Hendrik Buikema

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.

Glucagon-Like Peptide 1 Prevents Reactive Oxygen Species–Induced Endothelial Cell Senescence Through the Activation of Protein Kinase A

Objective—Endothelial cell senescence is an important contributor to vascular aging and is increased under diabetic conditions. Here we investigated whether the antidiabetic hormone glucagon-like peptide 1 (GLP-1) could prevent oxidative stress–induced cellular senescence in endothelial cells. Methods and Results—In Zucker diabetic fatty rats, a significant 2-fold higher level of vascular senescence was observed compared with control lean rats. Dipeptidyl-peptidase 4 (DPP-4) inhibition significantly increased GLP-1 levels in these animals and reduced senescence almost to lean animal levels. In vitro studies with human umbilical vein endothelial cells showed that GLP-1 had a direct protective effect on oxidative stress (H2O2)–induced senescence and was able to attenuate oxidative stress–induced DNA damage and cellular senescence. The GLP-1 analogue exendin-4 provided similar results, whereas exendin fragment 9–39, a GLP-1 receptor antagonist, abolished this effect. Intracellular signaling by the phosphoinositide 3-kinase (PI3K)/Akt survival pathway did not appear to be involved. Further analysis revealed that GLP-1 activates the cAMP response element-binding (CREB) transcription factor in a cAMP/protein kinase A (PKA)–dependent manner, and inhibition of the cAMP/PKA pathway abolished the GLP-1 protective effect. Expression analysis revealed that GLP-1 can induce the oxidative defense genes HO-1 and NQO1. Conclusion—Dipeptidyl-peptidase 4 inhibition protects against vascular senescence in a diabetic rat model. In vitro studies with human umbilical vein endothelial cells showed that reactive oxygen species–induced senescence was attenuated by GLP-1 in a receptor-dependent manner involving downstream PKA signaling and induction of antioxidant genes.

Effects of Quinapril on Clinical Outcome After Coronary Artery Bypass Grafting (The QUO VADIS Study)

The QUO VADIS study was designed to explore whether 1 year of angiotensin-converting enzyme inhibition with quinapril (40 mg/day) would decrease ischemia in patients who underwent coronary artery bypass grafting (CABG). Patients (n = 149) scheduled for CABG were randomized 4 weeks before surgery. Study medication was used from randomization up to 1 year after CABG. Exercise testing was performed at randomization; the exercise test was repeated 1 year after CABG and patients underwent 48-hour Holter monitoring. Clinical ischemic events were recorded and defined as death, revascularization, myocardial infarction, recurrence of angina pectoris, ischemic stroke, or transient ischemic attack. Baseline characteristics were similar between groups. Total exercise time increased overall by 75 +/- 76 seconds 1 year after CABG (placebo +79 +/- 75 seconds, quinapril +72 +/- 79 seconds, p = 0.6). All patients had ischemic ST-segment changes at randomization; 33% of patients had ischemic ST-segment changes 1 year after CABG (placebo 29%, quinapril 37%, p = 0.4). On Holter monitoring, the number of patients experiencing > or = 1 episodes of ischemia was equal in both groups. Treatment with quinapril significantly reduced clinical ischemic events after CABG: 15% in patients on placebo versus 4% of patients on quinapril (hazard ratio 0.23, 95% confidence interval 0.06 to 0.87, p = 0.02). Long-term quinapril treatment significantly reduced clinical ischemic events within 1 year after CABG, although ischemia at exercise testing and Holter monitoring was unchanged.

Comparison of zofenopril and lisinopril to study the role of the sulfhydryl-group in improvement of endothelial dysfunction with ACE-inhibitors in experimental heart failure

We evaluated the role of SH‐groups in improvement of endothelial dysfunction with ACE‐inhibitors in experimental heart failure. To this end, we compared the vasoprotective effect of chronic treatment with zofenopril (plus SH‐group) versus lisinopril (no SH‐group), or N‐acetylcysteine (only SH‐group) in myocardial infarcted (MI) heart failure rats. After 11 weeks of treatment, aortas were obtained and studied as ring preparations for endothelium‐dependent and ‐independent dilatation in continuous presence of indomethacin to avoid interference of vasoactive prostanoids, and the selective presence of the NOS‐inhibitor L‐NMMA to determine NO‐contribution. Total dilatation after receptor‐dependent stimulation with acetylcholine (ACh) was attenuated (−49%, P<0.05) in untreated MI (n=11), compared to control rats with no‐MI (n=8). This was in part due to impaired NO‐contribution in MI (−50%, P<0.05 versus no‐MI). At the same time the capacity for generation of biologically active NO after receptor‐independent stimulation with A23187 remained intact. Chronic treatment with n‐acetylcysteine (n=8) selectively restored NO‐contribution in total dilatation to ACh. In contrast, both ACE‐inhibitors fully normalized total dilatation to ACh, including the part mediated by NO (no significant differences between zofenopril (n=10) and lisinopril (n=8)). Zofenopril, but not lisinopril, additionally potentiated the effect of endogenous NO after A23187‐induced release from the endothelium (+100%) as well as that of exogenous NO provided by nitroglycerin (+22%) and sodium nitrite (+36%) (for all P<0.05 versus no‐MI). We conclude that ACE‐inhibition with a SH‐group has a potential advantage in improvement of endothelial dysfunction through increased activity of NO after release from the endothelium into the vessel wall. Furthermore, this is the first study demonstrating the selective normalizing effect of N‐actylcysteine on NO‐contribution to ACh‐induced dilatation in experimental heart failure.

Dual pathway for angiotensin II formation in human internal mammary arteries

1 Angiotensin converting enzyme (ACE) is thought to be the main enzyme to convert antiotensin I to the vasoactive angiotensin II. Recently, in the human heart, it was found that the majority of angiotensin II formation was due to another enzyme, identified as human heart chymase. In the human vasculature however, the predominance of either ACE or non‐ACE conversion of angiotensin I remains unclear. 2 To study the effects of ACE‐ and chymase‐inhibition on angiotensin II formation in human arteries, segments of internal mammary arteries were obtained from 37 patients who underwent coronary bypass surgery. 3 Organ bath experiments showed that 100 μM captopril inhibited slightly the response to angiotensin I (pD2 from 7.09±0.11–6.79±0.10, P<0.001), while 100 μM captopril nearly abolished the response to [pro10] angiotensin I, a selective substrate for ACE, and the maximum contraction was reduced from 83±19%–23±17% of the control response (P=0.01). A significant decrease of the pD2 of angiotensin I similar to captopril was observed in the presence of 50 μM chymostatin (pD2 from 7.36±0.13–6.99±0.15, P<0.039), without influencing the maximum response. In the presence of both inhibitors, effects were much more pronounced than either inhibitor alone, and a 300 times higher dose was needed to yield a significant contraction response to angiotensin I. 4 These results indicate the presence of an ACE and a non‐ACE angiontensin II forming pathway in human internal mammary arteries.

Overexpression of the human angiotensin II type 1 receptor in the rat heart augments load induced cardiac hypertrophy

Abstract. Angiotensin II is known to stimulate cardiac hypertrophy and contractility. Most angiotensin II effects are mediated via membrane bound AT1 receptors. However, the role of myocardial AT1 receptors in cardiac hypertrophy and contractility is still rarely defined. To address the hypothesis that increased myocardial AT1 receptor density causes cardiac hypertrophy apart from high blood pressure we developed a transgenic rat model which expresses the human AT1 receptor under the control of the α-myosin heavy-chain promoter specifically in the myocardium. Expression was identified and quantified by northern blot analysis and radioligand binding assays, demonstrating overexpression of angiotensin II receptors in the transgenic rats up to 46 times the amount seen in nontransgenic rats. Coupling of the human AT1 receptor to rat G proteins and signal transduction cascade was verified by sensitivity to GTP-γ-S and increased sensitivity of intracellular Ca2+ [Ca2+]i to angiotensin II in fluo-3 loaded transgenic cardiomyocytes. Transgenic rats exhibited normal cardiac growth and function under baseline conditions. Pronounced hypertrophic growth and contractile responses to angiotensin II, however, were noted in transgenic rats challenged by volume and pressure overload. In summary, we generated a new transgenic rat model that exhibits an upregulated myocardial AT1 receptor density and demonstrates augmented cardiac hypertrophy and contractile response to angiotensin II after volume and pressure overload, but not under baseline conditions.

Endothelial Dilatory Function Predicts Individual Susceptibility to Renal Damage in the 5/6 Nephrectomized Rat

In experimental animal models of renal disease the degree of renal damage varies between individuals. This could be caused by variation in the noxious event or by differences in individual susceptibility. Intact endothelial function is assumed to provide a defense mechanism against progressive renal damage. This study hypothesized that interindividual differences in renal endothelial function might be involved in individual susceptibility to renal damage, and it investigated whether endothelial function of small renal arteries before induction of 5/6 nephrectomy (5/6 Nx) in rats was related to development of renal damage after 5/6 Nx. Wistar rats underwent 5/6 Nx, and small renal arteries of the removed right kidney were investigated for endothelium-dependent relaxation to acetylcholine (ACh, 10(-8) to 10(-4) mol/L). The contribution of underlying endothelial dilative mediators, NO, prostaglandins (PG), and endothelium-derived hyperpolarizing factor (EDHF), was assessed using the inhibitors, L-NMMA, indomethacin, and charybdotoxin+apamin, respectively. After 5/6 Nx, proteinuria developed in each rat ranging from 22 to 278 (84 +/- 14) mg/24 h at week 5 (n = 23). Interestingly, a significant inverse correlation between individual ACh-relaxation (expressed as area under curve in arbitrary units) and proteinuria 5 wk after 5/6 Nx was found (r = -0.54; P = 0.008; n = 23). An inverse correlation was also found between individual NO contribution as well as PG contribution and proteinuria 5 wk after 5/6 Nx (r = -0.86, P = 0.001, n = 11; and r = -0.74, P = 0.01, n = 11, respectively). In addition, individual ACh-relaxation was positively correlated with GFR measured 6 wk after 5/6 Nx (r = 0.58; P = 0.016; n = 17). This study demonstrates for the first time that individual renal endothelial dilatory function of the healthy rat predicts susceptibility to renal damage after 5/6 Nx, which seems to depend on individual endothelial NO and PG activity.

Cardiovascular end-organ damage in Ren-2 transgenic rats compared to spontaneously hypertensive rats

Abstract To compare hypertensive end-organ damage in two genetic forms of hypertension we assessed cardiovascular function in two rat strains of genetic hypertension: transgenic rats overexpressing the mouse Ren-2 gene [(TGR(mREN2)27]) and blood pressure matched spontaneously hypertensive rats (SHR). Despite similarly elevated blood pressure, systolic dp/dt (mmHg/s) was more impaired in transgenic rats (3099±446) than in SHR (3571±272) and normals (4342±119; P<0.05). Left ventricular weight (mg/g body weight) increased more in the transgenic rats (40±3) than in SHR (31±2) and normals (26±2). Endothelium-dependent relaxation was significantly decreased only in the transgenic rats. This study shows significantly more cardiac and endothelial dysfunction in transgenic, hypertensive TGR (mREN2)27 than in age and blood pressure matched SHR. This supports the hypothesis that chronic activation of the renin-angiotensin system significantly contributes to hypertensive end-organ damage.

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.