Louis J. Dell’Italia

Angiotensin II formation from ACE and chymase in human and animal hearts: methods and species considerations.

The current study examined the contributions of angiotensin-converting enzyme (ACE) vs. chymase to angiotensin II (ANG II) generation in membrane preparations from left ventricles of humans, dogs, rabbits, and rats and from total heart of mice. ACE and chymase activity were measured in membrane preparations extracted with low or high detergent (LD and HD, respectively) concentrations. We hypothesized that ACE, which is membrane bound in vivo, would be preferentially localized to the HD preparation, whereas chymase, which is localized to the cytoplasm and cardiac interstitium, would be localized to the LD preparation. In human heart, ACE activity was 16-fold higher in the HD than in the LD preparation, whereas chymase activity was 15-fold higher in the LD than in the HD preparation. Total ANG II formation was greater in human heart [15.8 ± 3.4 (SE) μmol ANG II ⋅ g-1 ⋅ min-1] than in dog, rat, rabbit, and mouse hearts (3.90 ± 0.35, 0.41 ± 0.02, 0.61 ± 0.07, and 1.16 ± 0.08 μmol ANG II ⋅ g-1 ⋅ min-1, respectively, P < 0.05, by analysis of variance). ANG II formation from ACE was higher in mouse heart (1.09 ± 0.05 μmol ANG II ⋅ g-1 ⋅ min-1, P < 0.001) than in rabbit, human, dog, and rat hearts (0.55 ± 0.06, 0.34 ± 0.01, 0.32 ± 0.06, and 0.31 ± 0.02 μmol ANG II ⋅ g-1 ⋅ min-1, respectively). In contrast, chymase activity was higher in human heart (15.3 ± 3.4 μmol ANG II ⋅ g-1 ⋅ min-1) than in dog, rat, rabbit, and mouse hearts (3.59 ± 0.29, 0.10 ± 0.01, 0.06 ± 0.01, and 0.07 ± 0.01 μmol ANG II ⋅ g-1 ⋅ min-1, respectively). Our results demonstrate important species differences in the pathways of intracardiac ANG II generation. Chymase predominated over ACE activity in human heart, accounting for extremely high total ANG II formation in human heart compared with dog, rat, rabbit, and mouse hearts.

REFRACTORY HYPERTENSION: DEFINITION, PREVALENCE AND PATIENT CHARACTERISTICS

J Clin Hypertens (Greenwich).

Angiotensin II receptor blockade does not improve left ventricular function andremodeling in subacute mitral regurgitation in the dog

OBJECTIVES We hypothesized that angiotensin II type-1 (AT(1)) receptor blocker (AT(1)RB) would prevent adverse left ventricular (LV) remodeling and LV dysfunction when started at the outset of mitral regurgitation (MR). BACKGROUND Little is known regarding the efficacy of AT(1)RB treatment of MR. METHODS Mitral regurgitation was induced by chordal disruption in adult mongrel dogs. Six normal dogs (NLs) were compared to six untreated MR dogs (MR) and seven dogs treated with the receptor blocker irbesartan (MR+AT(1)RB) started 24 h after induction of MR (60 mg/kg p.o. b.i.d.) and continued for three months. RESULTS Treatment with AT(1)RB decreased systemic vascular resistance but did not significantly improve cardiac output, LV end-diastolic dimension (LVEDD) or LVEDD/wall thickness compared to untreated MR dogs. Resting isolated cardiomyocyte length increased in MR versus NLs and was further increased in AT(1)RB dogs. Left ventricular end-systolic dimension increased to a greater extent from baseline in AT(1)RB dogs versus untreated MR dogs (29 +/- 9% vs. 12 +/- 6%, p < 0.05), despite a significantly lower LV peak systolic pressure in AT(1)RB dogs. Plasma-angiotensin (ANG) II was elevated greater than threefold in both MR and MR+AT(1)RB versus NLs. In contrast, intracardiac ANG II was increased greater than twofold in MR dogs versus NLs, but was normalized by AT(1)RB. CONCLUSIONS The use of AT(1)RB decreased systemic vascular resistance and attenuated local expression of the renin-angiotensin system but did not prevent adverse LV chamber and cardiomyocyte remodeling. These results suggest that blockade of the AT(1) receptor does not improve LV remodeling and function in the early myocardial adaptive phase of MR.

Involvement of chymase-mediated angiotensin II generation in blood pressure regulation

Angiotensin I-converting enzyme (ACE) inhibitors are thought to lower blood pressure in hypertensive patients, mainly by decreasing angiotensin II (Ang II) formation. Chymase, a human mast cell protease, has recently been proposed to play a role in blood pressure regulation because of its Ang II-forming activity. Here we show that the predominant chymase mRNA species in the mouse aorta are those for types 4 and 5 isoforms, and that both are efficient Ang II-forming enzymes. Evaluation of ACE-dependent and ACE-independent Ang II-forming pathways in mast cell-deficient (Kit(w)/Kit(w-v)) mice and their mast cell-sufficient littermate (MC(+/+)) controls revealed that, in contrast to the latter, Kit(w)/Kit(w-v) mice fail to express chymase mRNAs in the vasculature and have almost no ACE-independent Ang II-forming activity in either isolated blood vessels or homogenates. Moreover, in MC(+/+) but not in Kit(w)/Kit(w-v) mice, a contribution of ACE-independent Ang II generation to blood pressure regulation was evident by a 1.6-fold greater maximal reduction in mean arterial pressure with acute ACE inhibition plus AT(1) receptor blockade than with ACE inhibition alone. Thus, mast cells are the source of the vascular ACE-independent pathway, and the antihypertensive benefit of combining ACE inhibitor therapy with AT(1) receptor antagonist therapy is most likely due to negation of chymase-catalyzed Ang II generation.

Evidence for Angiotensin-Converting Enzyme– and Chymase-Mediated Angiotensin II Formation in the Interstitial Fluid Space of the Dog Heart In Vivo

BACKGROUND We have previously demonstrated that angiotensin II (Ang II) levels in the interstitial fluid (ISF) space of the heart are higher than in the blood plasma and do not change after systemic infusion of Ang I. In this study, we assess the enzymatic mechanisms (chymase versus ACE) by which Ang II is generated in the ISF space of the dog heart in vivo. METHODS AND RESULTS Cardiac microdialysis probes were implanted in the left ventricular (LV) myocardium (3 to 4 probes per dog) of 12 anesthetized open-chest normal dogs. ISF Ang I and II levels were measured at baseline and during ISF infusion of Ang I (15 micromol/L, n=12), Ang I+the ACE inhibitor captopril (cap) (2.5 mmol/L, n=4), Ang I+the chymase inhibitor chymostatin (chy) (1 mmol/L, n=4), and Ang I+cap+chy (n=4). ISF infusion of Ang I increased ISF Ang II levels 100-fold (P<0.01), whereas aortic and coronary sinus plasma Ang I and II levels were unaffected and were 100-fold lower than ISF levels. Compared with ISF infusion of Ang I alone, Ang I+cap (n=4) produced a greater reduction in ISF Ang II levels than did Ang I+chy (n=4) (71% versus 43%, P<0.01), whereas Ang I+cap+chy produced a 100% decrease in ISF Ang II levels. CONCLUSIONS This study demonstrates for the first time a very high capacity for conversion of Ang I to Ang II mediated by both ACE and chymase in the ISF space of the dog heart in vivo.

Interaction of diet and diabetes on cardiovascular function in rats

Genetic rodent models of type 2 diabetes are routinely utilized in studies of diabetes-related cardiovascular disease; however, these models frequently exhibit abnormalities that are not consistent with diabetic complications. The aim of this study was to develop a model of type 2 diabetes that exhibits evidence of cardiovascular dysfunction commonly seen in patients with diabetes with minimal nondiabetes-related pathologies. Young male rats received either control (Con), high-fat (HF; 60%), or Western (Wes; 40% fat, 45% carbohydrate) diets for 2 wk after which streptozotocin (2 x 35 mg/kg ip 24 h apart) was administered to induce diabetes (Dia). Blood glucose levels were higher in Con + Dia and Wes + Dia groups compared with the HF + Dia group (25 +/- 1, 25 +/- 2, and 15 +/- 1 mmol/l, respectively; P < 0.05) group. Liver, kidney, and pancreatic dysfunction and cardiomyocyte lipid accumulation were found in all diabetic animals. Despite lower heart rates in Con + Dia and HF + Dia groups, arterial and left ventricular pressures were not different between any of the experimental groups. All three diabetic groups had diastolic dysfunction, but only HF + Dia and Wes + Dia groups exhibited elevated diastolic wall stress, arterial stiffness (augmentation index), and systolic dysfunction (velocity of circumferential shortening, systolic wall stress). Surprisingly, we found that left ventricular dysfunction and arterial stiffness were more pronounced in the HF + Dia than the Con + Dia group and was similar to the Wes + Dia group despite significantly lower levels of hyperglycemia compared with either group. In conclusion, the HF + Dia group exhibited a stable, modest level of hyperglycemia, which was associated with cardiac dysfunction comparable with that seen in moderate to advanced stages of human type 2 diabetes.

Short-Acting β-Adrenergic Antagonist Esmolol Given at Reperfusion Improves Survival After Prolonged Ventricular Fibrillation

Background—High catecholamine concentrations are cytotoxic to cardiac myocytes. We hypothesized that myocardial interstitial catecholamine levels are greatly elevated immediately after long-duration ventricular fibrillation (VF), defibrillation, and reperfusion and that the short-acting β-antagonist esmolol administered at reperfusion would protect against this catecholamine surge and improve survival. Methods and Results—In part 1 of this study, catecholamines from myocardial interstitial fluid (ISF) and aortic and coronary sinus plasma were quantified by use of 3H-labeled radioenzymatic assay in 8 open-chest, anesthetized pigs. Eight minutes of electrically induced VF was followed by internal defibrillation and reperfusion. By 4 minutes of VF, ISF norepinephrine increased significantly, from 1.3± 0.3 to 7.4± 2.4 ng/mL. Epinephrine increased significantly, from 0.4± 0.2 to 1.5± 0.7 ng/mL. ISF norepinephrine and epinephrine peaked at 219.2± 92.1 and 63.7± 25.1 ng/mL after defibrillation and reperfusion and decreased significantly to 12.2± 3.5 and 6.7± 3.1 ng/mL 23 minutes after defibrillation. Transcardiac catecholamine changes were similar. In part 2, 8 minutes of VF was followed by external defibrillation in anesthetized, closed-chest pigs. Animals received 1.0 mg/kg esmolol (n= 8) or saline (n= 8) intravenously at the start of cardiopulmonary resuscitation (CPR). Advanced cardiac life support, including CPR and epinephrine, was delivered to both groups. Esmolol before reperfusion improved return of spontaneous circulation and 4-hour survival (7/8 versus 3/8 survivors, χ2P < 0.05). Conclusions—Transcardiac and ISF norepinephrine and epinephrine levels are briefly massively elevated after 8 minutes of VF, defibrillation, and reperfusion. A short-acting β-antagonist administered immediately after defibrillation improves return of spontaneous circulation and 4-hour survival after this prolonged VF.

β1-Adrenergic Receptor Blockade Attenuates Angiotensin II–Mediated Catecholamine Release Into the Cardiac Interstitium in Mitral Regurgitation

Background—This study tested the hypothesis that &bgr;1-adrenoreceptor blockade modulates the angiotensin II (Ang II)–evoked neural release of norepinephrine (NE) and epinephrine (Epi) into the cardiac interstitial fluid (ISF) space in experimentally induced mitral regurgitation (MR) in the dog. Methods and Results—Normal dogs (n=8) were compared with dogs with MR of 2 (n=8) and 4 (n=6) weeks’ duration and with dogs with MR treated with &bgr;1-receptor blockade (RB; extended-release metoprolol succinate, 100 mg QD; MR+&bgr;1-RB) that was started 24 hours after MR induction for 2 (n=6) and 4 weeks (n=8). Left ventricular end-diastolic dimension increased 20% as plasma Ang II levels increased >5-fold in both MR and MR+&bgr;1-RB dogs at 2 and 4 weeks. Ang II infusion into the left atrium produced increases in ISF NE and Epi in normal dogs, which were further increased in 2- and 4-week MR dogs but were restored to normal in 4-week MR+&bgr;1-RB dogs. Ang II infusion produced 4-fold increases in circulating NE and Epi in 2- and 4-week MR dogs that returned to normal in 4-week+&bgr;1-RB dogs. Left ventricular angiotensin-converting enzyme activity and ISF Ang II were increased in 4-week MR dogs but were decreased in 4-week MR+&bgr;1-RB dogs. Conclusions—&bgr;1-RB decreases renin-angiotensin system sympathostimulation and activation by attenuating the Ang II–mediated NE and Epi release into the cardiac ISF and circulation and by decreasing left ventricular angiotensin-converting enzyme expression in the early phases of volume overload.

An evolving story of angiotensin-II-forming pathways in rodents and humans.

Lessons learned from the characterization of the biological roles of Ang-(1-7) [angiotensin-(1-7)] in opposing the vasoconstrictor, proliferative and prothrombotic actions of AngII (angiotensin II) created an underpinning for a more comprehensive exploration of the multiple pathways by which the RAS (renin-angiotensin system) of blood and tissues regulates homoeostasis and its altered state in disease processes. The present review summarizes the progress that has been made in the novel exploration of intermediate shorter forms of angiotensinogen through the characterization of the expression and functions of the dodecapeptide Ang-(1-12) [angiotensin-(1-12)] in the cardiac production of AngII. The studies reveal significant differences in humans compared with rodents regarding the enzymatic pathway by which Ang-(1-12) undergoes metabolism. Highlights of the research include the demonstration of chymase-directed formation of AngII from Ang-(1-12) in human left atrial myocytes and left ventricular tissue, the presence of robust expression of Ang-(1-12) and chymase in the atrial appendage of subjects with resistant atrial fibrillation, and the preliminary observation of significantly higher Ang-(1-12) expression in human left atrial appendages.

Activation of AKT by O-Linked N-Acetylglucosamine Induces Vascular Calcification in Diabetes Mellitus

Rationale: Vascular calcification is a serious cardiovascular complication that contributes to the increased morbidity and mortality of patients with diabetes mellitus. Hyperglycemia, a hallmark of diabetes mellitus, is associated with increased vascular calcification and increased modification of proteins by O-linked N-acetylglucosamine (O-GlcNAcylation). Objective: We sought to determine the role of protein O-GlcNAcylation in regulating vascular calcification and the underlying mechanisms. Methods and Results: Low-dose streptozotocin-induced diabetic mice exhibited increased aortic O-GlcNAcylation and vascular calcification, which was also associated with impaired aortic compliance in mice. Elevation of O-GlcNAcylation by administration of Thiamet-G, a potent inhibitor for O-GlcNAcase that removes O-GlcNAcylation, further accelerated vascular calcification and worsened aortic compliance of diabetic mice in vivo. Increased O-GlcNAcylation, either by Thiamet-G or O-GlcNAcase knockdown, promoted calcification of primary mouse vascular smooth muscle cells. Increased O-GlcNAcylation in diabetic arteries or in the O-GlcNAcase knockdown vascular smooth muscle cell upregulated expression of the osteogenic transcription factor Runx2 and enhanced activation of AKT. O-GlcNAcylation of AKT at two new sites, T430 and T479, promoted AKT phosphorylation, which in turn enhanced vascular smooth muscle cell calcification. Site-directed mutation of AKT at T430 and T479 decreased O-GlcNAcylation, inhibited phosphorylation of AKT at S473 and binding of mammalian target of rapamycin complex 2 to AKT, and subsequently blocked Runx2 transactivity and vascular smooth muscle cell calcification. Conclusions: O-GlcNAcylation of AKT at 2 new sites enhanced AKT phosphorylation and activation, thus promoting vascular calcification. Our studies have identified a novel causative effect of O-GlcNAcylation in regulating vascular calcification in diabetes mellitus and uncovered a key molecular mechanism underlying O-GlcNAcylation–mediated activation of AKT.