Schalekamp Ma

Arterial Stiffness and Risk of Coronary Heart Disease and Stroke The Rotterdam Study

Background— Arterial stiffness has been associated with the risk of cardiovascular disease in selected groups of patients. We evaluated whether arterial stiffness is a predictor of coronary heart disease and stroke in a population-based study among apparently healthy subjects. Methods and Results— The present study included 2835 subjects participating in the third examination phase of the Rotterdam Study. Arterial stiffness was measured as aortic pulse wave velocity and carotid distensibility. Cox proportional hazards regression analysis was performed to compute hazard ratios. During follow-up, 101 subjects developed coronary heart disease (mean follow-up period, 4.1 years), and 63 subjects developed a stroke (mean follow-up period, 3.2 years). The risk of cardiovascular disease increased with increasing aortic pulse wave velocity index. Hazard ratios and corresponding 95% CIs of coronary heart disease for subjects in the second and third tertiles of the aortic pulse wave velocity index compared with subjects in the reference category were 1.72 (0.91 to 3.24) and 2.45 (1.29 to 4.66), respectively, after adjustment for age, gender, mean arterial pressure, and heart rate. Corresponding estimates for stroke were 1.22 (0.55 to 2.70) and 2.28 (1.05 to 4.96). Estimates decreased only slightly after adjustment for cardiovascular risk factors, carotid intima-media thickness, the ankle-arm index, and pulse pressure. The aortic pulse wave velocity index provided additional predictive value above cardiovascular risk factors, measures of atherosclerosis, and pulse pressure. Carotid distensibility as measured in this study was not independently associated with cardiovascular disease. Conclusions— Aortic pulse wave velocity is an independent predictor of coronary heart disease and stroke in apparently healthy subjects.

Angiotensin-Converting Enzyme in the Human Heart Effect of the Deletion/Insertion Polymorphism

BACKGROUND An insertion (I)/deletion (D) polymorphism of the angiotensin-converting enzyme (ACE) gene has been associated with differences in the plasma levels of ACE as well as with myocardial infarction, cardiomyopathy, left ventricular hypertrophy, and coronary artery disease. METHODS AND RESULTS We determined the cardiac ACE activity and the ACE genotype in 71 subjects who died of noncardiac disorders. Cardiac ACE activity was significantly higher (P < .01) in subjects with the ACE DD genotype (12.7 +/- 1.9 mU/g wet wt) compared with subjects with the ID (8.7 +/- 0.8 mU/g) and the II (9.1 +/- 1.0 mU/g) genotypes. This difference was independent of sex, age, and the time required for tissue collection. CONCLUSIONS Cardiac ACE activity is highest in subjects with the DD genotype. Elevated cardiac ACE activity in these subjects may result in increased cardiac angiotensin II levels, and this may be a mechanism underlying the reported association between the ACE deletion polymorphism and the increased risk for several cardiovascular disorders.

Cardiac Renin and Angiotensins Uptake From Plasma Versus In Situ Synthesis

The existence of a cardiac renin-angiotensin system, independent of the circulating renin-angiotensin system, is still controversial. We compared the tissue levels of renin-angiotensin system components in the heart with the levels in blood plasma in healthy pigs and 30 hours after nephrectomy. Angiotensin I (Ang I)-generating activity of cardiac tissue was identified as renin by its inhibition with a specific active site-directed renin inhibitor. We took precautions to prevent the ex vivo generation and breakdown of cardiac angiotensins and made appropriate corrections for any losses of intact Ang I and II during extraction and assay. Tissue levels of renin (n = 11) and Ang I (n = 7) and II (n = 7) in the left and right atria were higher than in the corresponding ventricles (P < .05). Cardiac renin and Ang I levels (expressed per gram wet weight) were similar to the plasma levels, and Ang II in cardiac tissue was higher than in plasma (P < .05). The presence of these renin-angiotensin system components in cardiac tissue therefore cannot be accounted for by trapped plasma or simple diffusion from plasma into the interstitial fluid. Angiotensinogen levels (n = 11) in cardiac tissue were 10% to 25% of the levels in plasma, which is compatible with its diffusion from plasma into the interstitium. Like angiotensin-converting enzyme, renin was enriched in a purified cardiac membrane fraction prepared from left ventricular tissue, as compared with crude homogenate, and 12 +/- 3% (mean +/- SD, n = 6) of renin in crude homogenate was found in the cardiac membrane fraction and could be solubilized with 1% Triton X-100. Tissue levels of renin and Ang I and II in the atria and ventricles were directly correlated with plasma levels (P < .05), and in both tissue and plasma the levels were undetectably low after nephrectomy. We conclude that most if not all renin in cardiac tissue originates from the kidney. Results support the contentions that in the healthy heart, angiotensin production depends on plasma-derived renin and that plasma-derived angiotensinogen in the interstitial fluid is a potential source of cardiac angiotensins. Binding of renin to cardiac membranes may be part of a mechanism by which renin is taken up from plasma.

Determinants of interindividual variation of renin and prorenin concentrations: evidence for a sexual dimorphism of (pro)renin levels in humans

Background Plasma renin concentrations are an important factor in cardiovascular risk profiling. Objective To investigate the effects of sex, medication, and anthropometric factors that may contribute to the interindividual variation in the plasma concentrations of renin and its precursor prorenin. Design and methods Prorenin and renin levels in 327 men and 383 women, aged 52–69 years, who participated in a 1994 reexamination of a previous population survey in Bavaria, were measured by immunoradiometric assay. Results Prorenin and renin levels in men were significantly higher than those in women, those in women without estrogen replacement therapy were significantly higher than those in women with estrogen replacement therapy, and those in diabetics were significantly higher than those in nondiabetics. Prorenin level was correlated negatively to blood pressure and positively to age and the use of diuretics; it was normal in subjects using angiotensin converting enzyme inhibitors and β-adrenergic antagonists (β-blockers). Renin level was correlated negatively to atrial natriuretic peptide level and the use of b-blockers, and it was elevated above normal levels in subjects using angiotensin converting enzyme inhibitors and diuretics as well as in subjects who had previously suffered myocardial infarction. After exclusion of data for women being administered estrogen replacement therapy, multivariate analysis revealed that sex (P < 0.001), age (P < 0.02), blood pressure (P < 0.002), diabetes (P < 0.05), and the use of angiotensin converting enzyme inhibitors (P < 0.002), β-blockers (P < 0.001), and diuretics (P < 0.05) were independent determinants of plasma prorenin. Plasma renin was independently related to atrial natriuretic peptide level (P < 0.01) and the use of angiotensin converting enzyme inhibitors (P < 0.001), b-blockers (P < 0.001), and diuretics (P < 0.05). Conclusions These data demonstrate that there is a sexual dimorphism of prorenin levels in humans, suggesting that sex hormones affect the regulation of the renin gene. Data confirm previous reports of elevated prorenin levels in diabetics and older subjects, as well as of lower than normal prorenin levels in subjects with hypertension in smaller populations. Our findings may help to clarify the potential (patho)physiologic functions of prorenin and to identify the factors that influence the constitutive secretion and intracellular processing of this prohormone.

Angiotensin Production by the Heart A Quantitative Study in Pigs With the Use of Radiolabeled Angiotensin Infusions

BACKGROUND Beneficial effects of ACE inhibitors on the heart may be mediated by decreased cardiac angiotensin II (Ang II) production. METHODS AND RESULTS To determine whether cardiac Ang I and Ang II are produced in situ or derived from the circulation, we infused 125I-labeled Ang I or II into pigs (25 to 30 kg) and measured 125I-Ang I and II as well as endogenous Ang I and II in cardiac tissue and blood plasma. In untreated pigs, the tissue Ang II concentration (per gram wet weight) in different parts of the heart was 5 times the concentration (per milliliter) in plasma, and the tissue Ang I concentration was 75% of the plasma Ang I concentration. Tissue 125I-Ang II during 125I-Ang II infusion was 75% of 125I-Ang II in arterial plasma, whereas tissue 125I-Ang I during 125I-Ang I infusion was <4% of 125I-Ang I in arterial plasma. After treatment with the ACE inhibitor captopril (25 mg twice daily), Ang II fell in plasma but not in tissue, and Ang I and renin rose both in plasma and tissue, whereas angiotensinogen did not change in plasma and fell in tissue. Tissue 125I-Ang II derived by conversion from arterially delivered 125I-Ang I fell from 23% to <2% of 125I-Ang I in arterial plasma. CONCLUSIONS Most of the cardiac Ang II appears to be produced at tissue sites by conversion of in situ-synthesized rather than blood-derived Ang I. Our study also indicates that under certain experimental conditions, the heart can maintain its Ang II production, whereas the production of circulating Ang II is effectively suppressed.

A Clinical Prediction Rule for Renal Artery Stenosis

Renal artery stenosis impairs blood flow to the kidney and can consequently cause renovascular hypertension and renal failure [1, 2]. Although the prevalence of this condition among patients with hypertension is low, therapeutic options for relieving renal artery stenosis, such as renal angioplasty and stenting, make the search for renal artery stenosis worthwhile [2-4]. Renal angiography is the gold standard for diagnosing renal artery stenosis, but it is a costly and invasive procedure that can involve serious complications [5, 6]. To diagnose renal artery stenosis efficiently, angiography should be used selectively. Most physicians rely on captopril renal scintigraphy as a selection criterion, but the diagnostic accuracy of this test is low (sensitivity, 65% to 77%; specificity, 90%) [7, 8]. As an alternative, clinical characteristics can be used to select hypertensive patients for angiography [9]. Patients with normal renal function whose blood pressure can be controlled with one or two drugs can be excluded from angiography [9, 10]. In the remaining patients (those with drug-resistant hypertension), such clinical characteristics as atherosclerotic vascular disease, smoking history, and presence of an abdominal bruit can be used to estimate a patients probability of renal artery stenosis [11-14]. This estimate can then be used in selection for angiography. We analyzed the clinical characteristics of 477 patients with drug-resistant hypertension or an increase in serum creatinine concentration during therapy with angiotensin-converting enzyme (ACE) inhibitors who participated in the Dutch Renal Artery Stenosis Intervention Cooperative (DRASTIC) study [9]. We developed a clinical prediction rule for quantifying the probability of renal artery stenosis [15] and demonstrated the potential consequences of this rule for clinical practice by applying it to our patients. Methods Patients The DRASTIC study is a prospective cohort study conducted at 26 departments of internal medicine with an interest in hypertension throughout the Netherlands [9]. The diagnostic phase of the study was designed to find an optimal strategy for diagnosing renal artery stenosis. In the DRASTIC study, 1133 hypertensive patients 18 to 75 years of age with preserved renal function (serum creatinine concentration 200 mol/L [2.26 mg/dL]) were enrolled. These patients were referred for analysis of hypertension by general practitioners (55%) or hospital specialists (45%), in most cases because their hypertension was difficult to treat with antihypertensive drugs. Sixty percent of patients were from four hospitals. After giving written informed consent, patients were randomly assigned to one of two standard protocols with antihypertensive drugs: amlodipine, 10 mg, plus atenolol, 50 mg, in patients older than 40 years of age or enalapril, 20 mg, plus hydrochlorothiazide, 25 mg, in patients older than 40 years of age. Blood pressure was measured with a standard sphygmomanometer at three consecutive visits at least 1 week apart. Measurements were taken three times per visit after a 5-minute rest with the patient in the sitting position. Patients were selected for diagnostic workup if they had drug-resistant hypertension, defined as a mean diastolic blood pressure per visit of 95 mm Hg or more while receiving the standard drug regimen during all three visits or prescription of an additional drug regardless of blood pressure response. Patients were also selected if the serum creatinine concentration increased 20 mol/L (0.23 mg/dL) or more during therapy with ACE inhibitors. In these patients, intra-arterial digital subtraction angiography and other, noninvasive tests were performed. In accordance with the study protocol, patients who responded well to standard treatment were not evaluated further. The diagnostic phase of the study was followed by a therapeutic phase in which patients with atherosclerotic stenosis were randomly assigned to receive medication or renal angioplasty. Definitions After performing a literature study, we selected 12 clinical characteristics indicative of renovascular disease (predictors) [10, 11, 16-26]: age, sex, ethnicity (black or other), signs and symptoms of atherosclerotic vascular disease (femoral or carotid bruit, angina pectoris, claudication, myocardial infarction, cerebrovascular accident, or vascular surgery), recent onset of hypertension (within the past 2 years), family history of hypertension (parents, siblings, or children with hypertension), smoking history (ever or never), obesity (body mass index 25 kg/m2), abdominal bruit, advanced hypertensive retinopathy (fundus grade III or IV), serum creatinine concentration, and hypercholesterolemia (serum cholesterol level > 6.5 mmol/L [251.35 mg/dL] or use of cholesterol-lowering agents). These characteristics were used to predict the presence of renal artery stenosis. A patient was considered to have renal artery stenosis when the angiogram showed at least one stenosis of 50% or more in a renal artery according to the local-radiologist. Model Development Data are presented as a proportion or as the mean SD. The univariable association between clinical characteristics and presence of renal artery stenosis was studied by computing the value and 95% CI of the odds ratio. In a multivariable analysis, clinical characteristics were combined as predictor variables in a logistic regression model predicting the presence of renal artery stenosis (outcome) [27]. For each patient in the multivariable analysis, the probability of renal artery stenosis was calculated from the regression model (predicted probability). The reliability, discriminative ability, and validity of the model were assessed. The Appendix gives details on model development and evaluation. To enable the use of the regression model in clinical practice, a prediction rule was constructed for predicting renal artery stenosis in future patients with drug-resistant hypertension or an increase in serum creatinine concentration during therapy with ACE inhibitors. For the presence or level of each clinical characteristic in the regression model, a score was calculated on the basis of the regression coefficients (Appendix). These scores were added into a sum score. All possible sum scores and their corresponding predicted probabilities of renal artery stenosis were combined in a graph with 95% CIs of the predicted probabilities. Role of the Funding Source Our funding source had no role in the collection, analysis, or interpretation of the data or in the decision to submit the manuscript for publication. Results Statistical Analyses Angiography was performed in 439 patients with drug-resistant hypertension and 39 patients with an increase in serum creatinine concentration during therapy with ACE inhibitors. The procedure failed in 1 patient. For the remaining 477 patients, angiography showed renal artery stenosis in 107 patients (22%), of whom 90 (84%) had atherosclerotic stenosis and 17 (16%) had fibromuscular dysplasia. Bilateral stenoses were found in 27 of 107 affected patients (25%). Renal scintigraphy was performed in 458 patients; it had a sensitivity of 72% and a specificity of 90% for the diagnosis of renal artery stenosis. Table 1 shows the univariable distribution of the clinical characteristics for patients with renal artery stenosis and those with essential hypertension. Most clinical characteristics were indicative of renal artery stenosis (P < 0.05 or borderline significant) except sex, recent onset of hypertension, and presence of advanced hypertensive retinopathy. More young women without signs of atherosclerotic disease were found among patients with fibromuscular dysplasia than among those with atherosclerotic stenosis, but abdominal bruits occurred with the same frequency in both groups (29% and 27%, respectively). Table 1. Associations of Clinical Characteristics with Renal Artery Stenosis The results of multivariable analysis are also shown in Table 1. Advanced hypertensive retinopathy was not studied any further because this clinical characteristic was missing for 43% of the patients. Data on 11 clinical characteristics of 460 patients were considered predictive of renal artery stenosis. Ethnicity and family history of hypertension were removed from the regression model because their contribution to predicting renal artery stenosis was small. Because renal artery stenosis is believed to be more prevalent in young women and old men, interaction between age and sex was tested; this interaction was not statistically significant (P = 0.09). We included an interaction term between age and smoking because this was the only biologically plausible interaction term that was statistically significant (P = 0.01). This interaction term accounts for the fact that the predictive value of increasing age was stronger for patients who never smoked than for current and former smokers. Finally, the type of standard treatment did not provide additional diagnostic information when it was included in the regression model (P > 0.2). The multivariable odds ratios in Table 1 reflect the predictive effect of the individual clinical characteristics while correcting for the other predictors in the multivariable model. For example, the multivariable odds ratio for atherosclerotic vascular disease was lower than the univariable odds ratio because the model also accounted for the effects of age and smoking history. Model Performance Figure 1 shows the agreement between the predicted and the observed probabilities. For 204 patients (44%), the predicted probability of stenosis was 0% to 10%. The predicted probabilities of stenosis obtained from the model agreed well with the observed frequency of stenosis (goodness-of-fit test, P > 0.2). The model discriminated well between patients with renal artery stenosis (predicted probability, 49% 29%) and patients with essential hypertension (predicted probability, 15% 16%); the are

Metabolism and Production of Angiotensin I in Different Vascular Beds in Subjects With Hypertension

To study the metabolism and production of angiotensin I, highly purified monoiodinated [I25I] angiotensin I was given by constant systemic intravenous infusion, either alone (n=7) or combined with unlabeled angiotensin I (ra=5), to subjects with essential hypertension who were treated with the angiotensin converting enzyme inhibitor captopril (50 mg b.i.d.). Blood samples were taken from the aorta and the renal, antecubital, femoral, and hepatic veins. [l25I]Angiotensin I and angiotensin I were extracted from plasma, separated by highperformance liquid chromatography, and quantitated by gamma counting and radioimmunoassay. Plasma renin activity was measured at pH 7.4. The plasma decay curves after discontinuation of the infusions of [125I] angiotensin I and unlabeled angiotensin I were similar for the two peptides. The regional extraction ratio of [I2SI]angiotensin I was 47±4% (mean±SEM) across the forearm, 59±3% across the leg, 81±1% across the kidneys, and 96±1% across the hepatomesenteric vascular bed. These results were not different from those obtained for infused unlabeled angiotensin I. Despite the rapid removal of arterially delivered angiotensin I, no difference was found between the venous and arterial levels of endogenous angiotensin I across the various vascular beds, with the exception of the liver where angiotensin I in the vein was 50% lower than in the aorta. Thus, 50–90% of endogenous angiotensin I in the veins appeared to be derived from regional de novo production. The blood transit time is 0.1–0.2 minute in the limbs and in the kidneys and 0.3–0.5 minute in the hepatomesenteric vascular bed. This is too short for plasma renin activity to account for the measured de novo angiotensin I production. It was calculated that less than 20–30% in the limbs and in the kidneys and approximately 60% in the hepatomesenteric region of de novo-produced angiotensin I could be accounted for by circulating renin. These results indicate that a high percentage of plasma angiotensin I may be produced locally (i.e., not in circulating plasma).

Angiotensin II Type 1 (AT1) Receptor–Mediated Accumulation of Angiotensin II in Tissues and Its Intracellular Half-life In Vivo

Angiotensin II (Ang II) is internalized by various cell types via receptor-mediated endocytosis. Little is known about the kinetics of this process in the whole animal and about the half-life of intact Ang II after its internalization. We measured the levels of 125I-Ang II and 125I-Ang I that were reached in various tissues and blood plasma during infusions of these peptides into the left cardiac ventricle of pigs. Steady-state concentrations of 125I-Ang II in skeletal muscle, heart, kidney, and adrenal were 8% to 41%, 64% to 150%, 340% to 550%, and 680% to 2100%, respectively, of the 125I-Ang II concentration in arterial blood plasma (ranges of six experiments). The tissue concentrations of 125I-Ang I were less than 5% of the arterial plasma concentrations. 125I-Ang II accumulation seen in heart, kidney, and adrenal was almost completely blocked by a specific Ang II type 1 (AT1) receptor antagonist. Steady-state concentrations of 125I-Ang II were reached within 30 to 60 minutes in the tissues and within 5 minutes in blood plasma. The in vivo half-life of intact 125I-Ang II in heart, kidney, and adrenal was approximately 15 minutes, compared with 0.5 minute in the circulation. Thus, Ang II, but not Ang I, from the circulation is accumulated by some tissues, and this is mediated by AT1 receptors. The time course of this process and the long half-life of the accumulated Ang II support the contention that this Ang II has been internalized after its binding to the AT1 receptor, so that it is protected from rapid degradation by endothelial peptidases. The results of this study are in agreement with growing evidence of an important physiological role for internalized Ang II.

Prorenin, Renin, Angiotensinogen, and Angiotensin-Converting Enzyme in Normal and Failing Human Hearts Evidence for Renin Binding

BACKGROUND A local renin-angiotensin system in the heart is often invoked to explain the beneficial effects of ACE inhibitors in heart failure. The heart, however, produces little or no renin under normal conditions. METHODS AND RESULTS We compared the cardiac tissue levels of renin-angiotensin system components in 10 potential heart donors who died of noncardiac disorders and 10 subjects with dilated cardiomyopathy (DCM) who underwent cardiac transplantation. Cardiac levels of renin and prorenin in DCM patients were higher than in the donors. The cardiac and plasma levels of renin in DCM were positively correlated, and extrapolation of the regression line to normal plasma levels yielded a tissue level close to that measured in the donor hearts. The cardiac tissue-to-plasma concentration (T/P) ratios for renin and prorenin were threefold the ratio for albumin, which indicates that the tissue levels were too high to be accounted for by admixture with blood and diffusion into the interstitial fluid. Cell membranes from porcine cardiac tissue bound porcine renin with high affinity. The T/P ratio for ACE, which is membrane bound, was fivefold the ratio for albumin. Cardiac angiotensinogen was lower in DCM patients than in the donors, and its T/P ratio was half that for albumin, which is compatible with substrate consumption by cardiac renin. CONCLUSIONS These data in patients with heart failure support the concept of local angiotensin production in the heart by renin that is taken up from the circulation. Membrane binding may be part of the uptake process.

Mannose 6-Phosphate Receptor–Mediated Internalization and Activation of Prorenin by Cardiac Cells

The binding and internalization of recombinant human renin and prorenin (2500 microU/mL) and the activation of prorenin were studied in neonatal rat cardiac myocytes and fibroblasts cultured in a chemically defined medium. Surface-bound and internalized enzymes were distinguished by the addition of mannose 6-phosphate to the medium, by incubating the cells both at 37 degrees C and 4 degrees C, and by the acid-wash method. Mannose 6-phosphate inhibited the binding of renin and prorenin to the myocyte cell surface in a dose-dependent manner. At 37 degrees C, after incubation at 4 degrees C for 2 hours, 60% to 70% of cell surface-bound renin or prorenin was internalized within 5 minutes. Intracellular prorenin was activated, but extracellular prorenin was not. The half-time of activation at 37 degrees C was 25 minutes. Ammonium chloride and monensin, which interfere with the normal trafficking and recycling of internalized receptors and ligands, inhibited the activation of prorenin. Results obtained with cardiac fibroblasts were comparable to those in the myocytes. This study is the first to show experimental evidence for the internalization and activation of prorenin in extrarenal cells by a mannose 6-phosphate receptor-dependent process. Our findings may have physiological significance in light of recent experimental data indicating that angiotensin I and II are produced at cardiac and other extrarenal tissue sites by the action of renal renin and that intracellular angiotensin II can elicit important physiological responses.