Ym Pinto

Angiotensin II Type 1 Receptor A1166C Gene Polymorphism Is Associated With an Increased Response to Angiotensin II in Human Arteries

An adenine/cytosine (A/C) base substitution at position 1166 in the angiotensin II type 1 receptor (AT(1)R) gene is associated with the incidence of essential hypertension and increased coronary artery vasoconstriction. However, it is still unknown whether this polymorphism is associated with a difference in angiotensin II responsiveness. Therefore, we assessed whether the AT(1)R polymorphism is associated with different responses to angiotensin II in isolated human arteries. Furthermore, we evaluated whether inhibition of the renin-angiotensin system modifies the effect of the AT(1)R polymorphism. One hundred twelve patients who were undergoing coronary artery bypass graft surgery were prospectively randomized to receive an ACE inhibitor or a placebo for 1 week before surgery. Excess segments of the internal mammary artery were exposed to angiotensin II (0.1 nmol/L to 1 micromol/L) and KCl (60 mmol/L) in organ bath experiments. Patients homozygous for the C allele (n=17) had significantly greater angiotensin II responses (percentage of this maximal KCl-induced response) than did patients genotyped with AA+AC (n=95, P<0.05). Although ACE inhibition increased the response to angiotensin II, the difference in the response to angiotensin II, between CC and AA+AC patients remained intact in ACE inhibitor-treated patients. These results indicate increased responses to angiotensin II in patients with the CC genotype. The mechanism is preserved during ACE inhibition, which in itself also increased the response to angiotensin II. This reveals that the A1166C polymorphism may be in linkage disequilibrium with a functional mutation that alters angiotensin II responsiveness, which may explain the described relation between this polymorphism and cardiovascular abnormalities.

Angiotensin-(1–7) Is a Modulator of the Human Renin-Angiotensin System

The renin-angiotensin system is important for cardiovascular homeostasis. Currently, therapies for different cardiovascular diseases are based on inhibition of angiotensin-converting enzyme (ACE) or angiotensin II receptor blockade. Inhibition of ACE blocks metabolism of angiotensin-(1-7) to angiotensin-(1-5) and can lead to elevation of angiotensin-(1-7) levels in plasma and tissue. In animal models, angiotensin-(1-7) itself causes or enhances vasodilation and inhibits vascular contractions to angiotensin II. The function of angiotensin-(1-5) is unknown. We investigated whether angiotensin-(1-7) and angiotensin-(1-5) inhibit ACE or antagonize angiotensin-induced vasoconstrictions in humans. ACE activity in plasma and atrial tissue was inhibited by angiotensin-(1-7) up to 100%, with an IC(50) of 3.0 and 4.0 micromol/L, respectively. In human internal mammary arteries, contractions induced by angiotensin I and II and the non-ACE-specific substrate [Pro(11),D-Ala(12)]-angiotensin I were antagonized by angiotensin-(1-7) (10(-5) mol/L) in a noncompetitive way, with a 60% inhibition of the maximal response to angiotensin II. Contractions to ACE-specific substrate [Pro(10)]-angiotensin I were also inhibited, an effect only partly accounted for by antagonism of angiotensin II. Angiotensin-(1-5) inhibited plasma ACE activity with a potency equal to that of angiotensin I but had no effect on arterial contractions. In conclusion, angiotensin-(1-7) blocks angiotensin II-induced vasoconstriction and inhibits ACE in human cardiovascular tissues. Angiotensin-(1-5) only inhibits ACE. These results show that angiotensin-(1-7) may be an important modulator of the human renin-angiotensin system.

Myogenic constriction is increased in mesenteric resistance arteries from rats with chronic heart failure: instantaneous counteraction by acute AT1 receptor blockade

Increased vascular resistance in chronic heart failure (CHF) has been attributed to stimulated neurohumoral systems. However, local mechanisms may also importantly contribute to set arterial tone. Our aim, therefore, was to test whether pressure‐induced myogenic constriction of resistance arteries in vitro – devoid of acute effects of circulating factors – is increased in CHF and to explore underlying mechanisms. At 12 weeks after coronary ligation‐induced myocardial infarction or SHAM‐operations in rats, we studied isolated mesenteric arteries for myogenic constriction, determined as the active constriction (% of passive diameter) in response to stepwise increase in intraluminal pressure (20 – 160 mmHg), in the absence and presence of inhibitors of potentially involved modulators of myogenic constriction. We found that myogenic constriction in mesenteric arteries from CHF rats was markedly increased compared to SHAM over the whole pressure range, the difference being most pronounced at 60 mmHg (24±2 versus 4±3%, respectively, P<0.001). Both removal of the endothelium as well as inhibition of NO production (L‐NG‐monomethylarginine, 100 μM) significantly increased myogenic constriction (+16 and +25%, respectively), the increase being similar in CHF‐ and SHAM‐arteries (P=NS). Neither endothelin type A (ETA)‐receptor blockade (BQ123, 1 μM) nor inhibition of perivascular (sympathetic) nerve conduction (tetrodotoxin, 100 nM) affected the myogenic response in either group. Interestingly, increased myogenic constriction in CHF was fully reversed after angiotensin II type I (AT1)‐receptor blockade (candesartan, 100 nM; losartan, 10 μM), which was without effect in SHAM. In contrast, neither angiotensin‐converting enzyme (ACE) inhibition (lisinopril, 1 μM; captopril, 10 μM) or AT2‐receptor blockade (PD123319, 1 μM), nor inhibition of superoxide production (superoxide dismutase, 50 U ml−1), TXA2‐receptor blockade (SQ29,548, 1 μM) or inhibition of cyclooxygenase‐derived prostaglandins (indomethacin, 10 μM) affected myogenic constriction. Sensitivity of mesenteric arteries to angiotensin II (10 nM – 100 μM) was increased (P<0.05) in CHF (pD2 7.1±0.4) compared to SHAM (pD2 6.2±0.3), while the sensitivity to KCl and phenylephrine was not different. Our results demonstrate increased myogenic constriction in small mesenteric arteries of rats with CHF, potentially making it an important target for therapy in counteracting increased vascular resistance in CHF. Our results further suggest active and instantaneous participation of AT1‐receptors in increased myogenic constriction in CHF, involving increased sensitivity of AT1‐receptors rather than apparent ACE‐mediated local angiotensin II production.

Increased kallikrein expression protects against cardiac ischemia

Multiple indirect lines of evidence point at a cardioprotective role for enhanced bradykinin formation. In particular, the inhibition of angiotensin‐converting enzyme, also known as kininase II, can protect against cardiac ischemia, putatively via accumulation of bradykinin. To address whether an increase in kinin formation is sufficient to protect against cardiac ischemia, we studied transgenic rats harboring the human tissue kallikrein gene TGR(hKLK1) under the control of the metallothionein promoter, which drives expression of the transgene in various organs including the heart. We subjected the isolated hearts from transgenic rats and their transgene negative littermates to ex vivo regional cardiac ischemia and reperfusion. During the experiment, the hearts were treated with either vehicle or the specific bradykinin type 2 receptor antagonist HOE 140 (10‐9 M). In the transgenic rats, overflow of nucleotide breakdown products upon reperfusion was significantly less (455±54 nmol/min/g in transgene negative rats vs. 270±57 nmol/min/g in the transgenic rats, P<0.05). Systolic dP/dt at the end of ischemia was significantly higher (P<0.05) in the transgenic rats than in the transgene negative controls, despite a similar reduction in total coronary flow during ischemia. These differences were abolished in the rat hearts where HOE140 was co‐infused. This finding suggests that a chronic increase in tissue kallikrein can increase bradykinin levels sufficiently to protect against cardiac ischemia and that this effect is brought about by a rapid mechanism, rather than by long term structural alterations.

Dissociation of blood pressure reduction from end-organ damage in TGR(mREN2)27 transgenic hypertensive rats

Objective Since the biochemical disturbance underlying hypertension may be an important determinant of patient outcome, we compared the effects of early treatment with different antihypertensive drugs on end-organ damage in the TGR(mREN2)27 transgenic rat (REN-2). In these REN-2 rats, hypertension is primarily caused by increased activity of the tissue renin–angiotensin system. Design and methods Seven-week-old REN-2 rats were either untreated or treated orally with an optimal daily dose of carvedilol (30 mg/kg), hydralazine (30 mg/kg), losartan (10 mg/kg) or quinapril (15 mg/kg). Nontransgenic littermates served as normotensive controls. After 11 weeks of treatment, we determined plasma norepinephrine concentrations, left ventricular atrial natriuretic factor messenger RNA and cardiac and vascular function and hypertrophy. Results Chronic treatment with carvedilol and hydralazine significantly decreased blood pressure to a similar level but failed to normalize it, whereas both losartan and quinapril completely normalized blood pressure. Despite a blood pressure reduction in all treatment groups, only losartan, quinapril and hydralazine preserved endothelial function, while carvedilol did not. Furthermore, losartan and quinapril prevented cardiac and medial hypertrophy. The expression of atrial natriuretic factor messenger RNA paralleled the hemodynamic changes. Plasma norepinephrine levels were normalized by losartan or quinapril but remained increased after carvedilol and hydralazine treatment. Conclusions In REN-2 hypertensive rats, end-organ damage can be prevented by both inhibition of the angiotensin converting enzyme and blockade of the angiotensin II type 1 receptor, but not by merely lowering blood pressure. When blood pressure is not fully normalized, the effects on end-organs are clearly dissociated from the antihypertensive effects.

Increased risk for ischaemic events is related to combined RAS polymorphism

OBJECTIVE To determine whether the angiotensin converting enzyme (ACE) and the angiotensin II type 1 receptor (AT1R A1166C) gene polymorphism interact to increase the risk of ischaemic events, and whether this can be explained by the progression of angiographically defined coronary atherosclerosis. DESIGN Prospective defined substudy of the lipid lowering regression trial (REGRESS). SETTING University hospital. PATIENTS 885 male patients with stable coronary artery disease. MAIN OUTCOME MEASURES Incidence of ischaemic events during a two year follow up; serial quantitative coronary arteriography (mean segment diameter and minimum obstruction diameter) at baseline and after two years. RESULTS Patients who carried both the ACE-DD and AT1R-CC genotype had significantly more ischaemic events during the two year follow up than those carrying other genotype combinations (p = 0.035, Mantel-Haenszel test for linear association). There was no association between the two genotypes and mean segment diameter or minimum obstruction diameter at baseline or after two years. CONCLUSIONS The suggestion that ACE-DD and AT1R-CC genotypes interact to increase the risk of ischaemic events is confirmed. However, this increased risk was not accompanied by increased progression of angiographically defined coronary atherosclerosis.

beta-adrenergic signal transduction following carvedilol treatment in hypertensive cardiac hypertrophy

OBJECTIVE Treatment with the beta-blocker carvedilol leads to an improvement of outcome and ejection fraction in heart failure. These effects occur without affecting the number of beta-adrenergic receptors, as determined in right ventricular biopsies from patients with heart failure. This study was aimed at investigating the effects of carvedilol on beta-adrenergic signal transduction alterations in a model of left ventricular pressure overload, which is characterized by sympathetic activation and a desensitized beta-adrenergic signal transduction. METHODS Transgenic rats with overexpression of renin [TG(mREN2)27] were treated with carvedilol (30 micrograms/kg) or held under control conditions and were compared with Sprague-Dawley rats. Myocardial beta-adrenoceptors (125I-labeled iodocyanopindolol binding), Gi alpha (pertussis toxin labeling), Gs alpha-activity (reconstitution into cyc--S49 membranes) and adenylyl cyclase activity were measured. Blood pressure and heart rate, increase in heart rate during sacrifice and pressure rate products were determined. RESULTS beta-Adrenoceptors were downregulated and Gi alpha-protein levels were significantly increased, producing a desensitization of basal, isoprenaline- and guanine nucleotide-stimulated adenylyl cyclase activity compared to controls. Carvedilol reduced heart rate, blood pressure and pressure rate product in TG(mREN2)27. Carvedilol did not restore biochemical alterations, but even further reduced beta-adrenoceptor numbers and adenylyl cyclase. It exhibited a two affinity state, guanine nucleotide-sensitive binding to cardiac beta-adrenergic receptors similar to isoprenaline but different from metoprolol. CONCLUSIONS Carvedilol did not restore beta-adrenergic signal transduction at concentrations producing antiadrenergic effects in vivo. This effect might be due to an atypical guanine nucleotide-dependent interaction with beta-adrenergic receptors. Thus, ancillary properties could explain the recently reported beneficial effects in patients with heart failure independent from an upregulation of beta-adrenergic receptors.

Identification of a specific pattern of downregulation in expression of isoforms of vascular endothelial growth factor in dilated cardiomyopathy

Recent work suggests that myocardial hypoxia or ischaemia are also pathophysiologic factors in idiopathic dilated cardiomyopathy (IDC).1 Besides several other factors (increased wall stress, endothelial dysfunction, decreased coronary reserve), the observed decreased capillarisation in IDC, disproportionate to the rate of hypertrophy, may further contribute to this oxygen demand–supply mismatch. The reason for this seemingly decreased angiogenic capacity remains unclear, however a role for microvascular abnormalities in heart failure is now recognised.2 Hypoxia is the key factor in the induction of vascular endothelial growth factor (VEGF). Increased expression of VEGF causes angiogenesis, and expression level of VEGF could therefore mediate the capillarisation in IDC. Recently, data have been reported on this issue,3 and the authors found a downregulation of VEGF165 and VEGF189 isoforms. The VEGF121 however was not investigated, although it has been suggested that this isoform in particular has powerful angiogenic capacities. Additionally, it is unclear whether VEGF expression is related to the severity of the disease. We analysed 28 patients with IDC. Patients had enlarged left ventricular end diastolic and systolic diameters (LVEDD 69 (2.1) mm, LVESD 61 (2.6) mm), and decreased left ventricular ejection fraction (LVEF) (0.27 (0.03)), and elevated wedge (15 (1.9) mm Hg) and left ventricular end diastolic pressures (14.1 (2.7) mm Hg). Echocardiography did not reveal cardiac disease in the 10 brain dead subjects, who served as controls. Endomyocardial biopsy taken from the right ventricle showed cardiomyocyte hypertrophy and interstitial …

Recombinant Semliki Forest virus as a vector system for fast and selective in vivo gene delivery into balloon-injured rat aorta

Previously, we demonstrated that recombinant Semliki Forest virus (SFV) vector rapidly and selectively transfers genes into cultured vascular smooth muscle cells (VSMC), leaving endothelial cells (EC) unaffected. From this, we hypothesized that recombinant SFV in vivo only transfers genes into the media of balloon-injured but not intact vessel, that gene expression in VSMC is fast, and that the specificity of SFV for VSMC is caused by specific binding sites. To address these hypotheses, we studied the time course of in vivo SFV-LacZ and Ad-LacZ expression in balloon-injured rat aorta. In addition, the fusion characteristics of fluorescent pyrene-labeled SFV were explored in cultured VSMC and EC. In intact aorta, no LacZ expression was found in the intima or media at 24 h. In contrast, in denuded aorta, LacZ expression was detected in as early as 12 h after incubation. LacZ expression was predominantly present in the media. Ad-LacZ expression started after 12 h, but was predominantly present in the adventitia. Ad-LacZ expression in the media started after 72 h. In vitro transfection with SFV showed that fusion was higher and, moreover, saturable in VSMC as compared with EC, indicating the presence of specific SFV binding sites on VSMC, but not EC. From this we conclude that in vivo selectivity of SFV in balloon-injured vessels is based on the removal of the endothelium, which results in accessibility of VSMC in the media that carry specific binding sites for the SFV vector.

The ACE gene polymorphism: the good, the bad and the ugly.

See article by Perticone et al. ([3], pages 192–199) in this issue. After its first entry into the scientific stage [1] the deletion polymorphism of the Angiotensin Converting Enzyme presented cardiovascular researchers with a grateful opportunity to explore genetic mechanisms in cardiovascular disease. However, after a Good start, conflicting results gave the genotype a Bad reputation, and the stage now looks rather Ugly. After having discovered how promiscuously ACE behaves as an enzyme, it became apparent that the genotype was not much more faithful, being associated with an enormous variety of cardiovascular disorders, ranging from an association with myocardial infarction, vascular changes, even to issues like the ability to increase one’s physical performance [2]. In this issue of Cardiovascular Research Perticone et al. [3] report in a carefully executed study an association between the Angiotensin Converting Enzyme (ACE) deletion type gene and the prevalence of left ventricular hypertrophy in a population of untreated italian hypertensives. One of the most striking findings was that the deletion type allele of ACE was related to a more eccentric type of hypertrophy. This data further extends earlier observations suggesting a relation between the ACE genotype and left ventricular hypertrophy [4] and the relation between the ACE genotype and the … * Corresponding author. Tel.: +31-50-363-2810; fax: +31-50-363-2812.