensin</em> system (RAS) plays a major role in the pathophysiology of cardiovascular disorders. <em>Angiotensin</em> II (Ang-II), the final product of this pathway, is known for its vasoconstrictive and proliferative effects. <em>Angiotensin</em>-converting enzyme 2 (ACE2), a newly discovered homolog of ACE, plays a key role as the central negative regulator of the RAS. It diverts the generation of vasoactive Ang-II into the vasodilatory and growth inhibiting peptide <em>angiotensin</em>(<em>1</em>-<em>7</em>) [Ang(<em>1</em>-<em>7</em>)], thereby providing counter-regulatory responses to neurohormonal activation. There is substantial experimental evidence evaluating the role of ACE2/Ang(<em>1</em>-<em>7</em>) in hypertension, heart failure, and atherosclerosis. In this review, we aim to focus on the conceptual facts of the ACE2-Ang(<em>1</em>-<em>7</em>) axis with regards to clinical implications and therapeutic targets in cardiovascular disorders, with emphasis on the potential therapeutic role in cardiovascular diseases.

BACKGROUND

The renin-<em>angiotensin</em> system (RAS) plays a role in the pathogenesis of ARDS, <em>Angiotensin</em> II (Ang-II) contributing to the pathogenesis of inflammation and fibrogenesis. <em>Angiotensin</em>-(<em>1</em>-<em>7</em>) (Ang-(<em>1</em>-<em>7</em>)) may antagonize the effects of Ang-II. This study was aimed at evaluating the potential for Ang-(<em>1</em>-<em>7</em>) to reduce injury, inflammation and fibrosis in an experimental model of ARDS in the acute and late phases.

METHODS

Male Sprague Dawley rats underwent an instillation of 0.<em>1</em> M hydrochloric acid (HCl, 2.5 ml/kg) into the right bronchus. In an acute ARDS study, acid-injured rats were subjected to high stretch mechanical ventilation (<em>1</em>8 ml/kg) for 5 h and randomized to receive an intravenous infusion of either vehicle (saline), Ang-(<em>1</em>-<em>7</em>) at low dose(0.2<em>7</em> μg/kg/h) (ALD), or high dose (60 μg/kg/h) (AHD) starting simultaneously with injury or 2 h afterwards. Arterial blood gas analysis and bronchoalveolar lavage (BAL) were performed to assess the injury. For the late ARDS study, after HCl instillation rats were randomized to either vehicle or high dose Ang-(<em>1</em>-<em>7</em>) (300 μg/kg/day) infused by mini osmotic pumps for two weeks, and lung hydroxyproline content measured.

RESULTS

In the acute ARDS study, Ang-(<em>1</em>-<em>7</em>) led to a significant improvement in oxygenation (PaO2/FiO2 : vehicle 359 ± 86; ALD 436 ± <em>7</em>2; AHD 44 442 ± 56; ANOVA p = 0.00<em>7</em>) and reduced white blood cells counts (vehicle 4,5<em>1</em>9 ± 2,234; ALD 2,496 ± 62<em>1</em>; AHD 2,<em>7</em>44 ± <em>1</em><em>1</em>9/mm(3); ANOVA p = 0.004). Only treatment with high dose Ang-(<em>1</em>-<em>7</em>) reduced inflammatory cell numbers in BAL (vehicle <em>1</em>2<em>7</em> ± 34; AHD 96 ± 34/ μl; p = 0.033). Interestingly also delayed administration of Ang-(<em>1</em>-<em>7</em>) was effective in reducing injury. In later ARDS, Ang-(<em>1</em>-<em>7</em>) decreased hydroxyproline content (649 ± 202 and <em>1</em>,<em>1</em><em>1</em><em>7</em> ± 29<em>7</em> μg/lung; p < 0.05).

CONCLUSIONS

<em>Angiotensin</em>-(<em>1</em>-<em>7</em>), decreased the severity of acute lung injury and inflammation induced by combined acid aspiration and high stretch ventilation. Furthermore, continuous infusion of Ang-(<em>1</em>-<em>7</em>) reduced lung fibrosis 2 weeks following acid aspiration injury. These results call for further research on Ang-(<em>1</em>-<em>7</em>) as possible therapy for ARDS.

Histone deacetylases (HDACs) are transcriptional corepressors. Our recent study demonstrated that HDAC4 protein specifically increases in mesenteric artery from spontaneous hypertensive rats (SHR) compared with Wistar Kyoto rats (WKY). Vascular inflammation is important for pathogenesis of hypertension. We examined whether HDAC4 affects vascular inflammatory responses and promotes hypertension. In vivo, blood pressure, reactive oxygen species (ROS) production, and VCAM-<em>1</em> expression in isolated mesenteric artery were elevated in young SHR (<em>7</em> wk old) compared with age-matched WKY, which were prevented by long-term treatment of SHR with an HDACs inhibitor, trichostatin A (TSA; 500 μg·kg(-<em>1</em>)·day(-<em>1</em>) for 3 wk). In isolated mesenteric artery, the increased <em>angiotensin</em> II-induced contraction in SHR was reversed by TSA. The endothelium-dependent relaxation induced by ACh in SHR was augmented by TSA. In cultured rat mesenteric arterial smooth muscle cells (SMCs), expression of HDAC4 mRNA and protein was increased by TNF-α (<em>1</em>0 ng/ml). TSA (<em>1</em>0 μM, pretreatment for 30 min) inhibited VCAM-<em>1</em> expression and NF-κB phosphorylation induced by TNF (<em>1</em>0 ng/ml, 24 h or 20 min) in SMCs. HDAC4 small interfering RNA inhibited TNF-induced monocyte adhesion, VCAM-<em>1</em> expression, transcriptional activity of NF-κB, and ROS production in SMCs. The present results demonstrated that proinflammatory effects of HDACs may mediate the further development of hypertension in SHR. It is also suggested in cultured vascular SMCs that TNF-induced HDAC4 mediates vascular inflammation likely via VCAM-<em>1</em> induction through ROS-dependent NF-κB activation.

Many of the actions of <em>angiotensin</em> II (Ang II) are mediated by <em>angiotensin</em> type <em>1</em> receptors (AT<em>1</em>), of which there are 2 pharmacologically indistinguishable subtypes (AT<em>1</em>A and AT<em>1</em>B). The purpose of this study was to evaluate the effect of an AT<em>1</em>A homozygous deletion (AT<em>1</em>A-/-) on vascular reactivity. AT<em>1</em>A-/- mice and control littermates (AT<em>1</em>A+/+) were infused with vehicle (saline) or Ang II (<em>1</em>000 ng x kg(-<em>1</em>) x min(-<em>1</em>)) for <em>7</em> days by osmotic pumps. Systolic pressure was increased in AT<em>1</em>A+/+ mice (Delta45+/-8 mm Hg, P<0.000<em>1</em>) but unchanged in AT<em>1</em>A-/- mice (Delta5+/-3 mm Hg, P>0.<em>1</em>3) on day <em>7</em>. The carotid artery response to the vasodilators acetylcholine (ACh), nitroprusside, and papaverine and to the vasoconstrictors phenylephrine, U466<em>1</em>9, 5-hydroxytryptamine (5-HT), and KCl were not different between vehicle-infused AT<em>1</em>A+/+ and AT<em>1</em>A-/- animals. Carotid relaxation to ACh was impaired and contraction to 5-HT was increased in Ang II-infused AT<em>1</em>A+/+ mice. Ang II did not affect carotid responses in AT<em>1</em>A-/- mice. Superoxide, measured by lucigenin (5 micromol/L), and hydroethidine staining were not different between AT<em>1</em>A+/+ and AT<em>1</em>A-/- mice after vehicle or Ang II infusion, suggesting that it was not contributing to the altered ACh and 5-HT responses. The Rho-kinase inhibitor Y-2<em>7</em>632 (<em>1</em> micromol/L) attenuated the 5-HT response in both vehicle- and Ang II-infused AT<em>1</em>A+/+ mice. Moreover, concentration-dependent relaxation to Y-2<em>7</em>632 and RhoA protein expression were not different in vehicle- or Ang II-infused AT<em>1</em>A+/+. These data demonstrate that the AT<em>1</em>A receptor is required for Ang II-induced changes in carotid artery function.