Volume 1, Number 4, December 2000

POPULAR
TOPICPAPERThe losartan renal protection study – rationale, study design and baseline characteristics of RENAAL (Reduction of Endpoints in NIDDM with the Angiotensin II Antagonist Losartan)
Barry M Brenner, Mark E Cooper, Dick de Zeeuw, Jean-Pierre Grunfeld, William F Keane, Kiyoshi Kurokawa, Janet B McGill, William E Mitch, Hans Henrik Parving, Guiseppe Remuzzi, Arthur B Ribeiro, Mark D Schluchter, Duane Snavely, Zhongxin Zhang, Roger Simpson, Denise Ramjit, Shahnaz Shahinfar, for the RENAAL Study Investigators

The RENAAL Study is a double-blind, placebo-controlled trial to evaluate the renal protective effects of losartan in Type 2 diabetic patients with nephropathy. The study has enrolled 1513 patients and is expected to continue for 3.5 years after the last patient has been entered. Eligible patients must have a urinary albumin:creatinine ratio of at least 300 mg/g and serum creatinine between 1.3 to 3.0 mg/dL. Eligible hypertensive or normotensive patients are randomised to receive either losartan or placebo, in addition to their existing antihypertensive therapy. Medications that block angiotensin production or action, are excluded. The primary endpoint is a composite of the time to first event of doubling of serum creatinine, end-stage renal disease, or death; secondary endpoints include cardiovascular events, progression of renal disease, and changes in proteinuria; tertiary endpoints include quality of life, healthcare resource utilisation, and amputations. Patients include Caucasians (48.6%), Blacks (15.2%), Asians (16.7%), and Hispanics (18.2%). Baseline urinary albumin:creatinine ratio and serum creatinine levels average 1867 mg/g and 1.9 mg/dL, respectively. Mean systolic and diastolic blood pressures are 153 and 82 mmHg, respectively. RENAAL will document whether blockade of the AII receptor with losartan produces clinical benefits in patients with Type 2 diabetes and nephropathy.

JRAAS 2000;1:328-335.

POPULAR
TOPICPAPERThe pharmacological potency of various AT₁ antagonists assessed by Schild regression technique in man
Gustav G Belz, Kerstin Breithaupt-Grögler, Raunhild Butzer, Winfried Fuchs, Christian Hausdorf, Christian Mang

Rationale: A quantitative technique was used to compare the pharmacological potency in healthy volunteers of angiotensin II receptor antagonists (AIIA): candesartan cilexetil, losartan, irbesartan, valsartan, and telmisartan.
Methods: In a randomised, double-blind, parallel-group (4x12 subjects) study, single oral doses of candesartan cilexetil 4, 8 and 16 mg, losartan potassium 25, 50 and 100 mg, valsartan 40, 80 and 160 mg, and irbesartan 75, 150 and 300 mg were administered on three consecutive days. Telmisartan 20, 40 and 80 mg was similarly evaluated in 12 volunteers in an open amendment. Angiotensin II (Ang II) antagonistic effects were determined in vivo from rightward shifts in Ang II dose-response curves for diastolic blood pressure (BP) and dose ratios were calculated. Apparent K_i-doses, i.e. doses (in mg) required to induce a two-fold shift in Ang II dose-response curves (equivalent to approx. 50% blockade of receptors) were determined, using Schild regression analysis.
Results: All treatments dose-dependently attenuated increases in diastolic BP induced by infusion of exogenous Ang II. Candesartan cilexetil appeared to have a more pronounced increase in effect following cumulative dosing. At 24 hours, apparent K_i-doses were: candesartan cilexetil 6 mg, irbesartan 123 mg, valsartan 93.5 mg, and telmisartan 54 mg. It was not possible to determine an apparent K_i-dose for losartan at 24 hours.
Conclusion: Consistent with results from experimental pharmacology, candesartan cilexetil displayed the highest pharmacological potency (i.e. antagonistic activity per mg substance) of the AIIAs tested. Apparent K_i-doses at 24 hours were within the dose range recommended for clinical use in patients with hypertension.

JRAAS 2000;1:336-341.

POPULAR
TOPICPAPERTransforming growth factor-β₁ induces angiotensin-converting enzyme synthesis in rat cardiac fibroblasts during their differentiation to myofibroblasts
Victor V Petrov, Robert H Fagard, Paul J Lijnen

Objectives: Appearance of angiotensin-converting enzyme (ACE) in fibrotic tissue can be the result of the action either of one particular growth factor or of cross-talk between multiple factors. Transforming growth factor-β1 (TGF-β₁) is an effective inducor of the differentiation of cultured fibroblasts to myofibroblasts, which are heterogeneous cells with different phenotypes. The present study investigated whether TGF-β₁ is able to induce, in vitro, the differentiation of cultured fibroblasts to myofibroblasts with a phenotype containing ACE.
Design: Adult rat cardiac ventricular fibroblasts were obtained from hearts perfused with collagenase. Cells from second passage were always used. Rat cardiac ventricular fibroblasts were incubated with TGF-β₁ (10 ng/ml) for seven days. Cell characterisation was performed using light microscopy and indirect immunostaining. Presence of vimentin, desmin, factor VIII, α-smooth muscle actin, and ACE was checked with both immunostaining and Western blotting. ACE activity was measured fluorometrically with hippuryl-histidyl-leucine as substrate. Synthesis of DNA was measured as ³H-thymidine incorporation.
Results: Fibroblasts contained two types of activity of hip-his-leu degradation, namely a lisinopril-dependent activity (ACE activity) and a lisinopril-independent activity ('ACE-like' activity) which is performed by peptidase(s) other than ACE. The ACE activity constituted approximately 30% of the total activity. TGF-β₁ induced an increase in both ACE activity and ACE protein (detected by immunoblotting) by 4.5 ± 0.9- and 6.9 ± 2.0-fold, respectively (p<0.05). This induction of ACE was accompanied by a profound modification of the fibroblasts phenotype, which consisted of a change in cell morphology, an enlargement of cell volume and an increase in cell protein content. TGF-β₁ profoundly inhibited ³H-thymidine incorporation and the number of cells in growing cultures. The induction of α-smooth muscle actin by TGF-β₁ (6.8 ± 2.8-fold increase, p<0.05) simultaneously with these modifications in cell morphology and proliferation indicates the appearance of myofibroblasts. These myofibroblasts did not contain desmin.
Conclusion: TGF-β₁ is able to induce the appearance of ACE in cultures of adult rat cardiac ventricular fibroblasts. The appearance of the enzyme is accompanied by the differentiation of fibroblasts to myofibroblasts.

JRAAS 2000;1:342-352.

PAPERThe renin-angiotensin-aldosterone system is suppressed in adults with Type 1 diabetes
Mats Bojestig, Fredrik H Nystrom, Hans J Arnqvist, Johnny Ludvigsson, Bengt E Karlberg

Poor glycaemic control and high blood pressure are two important risk factors for the development of retinopathy and nephropathy in Type 1 diabetes. The renin-angiotensin-aldosterone system (RAAS) may be involved in this process, since treatment with angiotensin-converting enzyme (ACE) inhibitors postpones the development of these  complications. We investigated whether plasma renin activity (PRA), plasma angiotensin II (Ang II) and atrial natriuretic peptide (ANP) differed in Type 1 diabetic patients compared with healthy controls. We recruited 80 patients with Type 1 diabetes of more than 10 years' duration and 75 age-matched controls. We found that PRA and Ang II concentrations were significantly lower in patients than in the controls. The levels of ANP, on the other hand, were higher in patients than in controls. PRA correlated negatively to the mean value of HbA_1c during the previous five years. PRA and Ang II were significantly lower in patients with mean HbA_1c >8.4% compared with those with mean HbA_1c <7.2%. In summary, we found patients with Type 1 diabetes to have RAAS suppression and increased ANP levels, suggesting a state of fluid retention.

JRAAS 2000;1:353-356.

PAPEREffects of the mineralocorticoid fludrocortisone on fibrinolytic function in healthy subjects
Katja Lottermoser, Hans-Jörg Hertfelder, Martin Wehling, Beate Schiermeyer, Hans Vetter, Rainer Düsing

Recent evidence suggests that the renin-angiotensin-aldosterone system (RAAS) may participate in the regulation of fibrinolytic function. Angiotensin II (Ang II) is the primary candidate to mediate this inter-relationship, since this peptide is capable of stimulating plasminogen activator inhibitor-1 (PAI-1) in vitro and in vivo. It has been suggested that aldosterone may also modulate fibrinolysis, possibly by interacting with Ang II. The present study therefore investigates the effect of short-term treatment with the synthetic mineralocorticoid fludrocortisone (F) on fibrinolytic function. Ten healthy male volunteers, aged 25 to 30 years, on a constant intake of 160–180 mmol Na⁺ and 60–80 mmol K⁺, were studied on a control day (C1), after two days of oral administration of F (0.1 mg b.d.), and again three days after cessation of F (C2). F was associated with a marked decrease in plasma renin activity (PRA) from 0.91 ± 0.45 ng ml^-1 h^-1 to 0.34 ± 0.29 ng ml^-1 h^-1 (p=0.005), which returned to the baseline range at C2 (0.65 ± 0.45 ng ml^-1 h^-1; p=0.032). The experimental protocol was not associated with significant changes in the activity or antigen concentration of tissue plasminogen activator (t-PA). PAI-1 exhibited a circadian rhythm with  highest values at 0800 hours (41.8 ± 9.1 ng/ml), decreasing by 1230 hours (22.6 ± 5.9 ng/ml), with a further decrease at 1630 hours (12.3 ± 3.1 ng/ml). At all three time points, PAI-1 remained unchanged by the mineralocorticoid. Our results therefore do not support a major mineralocorticoid effect on PAI-1. However, our study does not exclude a modulatory role of F, since unchanged PAI-1 could be observed in spite of a marked suppression of the RAAS.

JRAAS 2000;1:357-360.

PAPERAcute effects of ACE inhibition on coronary endothelial dysfunction
Georg Nickenig, Alexander Stäblein, Sven Wassmann, Christoph Wyen, Cornelius Müller, Michael Böhm

The prerequisite of atherosclerosis, endothelial dysfunction, is characterised by impaired endothelium-dependent vasodilation caused by the reduced  bioavailibility of nitric oxide (NO). In order to assess the role of acute ACE inhibition in this setting, coronary arterial endothelial function was quantified following acute intracoronary administration of the angiotensin-converting enzyme (ACE) inhibitor quinapril.
Twenty-one patients with non-limiting coronary artery disease were studied before and after acute intracoronary administration of 10 mg quinapril. Nine patients received pre-treatment with the angiotensin AT₁-receptor antagonist losartan (2x50 mg, p.o.). Coronary cross-sectional diameter was measured via quantitative angiography and microvascular reaction was investigated by intracoronary Doppler flow measurement during intracoronary infusion of 0.1 to 10 μmol/l acetylcholine. Quinapril acutely improved endothelial dysfunction on the macro- as well as the microvascular level. Losartan did not alter macrovascular function but facilitated microvascular endothelial function. Acute quinapril application led to no further improvement of endothelial dysfunction in patients pre-treated with losartan.
Acute quinapril infusion improved endothelial  function in patients with coronary heart disease. Treatment with the AT₁-receptor antagonist losartan led to a slight improvement in microvascular endothelial function, but pre-treatment with losartan blunted the vascular effect of quinapril, suggesting that the combination of ACE inhibition and AT₁-receptor antagonism may not exert a synergistic benefical impact on the coronary vasculature.

JRAAS 2000;1:361-364.

PAPERShould the use of short acting angiotensin-converting enzyme inhibitors be abandoned?
Arie Erman, Geoffrey Boner, David J van Dijk

Background: Angiotensin-converting enzyme inhibitors (ACE-I) have different modes of action and different durations of inhibition. The effects of ACE-I on the various components of the renin-angiotensin system (RAS) at trough hours were studied in patients with diabetes mellitus receiving long-term ACE-I treatment.
Methods: Out of 86 Type 1 and 2 diabetic patients, 49 were untreated, 25 received captopril and 12 received enalapril as chronic treatment. Blood for the determination of plasma renin activity (PRA), serum ACE activity and plasma angiotensin II (Ang II) was drawn in the morning (0700–0900 hours) after an overnight fast, about 12 hours after the last dose. PRA and Ang II were measured by RIA and serum ACE activity was assayed by a radiometric assay using ³H-hippuryl-glycyl-glycine as a substrate.
Results: Mean age was significantly greater in the enalapril-treated patients. Systolic and diastolic blood pressures were not different between the captopril-treated and untreated groups. Serum ACE activity in the captopril-treated diabetic patients was 101.5±42.5 nmol/mL/min, values obtained in untreated diabetic patients (101.4±25.2 nmol/mL/min). In contrast, ACE activity in the enalapril-treated patients was significantly reduced (5.5±7.5 nmol/mL/min) compared with untreated and captopril-treated patients (p<0.00001). PRA values in the ACE-I treated patients were significantly increased. Plasma Ang II levels were significantly increased in the captopril-treated vs. untreated patients (65.1±50.2 vs. 36.2±31.7 pg/mL, p=0.006), whereas the values in the enalapril-treated patient were slightly, but not significantly, reduced (23.8±21.4 pg/mL).
Conclusion: Trough serum ACE activity is not suppressed in diabetic patients receiving captopril, compared with those receiving enalapril and we thus question the use of short acting ACE-I in these patients.

JRAAS 2000;1:365-368.

PAPERAngiotensin II binding and extracellular matrix remodelling in a rat model of myocardial infarction
Marwan E El-Sabban, Khaled A Hassan, Adel E Birbari, Khalil M Bitar, Anwar B Bikhazi

Clinical evidence points to a role for angiotensin II (Ang II) in the post-infarction remodelling of cardiac hypertrophy. The present study was designed to investigate the remodelling process in an animal model of myocardial infarction (MI) using the following criteria: 1) histological studies to examine the re-vascularisation process and collagen deposition in different regions of the myocardium; 2) histological evidence to investigate the cell type distribution using cell-specific markers; 3) histological and Western blot analysis to localise Ang II receptor subtypes (AT₁-receptor and AT₂-receptor) and to study their regulation; 4) kinetics of the binding of Ang II to its receptors in a heart perfusion model; and 5) to assess the effect of the Ang II antagonist (losartan) on these parameters.
MI was induced by ligation of the left anterior descending coronary artery of Sprague-Dawley rats. Four different animal groups were established: 1) sham-operated, non-treated; 2) sham-operated, treated with losartan; 3) myocardial infarct, non-treated; and 4) myocardial infarct, treated with losartan. In infarcted rat hearts, fibroblasts and collagen types I and III increased in the remnant viable region of the left ventricle compared with sham-operated rats. One month of losartan treatment in myocardial infarcted rats revealed insignificant changes in fibroblasts and collagen types I and III compared with sham controls. Also, myocardial infarction increased AT₁-receptor protein levels compared with sham-operated controls, as judged by Western blotting. In losartan-treated myocardial infarct animals, no changes were detected at the level of AT₁-receptor expression compared with non-treated myocardial infarct rats. Binding studies of Ang II on endothelial cell lining and directly on myocytes in sham-operated and infarcted perfused rat hearts revealed that, in myocardial infarcted-animals, Ang II binding affinity increased both in the endothelium and in myofibres. This may be considered a major putative effect of the peptide in potentiating the pharmacodynamics of hypertrophy. In losartan-treated myocardial infarcted-animals, a marked increase in the binding affinities of Ang II for the AT₂-receptor subtype was observed. Hence, potential cardioprotective effects of the AT₁-receptor antagonist are proposed.

JRAAS 2000;1:369-378.

PAPERThe effect of angiotensin II on mitogen-activated protein kinase in human cardiomyocytes
Chiming Wei, Marcelo G Cardarelli, Stephen W Downing, Joseph S McLaughlin

The role of angiotensin II (Ang II)-receptors on mitogen-activated protein kinase (MAPK) activation in cardiomyocytes remains controversial. Therefore, the current study was designed to investigate the actions of AT₁- and AT₂-receptors on Ang II-induced extracellular signal-regulated kinase (ERK), p38 and the c-Jun N-terminal kinase (JNK) MAPK activities in human cardiomyocytes. Human cardiac tissue was obtained from open-heart surgery (n=6). The cardiac tissue was minced and incubated in the special tissue culture system for 24 hours in the absence or presence of Ang II (10^-7 M). These studies were repeated with the AT₁-receptor antagonist losartan (10^-6 M) or the AT₂-receptor antagonist PD-123319 (10^-6 M). Immunohistochemical staining and Western blot analysis with phospho-antibodies were performed to determine ERK, JNK and p38 activities. Ang II increased ERK and p38 activities in human cardiomyocytes. The effects of Ang II were abolished by losartan and enhanced by PD-123319. Co-incubation with both losartan and PD-123319 resulted in a decrease of ERK and p38 activities in cardiomyocytes. The immunohistochemical staining of JNK showed no significant differences between Ang II alone, Ang II plus losartan and Ang II plus PD-123319 groups. In conclusion, Ang II has a potent effect on ERK and p38 MAPK activities in cardiomyocytes, by acting through AT₁-receptors. This effect of Ang II is modified by AT₂-receptors. Therefore, Ang II, via AT₁- and AT₂-receptor stimulation, has a distinct effect on MAPK activity in cardiomyocytes.

JRAAS 2000;1:379-384.

EDITORIAL REVIEWRecruitable ACE and tissue repair in the infarcted heart
Karl T Weber, Yao Sun

Constitutive angiotensin-converting enzyme (ACE) is bound to endothelial cells where it serves to regulate circulating concentrations of angiotensin II (Ang II) that normally contribute to circulatory homeostasis. Recruitable ACE, bound to macrophage and myofibroblast cell membrane, regulates local concentrations of Ang II involved in tissue repair. De novo generation of Ang II modulates expression of TGF-β1 whose autocrine/paracrine properties regulate collagen turnover at sites of fibrous tissue formation that appear in response to various forms of injury in diverse tissues. Persistent myofibroblasts and their ACE activity at the infarct site contribute to a sustained metabolic activity that can account for a progressive fibrosis at, and remote to, sites of myocardial infarction. Activation of the circulating renin-angiotensin-aldosterone system with sustained elevations in plasma Ang II and aldosterone induce a pro-inflammatory vascular phenotype of small arteries and arterioles. This further promotes the appearance of recruitable ACE bound to macrophages and myofibroblasts involved in vascular remodelling. Locally produced Ang II from these vascular sites leads to perivascular fibrosis of intramural coronary vasculature of non-infarcted myocardium. At these sites, remote to the infarct, such adverse structural remodelling by fibrous tissue eventuates in ICM, a major aetiologic factor involved in the appearance of chronic cardiac failure and contributes to its progressive nature.

JRAAS 2000;1:295-303.

POPULAR
TOPICEDITORIAL REVIEWPractical implications of current natriuretic peptide research
Giuseppe A Sagnella

Since the original discovery of atrial natriuretic peptide (ANP) nearly 20 years ago and the subsequent realisation of the existence of a family of natriuretic peptides, there has been considerable progress in the elucidation of the physiological and pathophysiological significance of these peptides. This review has examined two potentially important practical aspects arising from natriuretic peptide research - the significance of measurement of plasma levels of ANP and of brain natriuretic peptide BNP for cardiovascular disease and the therapeutic potential of targeting the natriuretic peptide system.
Several situations where the measurement of plasma ANP and BNP may be of benefit in the overall assessment and prognosis of cardiac disease have been discussed. The measurement of plasma levels of these peptides appears to have limited value as a specific diagnostic tool and is unlikely to replace well-established procedures to assess cardiac function. Nevertheless, given the strong negative predictive value, the value of the measurement of plasma natriuretic peptides particularly BNPs, in people with suspected heart disease, rests on the evidence that a normal value indicates a low risk of cardiac impairment. Moreover, a consistently elevated plasma level of BNP after myocardial infarction is associated with a distinctly poor prognosis. In turn, this may help to select those with high plasma levels for subsequent detailed investigation of cardiac dysfunction. This may be an important option, especially where the facilities for the more invasive cardiological procedures are not available. Intriguingly, recent research also suggests the possibility that plasma levels of natriuretic peptides may have an important role in guiding more effective therapy for heart failure.
The potent cardiovascular and renal effects of ANP and BNP provide an important therapeutic potential for hypertension and for conditions associated with volume overload. A number of approaches which have been used to enhance endogenous activity of these peptides have been highlighted. The use of the native peptides ANP and BNP may well be valuable in some circumstances, such as in critically ill individuals with congestive heart failure or renal failure. However, the limitations of the use of peptides, especially for long-term treatment, are obvious. In view of this, considerable effort has been devoted to the development of orally active agents to enhance endogenous natriuretic peptides by inhibition of breakdown by neutral endopeptidase. This research has led to the development of vasopeptidase inhibitors - dual inhibitors of both endopeptidase and angiotensin-converting enzyme - to enhance endogenous natriuretic peptide function on a background of reduced angiotensin II activity. The broad spectrum of action and the potentially important target-organ protection of these inhibitors offer potential benefits which may well go beyond existing treatment of hypertension and of conditions associated with overt volume overload.

JRAAS 2000;1:304-315.

EDITORIAL REVIEWGenetic basis of cardiovascular disease – the renin-angiotensin-aldosterone system as a paradigm
Neal Padmanabhan, Sandosh Padmanabhan, John MC Connell

There is considerable evidence that genetic variation in the renin-angiotensin-aldosterone system (RAAS) may influence cardiovascular structure and risk. However, the strength of the associations remain uncertain and it is likely that their importance (and particularly that of the angiotensin-converting enzyme [ACE] inhibitor insertion/deletion [I/D] polymorphism) has been over-stated. The existence of numerous contradictory studies engenders scepticism, which is compounded by lack of evidence of an intermediate phenotype to link genotype and phenotype. In addition, mechanisms by which polymorphisms in non-coding regions of these genes can influence their expression have not been identified. Authors have consistently suggested that the ACE I/D and AT₁-receptor A1166C polymorphisms, for example, may be in linkage disequilibrium with regulatory elements, but these have yet to be identified or characterised at a molecular level. It is equally possible that they are also in linkage disequilibrium with other loci. Of the available evidence, the most persuasive comes from data on aldosterone synthase. This locus is implicated in animal models of raised blood pressure, rare monogenic syndromes and may also be involved in patients with essential hypertension. Finally, further work is clearly required to determine whether specific polymorphisms, or combinations of polymorphisms, will be useful in quantifying cardiovascular risk or guiding treatment. Future studies will need to consider other candidate genes, both within the RAAS and distinct from it. Other studies are already being performed which do not depend on the knowledge of a candidate gene, but instead use the tools of population genetics to identify useful markers. In this regard, the possibility of gene-gene and gene-environment interactions adds another layer of complexity to an already confusing subject.

JRAAS 2000;1:316-324.

POPULAR
TOPICEDITORIAL REVIEWTo block the renin system or the calcium channel in hypertension – where do we stand?
Peter S Sever

Early trials of intervention in hypertensive patients failed to show that antihypertensive drugs protected against myocardial infarction (MI). Individual trials were underpowered to address this question and meta-analyses of pooled data suggested some protection (16% relative risk reduction). This represents a shortfall, since observational data predicted a 25% reduction in MI for a similar fall in blood pressure.
Comparisons of newer agents (calcium channel blockers [CCBs] and angiotensin-converting enzyme inhibitors [ACE-I]) with older agents challenged the hypothesis that drugs free of adverse metabolic sequelae would improve outcome, particularly with respect to coronary heart disease (CHD). Regrettably, these new trials were designed with unrealistic objectives, and were underpowered to establish whether CCBs or ACE-Is improve CHD outcome compared with diuretics or beta-blockers.
New meta-analyses, involving 16 trials and more than 70,000 patients, have recently been published. In trials comparing different active treatments, small differences of marginal significance were observed, and no clear discrimination between treatments can be concluded. Two large ongoing trials (The Anglo Scandinavian Cardiac Outcomes Trial [ASCOT] and Antihypertensive and Lipid-Lowering treatment to prevent Heart Attack Trial [ALLHAT]) and further pooled analyses will be required to define optimal treatment strategies in hypertension, for the hypertensive population as a whole and for important high-risk subgroups.

JRAAS 2000;1:325-327.