Comparative effects of a vasopeptidase inhibitor vs. an angiotensin convertin enzyme inhibitor on cardiomyocyte apoptosis in rats with heart failure

Apoptosis is involved in ventricular remodeling after myocardial infarction (MI). We investigated the effects of the vasopeptidase inhibitor (VPI) omapatrilat on cardiomyocyte apoptosis and compared it to the angiotensin converting enzyme inhibitor (ACEI) captopril in the rat post-MI model and in cultured neonatal rat cardiomyocytes. Wistar males rats surviving 4 h post-MI were assigned to omapatrilat (40 or 80 mg/kg/day), captopril (160 mg/kg/day) or no treatment. After 56 days, hemodynamic measurements were performed (n = 96) and rats were sacrificed. One group had assessment of cardiac remodeling and detection of DNA fragments by in situ end labelling method (ISEL), while the other had morphologic measurements and DNA laddering assessed. In addition, cultured neonatal rat cardiomyocytes (n = 6) were treated for 72 h with vehicle, captopril or omapatrilat in the presence or absence of the apoptosis inducing agent H₂O₂. Omapatrilat and captopril resulted in similar improvements of hemodynamic measurements, ventricular weight and dilatation, cardiac fibrosis and myocardial cell cross-section in large MI rats. Omapatrilat increased scar thickness more than did captopril. All sham-operated groups had little evidence of apoptosis. In the large MI group, there was a significant increase in ISEL-positive cells in the control (0.095 ± 0.016%) and captopril (0.124 ± 0.024%) groups in comparison with control sham-operated (0.006 ± 0.006%), but this increase was limited to the peri-MI area. Omapatrilat (0.012 ± 0.012% for both doses) prevented the increase in apoptosis in the peri-MI area. Also, omapatrilat but not captopril reduced DNA laddering in large MI. Moreover, in cultured neonatal rat cardiomyocytes, omapatrilat but not captopril reduced apoptosis as assessed by DNA laddering. The VPI omapatrilat, with its combination of NEP and ACE inhibition, suppresses cardiomyocyte apoptosis post-MI and in neonatal cultured rat cardiomyocytes more than the ACEI captopril, but this does not result in significant hemodynamic or morphologic differences between omapatrilat and captopril.     

Effects of captopril and omapatrilat on early post-myocardial infarction survival and cardiac hemodynamics in rats: interaction with cardiac cytokine expression

The purpose of this study was to evaluate and compare the effects of simultaneous angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) inhibition by the vasopeptidase inhibitor omapatrilat (10 and 40 mg x kg(-1) x day(-1)) with those of the selective ACE inhibitor captopril (160 mg x kg(-1) x day(-1)) on survival, cardiac hemodynamics, and cytokine mRNA expression in left ventricular (LV) tissues 4 days after myocardial infarction (MI) in rats. The effects of the co-administration of both B1 and B2 kinin receptor antagonists (2.5 mg x kg(-1) x day(-1) each) with and without omapatrilat were also evaluated to assess the role of bradykinin (BK) during this post-MI period. Both omapatrilat and captopril treatments improve early (4 days) post-MI survival when started 4 h post-MI. The use of kinin receptor antagonists had no significant effect on survival in untreated MI rats and omapatrilat-treated MI rats. This improvement in survival with omapatrilat and captopril is accompanied by a reduced LV end-diastolic pressure (LVEDP) and pulmonary congestion. The use of kinin receptor antagonists had little effect on cardiac hemodynamics or morphologic measurements. Acute MI significantly increased the expression of cardiac cytokines (TNF-alpha, TGF-beta1, and IL-10). Captopril significantly attenuated this activation, while omapatrilat had variable effects: sometimes increasing but generally not changing activation depending on the cytokine measured and the dose of omapatrilat used. The co-administration of both kinin receptor antagonists attenuates the increase in expression of cardiac TNF-alpha and TGF-beta1 after omapatrilat treatment. Taken together, these results would suggest that despite very marked differences in the way these drugs modified the expression of cardiac cytokines, both omapatrilat and captopril improved early (4 days) post-MI survival and cardiac function to a similar extent.     

Effects of the vasopeptidase inhibitor omapatrilat on cardiac endogenous kinins in rats with acute myocardial infarction

The purposes of this study were to evaluate and to compare the effects of simultaneous angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) inhibition by the vasopeptidase inhibitor omapatrilat (1 mg. kg(-1). day(-1)) with those of the selective ACE inhibitor enalapril (1 mg. kg(-1). day(-1)) on survival, cardiac hemodynamics, and bradykinin (BK) and des-Arg(9)-BK levels in cardiac tissues 24 h after myocardial infarction (MI) in rats. The effect of the co-administration of both B(1) and B(2) kinin receptor antagonists (2.5 mg. kg(-1). day(-1) each) with metallopeptidase inhibitors was also evaluated. The pharmacological treatments were infused subcutaneously using micro-osmotic pumps for 5 days starting 4 days before the ligation of the left coronary artery. Immunoreactive kinins were quantified by highly sensitive and specific competitive enzyme immunoassays. The post-MI mortality of untreated rats with a large MI was high; 74% of rats dying prior to the hemodynamic study. Mortality in the other MI groups was not significantly different from that of the untreated MI rats. Cardiac BK levels were not significantly different in the MI vehicle-treated group compared with the sham-operated rats. Both omapatrilat and enalapril treatments of MI rats significantly increased cardiac BK concentrations compared with the sham-operated group (P < 0.05). However, cardiac BK levels were significantly increased only in the MI omapatrilat-treated rats compared with the MI vehicle-treated group (P < 0.01). Cardiac des-Arg(9)-BK concentrations were not significantly modified by MI, and MI with omapatrilat or enalapril treatment compared with the sham-operated group. The co-administration of both kinin receptor antagonists with MI omapatrilat- and enalapril-treated rats had no significant effect on cardiac BK and des-Arg(9)-BK levels. Thus, the significant increase of cardiac BK concentrations by omapatrilat could be related to a biochemical or a cardiac hemodynamic parameter on early (24 h) post-MI state.     

Endothelial permeability in vitro and in vivo: Protective actions of ANP and omapatrilat in experimental atherosclerosis

Increased arterial endothelial cell permeability (ECP) is considered an initial step in atherosclerosis. Atrial natriuretic peptide (ANP) which is rapidly degraded by neprilysin (NEP) may reduce injury-induced endothelial cell leakiness. Omapatrilat represents a first in class of pharmacological agents which inhibits both NEP and angiotensin converting enzyme (ACE). We hypothesized that ANP prevents thrombin-induced increases of ECP in human aortic ECs (HAECs) and that omapatrilat would reduce aortic leakiness and atherogenesis and enhance ANP mediated vasorelaxation of isolated aortas. Thrombin induced ECP determined by I¹²⁵ albumin flux was assessed in HAECs with and without ANP pretreatment. Next we examined the effects of chronic oral administration of omapatrilat (12 mg/kg/day, n = 13) or placebo (n = 13) for 8 weeks on aortic leakiness, atherogenesis and ANP-mediated vasorelaxation in isolated aortas in a rabbit model of atherosclerosis produced by high cholesterol diet. In HAECs, thrombin-induced increases in ECP were prevented by ANP. Omapatrilat reduced the area of increased aortic leakiness determined by Evans-blue dye and area of atheroma formation assessed by Oil-Red staining compared to placebo. In isolated arterial rings, omapatrilat enhanced vasorelaxation to ANP compared to placebo with and without the endothelium. ANP prevents thrombin-induced increases in ECP in HAECs. Chronic oral administration of omapatrilat reduces aortic leakiness and atheroma formation with enhanced endothelial independent vasorelaxation to ANP. These studies support the therapeutic potential of dual inhibition of NEP and ACE in the prevention of increased arterial ECP and atherogenesis which may be linked to the ANP/cGMP system.     

Investigation of bradykinin metabolism in human and rat plasma in the presence of the dual ACE/NEP inhibitors GW660511X and omapatrilat.

Several studies have suggested that the accumulation of bradykinin, or that of one its metabolites BK1-8, is involved in the occurrence of side effects such as AE associated with the use of various ACEi. In this work a novel approach combining HPLC-UV on-line with oaTOF-MS and ICPMS was applied to investigate in human and rat plasma the metabolism of labelled BK (79/81 Br-Phe5) BrBK in the presence of two new dual ACE/NEP inhibitors (GW660511X and omapatrilat) currently under clinical trial. In human plasma the BrBK half-life values in the absence or in the presence of GW660511X (3.8 microM) or omapatrilat (32 nM) were 38.7 +/- 2.4, 51.2 +/- 4.7 and 114.7 +/- 9.3 min, respectively and BrBK was degraded into BrBK1-8, BrBK1-7, BrBK1-5 and Br-Phe. In the presence of inhibitors, however, the levels of these resultant metabolites were different. Unlike GW660511X, omapatrilat abolished the production of BrBK1-5 and BrBK1-7, suggesting a better ACE inhibition effect over GW660511X as no NEP activity was found. In addition the production of BrBK1-8 was enhanced in the presence of these inhibitors with a greater accumulation being observed with omapatrilat. The production of Br-Phe5 was reduced with GW660511X while no significant change was observed with omapatrilat after 4 h of incubation. In rat plasma the BrBK half-life values in the absence or in the presence of GW660511X (530 nM) or omapatrilat (50 nM) were 9.31 +/- 1.7, 22.06 +/- 3.1 and 25.3 +/- 1.7 min, respectively and BrBK was degraded into BrBK1-8, BrBK1-7, BrBK1-5 and Br-Phe5 plus BrBK2-9, BrBK4-8 and BrBK2-8 metabolites not found in human plasma. GW660511X and omapatrilat reduced the production of BrBK1-5 and BrBK1-7 with more effect being observed with omapatrilat. GW660511X and omapatrilat increased the production of both BrBK1-8 and Br-Phe5 but not that of BrBK4-8 and BrBK2-8. This study shows that the potency of GW660511X in comparison with omapatrilat is more than 100-fold lower in human, but less than 10-fold lower in rat plasma, suggesting that rat may not be a suitable in vivo model for the evaluation of ACE/NEP inhibition in relation to effects in humans.     

Bradykinin metabolism in rat hearts with left-ventricular hypertrophy following myocardial infarction

The aim of the present study was to assess the contribution of angiotensin I converting enzyme (ACE)and neutral endopeptidase (NEP) in the coronary degradation of bradykinin (BK) after left-ventricular hypertrophy following myocardial infarction (MI) in rats. Myocardial infarction was induced by left descendant coronary artery ligation, and the contribution of ACE and NEP in the degradation of exogenous BK after a single passage through the coronary bed was assessed at 2, 5, and 36 days post-MI. BK degradation rate (V(max)/Km) was found to be significantly lower in hearts at 36 days (3.30 +/- 0.28 min(-1)) compared with 2 days (4.39 +/- 0.32 min(-1)) for noninfarcted hearts, but this reduction was just above the statistical level of significance for post-MI hearts. In infarcted hearts, V(max)/Km was increased significantly 5 days post-MI (4.91 +/- 0.28 min(-1)) compared with the 2 and 36 day-groups (3.43 +/- 0.20 and 2.78 +/- 0.16 min(-1), respectively). The difference between noninfarcted and MI was significant only 2 days post-MI. Treatment with the vasopeptidase inhibitor, omapatrilat, showed that the relative contribution of ACE and NEP combined increased over time in infarcted hearts and became significantly higher 36 versus 2 days post-MI. Finally, the treatment with an ACE inhibitor (enalaprilat) and a NEP inhibitor (retrothiorphan) in the 36-day infarcted and noninfarcted hearts showed that the relative contribution of ACE in infarcted hearts was comparable with that of noninfarcted hearts, whereas the relative contribution of NEP was increased significantly in infarcted hearts. In conclusion, experimental MI in rats induces complex changes in the metabolism of exogenous BK. The changes resulted in an increased relative contribution of NEP 36 days after infarction.     

Effect of the NEP inhibitor SCH32615 on airway responses to intravenous substance P in guinea pigs.

We examined the effects of the selective neutral endopeptidase (NEP) inhibitor SCH32615 on airway responses to rapid intravenous infusions of substance P (SP) and neurokinin A (NKA) and on recovery of administered tachykinins from arterial blood in anesthetized mechanically ventilated guinea pigs. SCH32615, in doses that cause a marked increase in the magnitude of bronchoconstriction induced by infused NKA, had little effect on the changes in pulmonary conductance (GL) or dynamic compliance induced by SP. In animals in which SCH32615 (1 mg/kg) was administered in combination with the angiotensin-converting enzyme (ACE) inhibitor captopril (5.7 mg/kg), the dose of SP required to decrease GL by 50% was fourfold less than in animals that received captopril alone (P < 0.005). SP measured in arterial blood withdrawn within 45 s of intravenous administration of this tachykinin was not different in control and SCH32615-treated animals, whereas captopril caused an approximately threefold increase in SP concentrations (P < 0.005). When SCH32615 and captopril were administered together, significantly more SP was recovered than when captopril or SCH32615 was administered alone (P < 0.0005). Our results are consistent with the hypothesis that both NEP and ACE contribute to the degradation of intravenously infused SP. ACE degradation of SP is sufficient to limit SP-induced bronchoconstriction even in the presence of specific NEP inhibition.     

N-formyl hydroxylamine containing dipeptides: generation of a new class of vasopeptidase inhibitors

Four primary zinc-binding pharmacophores (thiols, carboxylates, phosphorus acids, and hydroxamates) have been utilized in generating inhibitors of zinc metalloproteases such as ACE, NEP, the MMPs, and ECE. Although compounds which inhibit the activity of both ACE and NEP (vasopeptidase inhibitors, VPIs) have been reported which incorporate a thiol, carboxylate, or phosphorus acid pharmacophore, the generation of hydroxamate based vasopeptidase inhibitors has remained elusive. Herein we report the first potent vasopeptidase inhibitors which were generated from the incorporation of conformationally restricted dipeptide mimetics to an N-formyl hydroxylamine zinc-binding group. Compounds such as 13c and 13d are among the most potent in this series, exhibiting in vitro activity comparable to other classes of inhibitors.     

The ACE inhibitors enalapril and captopril modulate cytokine responses in Balb/c and C57Bl/6 normal mice and increase CD4CD103CD25 splenic T cell numbers

Increasing evidence implies beneficial effects of angiotensin-converting enzyme (ACE) inhibitors beyond those of their original indications to control hypertension. One of the most attractive non-hemodynamic properties of ACE inhibitors is their ability to regulate cytokine production. The mechanism(s) underlying the role of ACE inhibitors on cytokine synthesis are not well understood but they have traditionally been attributed to the inhibition of angiotensin (Ang) II formation. In fact, it has been extensively demonstrated that ACE inhibitors decrease Ang II-induced production of proinflammatory cytokines and chemokines. However, it is not well described if inhibition of endogenous Ang II generation by ACE inhibitors modulates systemic cytokine production in mice. To verify that, in this work, we investigated the effects of treatment with the ACE inhibitors enalapril and captopril on cytokine synthesis in C57Bl/6 and Balb/c mice. Our results show that enalapril up regulates IL-10 produced by splenocytes from Balb/c and C57Bl/6 mice and captopril increased it only in Balb/c mice. Furthermore, CD4⁺CD103⁺ presented increased IL-10 production after enalapril treatment. Enalapril as well as captopril short-term treatment enhanced IL-2 synthesis in Balb/c mice. Besides, enhanced IL-2 and IL-10 levels correlates with increased CD4⁺CD103⁺CD25^negative T cells numbers in spleens from enalapril-treated mice.     

Effects of pre-, peri-, and postmyocardial infarction treatment with omapatrilat in rats: survival,arrhythmias, ventricular function,and remodeling

We showed previously that the vasopeptidase inhibitor (VPI) omapatrilat improves peri-myocardial infarction (MI) survival, but the mechanisms involved and whether these effects are sustained remained to be determined, and are the subject of this study. Rats (n = 272) received omapatrilat (20 mg x kg-1x day-1) starting 7 days before MI and continued peri- and post-MI, or no treatment (control). One group of rats had continuous ambulatory ECG and blood pressure recordings started 6 h before MI and continued until 24 h after MI, when survival was evaluated, and the rats were killed, and MI size was evaluated. A second group had left ventricular (LV) remodeling evaluated by echocardiography at 30 days and, at 38 days, had cardiac hemodynamics and morphology done and survival evaluated. Survival 24 h after MI (n = 255) improved with omapatrilat (60% vs. 46% for control; P = 0.0378). Over the next 37 days, there was no further improvement with omapatrilat but the early benefit was sustained. Omapatrilat reduced MI size 24 h after MI (36 +/- 2 vs. 42 +/- 2 mm2 for controls; P = 0.034). Omapatrilat reduced ventricular arrhythmia score 1-12 h after MI. Omapatrilat decreased blood pressure, but not during the first 24 h after MI. Omapatrilat reduced LV diastolic and systolic dimensions and LV and right ventricular weights compared with control large MI, indicating a decrease in reactive hypertrophy. Improvement in cardiac remodeling was accompanied by improved cardiac hemodynamics. Thus this study indicates that pre-, peri-, and post-MI treatment with the VPI omapatrilat is beneficial in survival, ventricular arrhythmias, LV remodeling, and cardiac function.     

Bradykinin (B2 independent effect of captopril on the development of pressure overload cardiac hypertrophy

Besides the reduction of angiotensin II formation, locally increased kinins may play a role in the cardiovascular action of angiotensin converting enzyme (ACE) inhibitors.To characterize the contribution of bradykinin to the effects of ACE inhibition by captopril on the development of pressure overload hypertrophy, sham-operated rats and rats with ascending aortic constriction were treated with captopril (80 mg/kg/day) or captopril and B₂-kinin receptor antagonist HOE 140 (0.5 mg/kg/day) for 7 weeks. Left ventricular mass and geometry, hydroxyproline concentration and myosin isozymes (marker of a fetal phenotype) were assessed. Rats with aortic constriction exhibited a marked increase in left ventricular weight and diastolic pressure-volume relationship was shifted to smaller volumes. Signs of congestive heart failure were not apparent. The hydroxyproline concentration remained unaltered. However, the proportion of isomyosin V3 was increased (p < 0.05). Administration of captopril reduced (p < 0.05) systolic blood pressure, body and cardiac weight in all treated rats. The reduction of left ventricular weight was disproportionally higher in pressure overloaded rats, thus the relative left ventricular weight decreased by 15% (p < 0.05). Captopril augmented the isomyosin V1 expression (p < 0.05) in sham operated as well as pressure overloaded rats. The isomyosin V1 percentage was inversely related to the relative left ventricular weight. Two different (p < 0.05) correlation lines were detected for untreated and captopril treated rats. None of captopril associated effects were removed by simultaneously administered B₂ kinin receptor antagonist HOE 140.Thus, stimulation of bradykinin B2 receptor appears not to mediate the effects of captopril on cardiac growth and contractile proteins during the development of pressure overload hypertrophy.     

Angiotensin converting enzyme has an inhibitory role in CGRP metabolism in human skin

The neutral endopeptidase (NEP) is important for calcitonin gene related peptide (CGRP) degradation, while the role of angiotensin converting enzyme (ACE) remains unclear. By using dermal microdialysis we explored the effect of phosphoramidon (NEP blocker), captopril (ACE blocker) and a mixture of both drugs on the intensity of electrically-induced CGRP-mediated neurogenic flare. The results reveal that phosphoramidon elevated flare intensity, but that this was not further increased by adding captopril. In contrast, neurogenic flare was decreased when the drug mixture was applied in compared to NEP only. Electrically released CGRP levels could be measured directly in perfusates containing phosphoramidon and the mixture. Again, CGRP levels were elevated in phosphoramidon treated sites, and significantly reduced upon adding captopril. These findings suggest that NEP and ACE do not have additive effects regarding neuropeptide degradation. In contrast, inhibition of ACE seems to augment CGRP catabolism.     

Focus: The Science of Stress: Resveratrol Supplants Captopril’s Protective Effect on Cardiac Remodeling in a Hypertension Model Elicited by Renal Artery Stenosis

Background: Renovascular hypertension elicits cardiac damage and remodeling. Two-kidney, one-clip (2K1C) is an experimental model used to study hypertension pathophysiology. In this model, the renin-angiotensin-system (RAS) is overactive due to renal artery stenosis, leading to cardiac remodeling. Redox mechanisms underlying RAS activation mediate hypertension-induced cardiovascular damage. Preclinical studies and clinical trials demonstrated resveratrol’s protective effects in cardiovascular diseases, mainly attributed to its antioxidant properties. We hypothesized resveratrol alone or in combination with an angiotensin-converting enzyme (ACE) inhibitor would be beneficial against cardiac damage caused by renovascular hypertension. Objective: We investigated the benefits of resveratrol against cardiac remodeling in 2K1C rats compared with captopril. Methods: Male Wistar rats underwent unilateral renal stenosis – 2K1C Goldblatt model of hypertension. Systolic Blood Pressure (SBP) was measured before and 6 weeks after surgery. Hypertensive 2K1C rats presented SBP≥160 mmHg. From the 6^th week after the surgery, the animals received oral resveratrol (20 mg/kg), captopril (12 mg/kg), or their combination for 3 times per week for 3 weeks. Whole heart hypertrophy was evaluated. Histological assays assessed left ventricle hypertrophy and fibrosis. Results: Renovascular hypertension caused cardiac hypertrophy, accompanied by increased myocyte diameter and collagen deposition. Resveratrol reduced 2K1C rats’ SBP and whole heart hypertrophy, independently of captopril. Resveratrol caused a higher reduction in ventricular hypertrophy than captopril. Collagen deposition was greater reduced by 2K1C treated only with resveratrol than with captopril alone or combined with resveratrol. Conclusion: Independent of captopril, resveratrol prompts cardioprotective effects on cardiomyocyte remodeling and fibrosis resulting from renovascular hypertension in 2K1C rats.     

Effect of acute oral administration of captopril and MK-421 on vascular angiotensin converting enzyme activity in the spontaneously hypertensive rat

Vascular angiotensin converting enzyme (ACE) may be important as a potential site of action for the antihypertensive effect of ACE inhibitors. ACE activity, estimated by hydrolysis of [³H] HipGlyGly, was similar in the aorta, mesenteric and carotid arteries of SHR. ACE activity in veins was not as consistent and was significantly lower in the jugular veins and vena cava of SHR than in the arteries. Nevertheless, ACE activity in all the blood vessels examined, although less than lung ACE activity, was higher than ACE activity found in other tissues such as brain, heart and kidney. Equieffective antihypertensive doses of captopril (10 mg/kg p.o.) and MK-421 (1.0 mg/kg p.o.) dramatically inhibited ACE activity in all the arteries and veins examined. Maximal ACE inhibition occured within 15 minutes after the oral administration of captopril. In contrast, maximal ACE inhibition was slower in onset and of longer duration after MK-421 than after captopril for all the vessels. Thus, relatively high ACE activity can be measured in both arteries and veins from the spontaneously hypertensive rat (SHR) and ACE was dramatically inhibited after antihypertensive oral doses of captopril or MK-421. Furthermore, vascular ACE inhibition can be used to compare the onset and duration of activity of ACE inhibitors; MK-421 has a longer onset and duration in SHR than captopril based on inhibition of ACE in blood vessels.     

Capsaicin-induced release of tachykinins: effects of enzyme inhibitors.

We studied the effects of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) inhibition on the airway responses and the recovery of endogenously released substance P- and neurokinin A-like immunoreactivities (SP-LI and NKA-LI) after tracheal injection of capsaicin in isolated guinea pig lungs superfused through the trachea. Capsaicin in doses from 10(-10) to 10(-7) mol induced a dose-dependent increase in airway opening pressure and release of SP-LI and NKA-LI. Airway opening pressure changes and the recovery of SP-LI and NKA-LI were significantly greater in lungs superfused with the NEP inhibitor SCH 32615 than in control lungs. ACE inhibition with captopril did not increase the mechanical response or the recovery of SP-LI compared with lungs not receiving captopril. In lungs from guinea pigs pretreated with high doses of capsaicin 7-10 days before study, a regimen designed to deplete endogenous tachykinins, there was a significant decrease in the content and release of NKA-LI and SP-LI. There were no detectable airway effects of acute capsaicin infusion even after doses of 10(-5) mol. Because NEP is important in modulating the airway effects of endogenously released tachykinins after tracheal infusion of capsaicin, but ACE is not, it seems likely that tracheal administration of capsaicin releases tachykinins from epithelial rather than endothelial loci.     

Pharmacological enhancement of the kallikrein-kinin system promotes anti-fibrotic responses in human mesangial cells.

The aim of the present study was to investigate whether pharmacological enhancement of the renal kallikrein-kinin system using the vasopeptidase inhibitor omapatrilat plays a direct role in modulating the fibrotic responses of human mesangial cells to injury. Treatment with 40 micromol/L omapatrilat was able to reduce macrophage-conditioned medium (MPCM)-induced fibronectin levels without affecting mRNA expression. MPCM injury also suppressed kallikrein and low molecular weight kininogen mRNA. Omapatrilat was able to attenuate this suppression. Bradykinin levels in contrast were increased by MPCM and treatment with omapatrilat further augmented levels. Co-incubation with the bradykinin B2 receptor antagonist HOE 140 attenuated the omapatrilat-induced lowering of fibronectin. Moreover, inhibition of cGMP release had a similar effect. Paradoxically, RT-PCR and Southern blotting demonstrated that bradykinin B2 receptor mRNA levels were down regulated in response to omapatrilat. Western blotting supported this data. Supernatant levels of tissue plasminogen activator (tPA), a product of bradykinin stimulation, were decreased by omapatrilat while cell associated tPA levels were increased. Matrix metalloproteinase-9 (MMP-9) mRNA expression was up regulated by omapatrilat treatment, although no difference in active zymogen levels was observed. In conclusion enhancement of kallikrein-kinin system appears to play a direct role in promoting anti-fibrotic responses in MPCM-injured human mesangial cells.     

Peptidase modulation of noncholinergic vagal bronchoconstriction and airway microvascular leakage

We investigated whether inhibition of neutral endopeptidase 24.11 (NEP) and/or angiotensin-converting enzyme (ACE) modifies vagally induced nonadrenergic noncholinergic (NANC) airflow obstruction and airway microvascular leakage as measured by extravasation of Evans blue dye (intravenous) in anesthetized guinea pigs. We gave phosphoramidon to inhibit NEP and enalapril maleate or captopril to inhibit ACE. Animals pretreated with inhaled phosphoramidon (7.5 or 75 nmol), enalapril maleate (87 or 870 nmol), or captopril (350 nmol) reached higher peak lung resistance (RL) values (14.3 +/- 2.7, 15.7 +/- 3.8, 16.7 +/- 3.8, 11.4 +/- 1.6, and 24.6 +/- 3.5 cmH2O.ml-1.s, respectively) than saline-treated animals (5.9 +/- 1.1; P less than 0.05) after bilateral vagus nerve stimulation (5 Hz, 10 V, 10 ms, 150 s). Intravenous phosphoramidon (1 mg/kg), but not intravenous captopril (6 mg/kg), potentiated peak RL (22.9 +/- 6.9 and 7.1 +/- 1.5 cmH2O.ml-1.s, respectively). Vagal nerve stimulation (1 and 5 Hz) increased the extravasation of Evans blue dye in tracheobronchial tissues compared with sham-stimulated animals, but this was not potentiated by inhaled enzyme inhibitors or intravenous captopril. However, intravenous phosphoramidon significantly augmented the extravasation of Evans blue dye in main bronchi and intrapulmonary airways. We conclude that degradative enzymes regulate both NANC-induced airflow obstruction and airway microvascular leakage.     

Isothiocyanates sensitize the effect of chemotherapeutic drugs via modulation of protein kinase C and telomerase in cervical cancer cells

We determined effects of the vasopeptidase inhibitor (VPI) omapatrilat and angiotensin II type 1 receptor (AT(1)R) blocker (ARB) candesartan in rats during healing between day-2 and day-21 after reperfused myocardial infarction (RMI) on left ventricular (LV) remodeling and function, and regional matrix metalloproteinase (MMP)-9, tissue inhibitor of MMP (TIMP)-3, inducible-nitric-oxide-synthase (iNOS), oxidant-generating myeloperoxidase (MPO), and cytokines tumor-necrosis-factor (TNF)-alpha, interleukin (IL)-6 and IL-10, and transforming-growth-factor (TGF)-beta(1), and collagens. Compared to RMI-placebo, both agents reversed adverse LV remodeling and systolic and diastolic dysfunction, improved collagen remodeling, and normalized MMP-9 (activity, protein, and mRNA), TIMP-3 (protein and mRNA), and iNOS, MPO, TNF-alpha, IL-6, and TGF-beta(1) proteins, and improved MMP-9/TIMP-3 balance and IL-10 levels in previously ischemic zones. The results suggest that modulation of matrix proteases, oxidants, cytokines, and NOSs with omapatrilat and candesartan contribute to reversal of adverse collagen and LV remodeling and attenuation of LV dysfunction during healing after RMI.     

Upregulation of cardiac insulin-like growth factor-I receptor by ACE inhibition after myocardial infarction: potential role in remodeling.

This study evaluated the effects of angiotensin-converting enzyme (ACE) inhibition after myocardial infarction (MI) on cardiac remodeling and gene expression and localization of components (ligands, receptors, and binding proteins) of the cardiac insulin-like growth factor (IGF) system. After ligation of the coronary artery, rats were randomized to vehicle or ACE inhibitor (Captopril, 50 mg/kg/day) for 4 weeks. Blood pressure, cardiac remodeling, and components of the IGF system were localized in the heart using in situ hybridization (ISH) and immunohistochemistry (IHC). The average infarct size was 42%. There were regional differences in the expression of the IGF system after MI, with increased IGF-I mRNA abundance in the border (24-fold) and infarct (12-fold) and increased IGF-binding protein (IGFBP)-3 mRNA in all areas of the failing left ventricle (threefold). Captopril reduced blood pressure, attenuated cardiac remodeling, and caused a threefold increase in IGF-I receptor mRNA and protein in infarct, border zone, and viable myocardium (p<0.01). Captopril had no effect on IGF-I mRNA but further increased IGFBP-3 mRNA and protein in the border zone, (p<0.05). The changes in the cardiac IGF system following MI are highly localized, persist for at least 4 weeks, and can be modulated by ACE inhibition. These data suggest that the benefits of ACE inhibitors in attenuation of cardiac remodeling may be mediated in part through increased expression of the IGF-I receptor sensitizing the myocardium to the positive effects of endogenous IGF-I.     

The cardioprotective effect of dual metallopeptidase inhibition: respective roles of endogenous kinins and natriuretic peptides

The objective of the present study was to assess the cardioprotective effect of dual NEP-ACE inhibition in relation to endogenous cardiac bradykinin (BK), its active metabolite des-Arg9-BK, endogenous brain natriuretic peptides (BNP), and cGMP. Rats were treated with the dual metallopeptidase inhibitor, omapatrilat, or the ACE inhibitor, ramipril, for 7 d (1 mg.kg(-1).d(-1)). Hearts were then isolated and subjected to a zero-flow ischemia and reperfusion (except controls), in the absence or presence of either a B2-receptor antagonist (Hoe-140), a B1-receptor antagonist (Lys-Leu8-des-Arg9-BK), or the GC-A/GC-B-receptor antagonist (HS-142-1). Chronic omapatrilat and ramipril increased the amount of endogenous BK collected upon reperfusion, but only ramipril increased that of des-Arg9-BK. Only omapatrilat increased both peak BNP and peak cGMP upon reperfusion, those increases being blocked by Hoe-140. Chronic omapatrilat (but not ramipril) decreased the total noradrenaline and lactate dehydrogenase release during the reperfusion period. Importantly, only omapatrilat improved the functional recovery of the ischemic reperfused heart, with a reduced left ventricular end-diastolic pressure, and improved developed left ventricular pressure. All cardio protective effects of omapatrilat were blocked by Hoe-140 and by HS-142-1, but not by the B1-receptor antagonist. In conclusion, a chronic treatment with a dual metallopeptidase inhibitor demonstrated a cardioprotective action not observed with an ACE inhibitor in a context of severe ischemia in rat isolated hearts, which was mediated by both endogenous BK and BNP.