Andrea Daragó

New perspectives in the renin-angiotensin-aldosterone system (RAAS) I: endogenous angiotensin converting enzyme (ACE) inhibition.

Angiotensin-converting enzyme (ACE) inhibitors represent the fifth most often prescribed drugs. ACE inhibitors decrease 5-year mortality by approximately one-fifth in cardiovascular patients. Surprisingly, there are reports dating back to 1979 suggesting the existence of endogenous ACE inhibitors, which endogenous inhibitory effects are much less characterized than that for the clinically administered ACE inhibitors. Here we aimed to investigate this endogenous ACE inhibition in human sera. It was hypothesized that ACE activity is masked by an endogenous inhibitor, which dissociates from the ACE when its concentration decreases upon dilution. ACE activity was measured by FAPGG hydrolysis first. The specific (dilution corrected) enzyme activities significantly increased by dilution of human serum samples (23.2±0.7 U/L at 4-fold dilution, 51.4±0.3 U/L at 32-fold dilution, n = 3, p = 0.001), suggesting the presence of an endogenous inhibitor. In accordance, specific enzyme activities did not changed by dilution when purified renal ACE was used, where no endogenous inhibitor was present (655±145 U/L, 605±42 U/L, n = 3, p = 0.715, respectively). FAPGG conversion strongly correlated with angiotensin I conversion suggesting that this feature is not related to the artificial substrate. Serum samples were ultra-filtered to separate ACE (MW: 180 kDa) and the hypothesized inhibitor. Filtering through 50 kDa filters was without effect, while filtering through 100 kDa filters eliminated the inhibiting factor (ACE activity after <100 kDa filtering: 56.4±2.4 U/L, n = 4, control: 26.4±0.7 U/L, n = 4, p<0.001). Lineweaver-Burk plot indicated non-competitive inhibition of ACE by this endogenous factor. The endogenous inhibitor had higher potency on the C-terminal active site than N-terminal active site of ACE. Finally, this endogenous ACE inhibition was also present in mouse, donkey, goat, bovine sera besides men (increasing of specific ACE activity from 4-fold to 32-fold dilution: 2.8-fold, 1.7-fold, 1.5-fold, 1.8-fold, 2.6-fold, respectively). We report here the existence of an evolutionary conserved mechanism suppressing circulating ACE activity, in vivo, similarly to ACE inhibitory drugs.

New Perspectives in the Renin-Angiotensin-Aldosterone System (RAAS) II: Albumin Suppresses Angiotensin Converting Enzyme (ACE) Activity in Human

About 8% of the adult population is taking angiotensin-converting enzyme (ACE) inhibitors to treat cardiovascular disease including hypertension, myocardial infarction and heart failure. These drugs decrease mortality by up to one-fifth in these patients. We and others have reported previously that endogenous inhibitory substances suppress serum ACE activity, in vivo, similarly to the ACE inhibitor drugs. Here we have made an effort to identify this endogenous ACE inhibitor substance. ACE was crosslinked with interacting proteins in human sera. The crosslinked products were immunoprecipitated and subjected to Western blot. One of the crosslinked products was recognized by both anti-ACE and anti-HSA (human serum albumin) antibodies. Direct ACE-HSA interaction was confirmed by binding assays using purified ACE and HSA. HSA inhibited human purified (circulating) and human recombinant ACE with potencies (IC50) of 5.7±0.7 and 9.5±1.1 mg/mL, respectively. Effects of HSA on the tissue bound native ACE were tested on human saphenous vein samples. Angiotensin I evoked vasoconstriction was inhibited by HSA in this vascular tissue (maximal force with HSA: 6.14±1.34 mN, without HSA: 13.54±2.63 mN), while HSA was without effects on angiotensin II mediated constrictions (maximal force with HSA: 18.73±2.17 mN, without HSA: 19.22±3.50 mN). The main finding of this study is that HSA was identified as a potent physiological inhibitor of the ACE. The enzymatic activity of ACE appears to be almost completely suppressed by HSA when it is present in its physiological concentration. These data suggest that angiotensin I conversion is limited by low physiological ACE activities, in vivo.

Vascular metabolism of anandamide to arachidonic acid affects myogenic constriction in response to intraluminal pressure elevation

AIMS We hypothesized that arachidonic acid produced by anandamide breakdown contributes to the vascular effects of anandamide. MAIN METHODS Isolated, pressurized rat skeletal muscle arteries, which possess spontaneous myogenic tone, were treated with anandamide, arachidonic acid, capsaicin (vanilloid receptor agonist), WIN 55-212-2 (cannabinoid receptor agonist), URB-597 (FAAH inhibitor), baicalein (lipoxygenase inhibitor), PPOH (cytochrome P450 inhibitor), and indomethacin (cyclooxygenase inhibitor). Changes in the arteriolar diameter in response to the various treatments were measured. To assess the effect of anandamide metabolism, anandamide was applied for 20 min followed by washout for 40 min. This protocol was used to eliminate other, more direct effects of anandamide in order to reveal how anandamide metabolism may influence vasodilation. KEY FINDINGS Anandamide at a low dose (1μM) evoked a loss of myogenic tone, while a high dose (30 μM) not only attenuated the myogenic response but also evoked acute dilation. Both of these effects were inhibited by the FAAH inhibitor URB-597 and were mimicked by arachidonic acid. The CB1 and CB2 agonist R-WIN 55-212-2 and the vanilloid receptor agonist capsaicin were without effect on the myogenic response. The inhibition of the myogenic response by anandamide was blocked by indomethacin and PPOH, but not by baicalein or removal of the endothelium. FAAH expression in the smooth muscle cells of the blood vessels was confirmed by immunohistochemistry. SIGNIFICANCE Anandamide activates the arachidonic acid pathway in the microvasculature, affecting vascular autoregulation (myogenic response) and local perfusion.

New perspectives in the renin-angiotensin-aldosterone system (RAAS) III: endogenous inhibition of angiotensin converting enzyme (ACE) provides protection against cardiovascular diseases.

ACE inhibitor drugs decrease mortality by up to one-fifth in cardiovascular patients. Surprisingly, there are reports dating back to 1979 suggesting the existence of endogenous ACE inhibitors. Here we investigated the clinical significance of this potential endogenous ACE inhibition. ACE concentration and activity was measured in patients serum samples (n = 151). ACE concentration was found to be in a wide range (47–288 ng/mL). ACE activity decreased with the increasing concentration of the serum albumin (HSA): ACE activity was 56±1 U/L in the presence of 2.4±0.3 mg/mL HSA, compared to 39±1 U/L in the presence of 12±1 mg/mL HSA (values are mean±SEM). Effects of the differences in ACE concentration were suppressed in human sera: patients with ACE DD genotype exhibited a 64% higher serum ACE concentration (range, 74–288 ng/mL, median, 155.2 ng/mL, n = 52) compared to patients with II genotype (range, 47–194 ng/mL, median, 94.5 ng/mL, n = 28) while the difference in ACE activities was only 32% (range, 27.3–59.8 U/L, median, 43.11 U/L, and range 15.6–55.4 U/L, median, 32.74 U/L, respectively) in the presence of 12±1 mg/mL HSA. No correlations were found between serum ACE concentration (or genotype) and cardiovascular diseases, in accordance with the proposed suppressed physiological ACE activities by HSA (concentration in the sera of these patients: 48.5±0.5 mg/mL) or other endogenous inhibitors. Main implications are that (1) physiological ACE activity can be stabilized at a low level by endogenous ACE inhibitors, such as HSA; (2) angiotensin II elimination may have a significant role in angiotensin II related pathologies.

Plasma homocysteine levels are related to medium-term venous graft degeneration in coronary artery bypass graft patients

Objective: Saphenous venous grafts (SVGs) are established choices for coronary artery bypass grafting (CABG); however, their lumen patency is limited. Our goal was to investigate the risk factors of SVG degeneration. Methods: Seventy-five patients (mean age, 57.5±10.4 years) with 133 SVG conduits who had cardiac catheterization ≥1 year after CABG were selected; follow-up period was 67.6±36.8 months. Patients were divided into 3 groups according to angiographic status at follow up [intact: <20% (n=23); narrowed: 20–99% (n=24); and occluded (n=28)]. Baseline clinical conditions were evaluated in relation to follow-up angiography. As onset date of chronic total occlusions is usually uncertain, they arise typically from thrombotic lesions; thus, their value in evaluation is limited. Results: There were no significant differences between the 3 groups in clinical parameters. Linear correlation analysis found significant (p<0.01) positive connection of SVG disease (luminal diameter reduction 20–99%) with C-reactive protein (CRP) and homocysteine (Hcy), as well as between CRP and Hcy. Multiple regression analysis showed plasma Hcy level to be significantly related to graft diameter reduction normalized to time elapsed until angiography in narrowed grafts: 1 µmol/L increase of Hcy was associated with 0.053%/month decrease in lumen diameter (p<0.01; R2=0.428); extrapolating: +10 µmol/L higher Hcy level during 5 years is associated with 32.1% lumen reduction. Conclusion: Medium- to long-term SVG degeneration is related to elevated plasma total Hcy in patients with sub-occlusive graft stenosis, while in cases with intact SVGs, the beneficial local flow conditions may protect the grafts from degeneration.

Differences in Angiotensin Convertase Enzyme (ACE) Activity and Expression May Contribute to Shorter Event Free Period After Coronary Artery Bypass Graft Surgery

AIMS The goal of this study was to investigate the importance of the vascular angiotensin convertase enzyme (ACE) in coronary artery bypass graft surgery (CABG) patients. METHODS Vascular tissue (distal saphenous vein [n= 163] and/or radial artery [n= 120] segments) and blood samples were collected from CABG patients (n= 81). We studied (i) the potency of angiotensin I (AngI) and angiotensin II (AngII) to evoke vascular contractions; (ii) vascular and plasma ACE concentrations; and (iii) ACE genotype of the patients enrolled. RESULTS The ratio of the potencies (EC(50) ) of AngII and AngI was significantly lower in radial artery compared to the saphenous vein (0.17 ± 0.03 nM and 0.51 ± 0.14 nM, respectively, P= 0.003), suggesting a 3-fold more effective AngI conversion in saphenous vein samples. Angiotensin constrictions were inhibited with telmisartan and captopril in both saphenous veins and radial arteries. Vascular ACE expression was significantly higher in saphenous vein compared to radial artery (9.7 ± 1.0 ng/mg and 5.3 ± 0.7 ng/mg, respectively, P= 0.01). Serum but no tissue ACE concentration was determined by ACE insertion/deletion polymorphism. Accordingly, no relation was found between serum and tissue ACE expression. CONCLUSION ACE-inhibitor therapy targeting tissue located ACE may be beneficial to patients with saphenous vein grafts after CABG surgery.