Oscar A. Carretero

Effects of angiotensin-converting enzyme inhibitors and angiotensin II type 1 receptor antagonists in rats with heart failure. Role of kinins and angiotensin II type 2 receptors.

Angiotensin-converting enzyme inhibitors (ACEi) improve cardiac function and remodeling and prolong survival in patients with heart failure (HF). Blockade of the renin-angiotensin system (RAS) with an angiotensin II type 1 receptor antagonist (AT1-ant) may have a similar beneficial effect. In addition to inhibition of the RAS, ACEi may also act by inhibiting kinin destruction, whereas AT1-ant may block the RAS at the level of the AT1 receptor and activate the angiotensin II type 2 (AT2) receptor. Using a model of HF induced by myocardial infarction (MI) in rats, we studied the role of kinins in the cardioprotective effect of ACEi. We also investigated whether an AT1-ant has a similar effect and whether these effects are partly due to activation of the AT2 receptor. Two months after MI, rats were treated for 2 mo with: (a) vehicle; (b) the ACEi ramipril, with and without the B2 receptor antagonist icatibant (B2-ant); or (c) an AT1-ant with and without an AT2-antagonist (AT2-ant) or B2-ant. Vehicle-treated rats had a significant increase in left ventricular end-diastolic (LVEDV) and end-systolic volume (LVESV) as well as interstitial collagen deposition and cardiomyocyte size, whereas ejection fraction was decreased. Left ventricular remodeling and cardiac function were improved by the ACEi and AT1-ant. The B2-ant blocked most of the cardioprotective effect of the ACEi, whereas the effect of the AT1-ant was blocked by the AT2-ant. The decreases in LVEDV and LVESV caused by the AT1-ant were also partially blocked by the B2-ant. We concluded that (a) in HF both ACEi and AT1-ant have a cardioprotective effect, which could be due to either a direct action on the heart or secondary to altered hemodynamics, or both; and (b) the effect of the ACEi is mediated in part by kinins, whereas that of the AT1-ant is triggered by activation of the AT2 receptor and is also mediated in part by kinins. We speculate that in HF, blockade of AT1 receptors increases both renin and angiotensins; these angiotensins stimulate the AT2 receptor, which in turn may play an important role in the therapeutic effect of the AT1-ant via kinins and other autacoids.

Echocardiographic assessment of cardiac function in conscious and anesthetized mice.

Using a high-frequency linear transducer (15L8), we studied 1) the feasibility of performing echocardiography in nonanesthetized mice compared with mice given pentobarbital sodium (Pento) or a mixture of ketamine and xylazine and 2) the feasibility of echocardiographic evaluation of left ventricular (LV) hypertrophy, dilatation, and function in mice with two-kidney, one-clip hypertension or myocardial infarction (MI). Heart rate (HR) in awake mice was 658 +/- 9 beats/min; Pento and ketamine plus xylazine reduced HR to 377 +/- 11 and 293 +/- 19 beats/min, respectively, associated with a significant decrease in shortening fraction (SF), ejection fraction (EF), and cardiac output (CO) and an increase in LV end-diastolic (LVEDD) and end-systolic dimensions (LVESD). Mice with 4 wk of two-kidney, one-clip hypertension had increased LV mass (15.62 +/- 0. 62 vs. 22.17 +/- 1.79 mg) without altered LV dimensions, SF, EF, or CO. Mice studied 4 wk post-MI exhibited obvious LV dilatation and systolic dysfunction, as evidenced by increased LVEDD and LVESD and decreased SF, EF, and CO. Our findings clearly show the adverse impact of anesthesia on basal cardiac function and the difficulty in interpreting data obtained from anesthetized mice. We believe this is the first study to demonstrate the feasibility of using echocardiography to assess cardiovascular function in the nonanesthetized mouse.Using a high-frequency linear transducer (15L8), we studied 1) the feasibility of performing echocardiography in nonanesthetized mice compared with mice given pentobarbital sodium (Pento) or a mixture of ketamine and xylazine and 2) the feasibility of echocardiographic evaluation of left ventricular (LV) hypertrophy, dilatation, and function in mice with two-kidney, one-clip hypertension or myocardial infarction (MI). Heart rate (HR) in awake mice was 658 ± 9 beats/min; Pento and ketamine plus xylazine reduced HR to 377 ± 11 and 293 ± 19 beats/min, respectively, associated with a significant decrease in shortening fraction (SF), ejection fraction (EF), and cardiac output (CO) and an increase in LV end-diastolic (LVEDD) and end-systolic dimensions (LVESD). Mice with 4 wk of two-kidney, one-clip hypertension had increased LV mass (15.62 ± 0.62 vs. 22.17 ± 1.79 mg) without altered LV dimensions, SF, EF, or CO. Mice studied 4 wk post-MI exhibited obvious LV dilatation and systolic dysfunction, as evidenced by increased LVEDD and LVESD and decreased SF, EF, and CO. Our findings clearly show the adverse impact of anesthesia on basal cardiac function and the difficulty in interpreting data obtained from anesthetized mice. We believe this is the first study to demonstrate the feasibility of using echocardiography to assess cardiovascular function in the nonanesthetized mouse.

Modulation of angiotensin II-induced vasoconstriction by endothelium-derived relaxing factor in the isolated microperfused rabbit afferent arteriole.

Although endothelium-derived relaxing factor (EDRF) has been studied extensively in large vessels, little is known about its role in the preglomerular afferent arteriole (Af-Art). We tested the hypothesis that EDRF, which is produced locally in the Af-Art, modulates arteriolar responses to angiotensin II (AII). A single rabbit Af-Art with its glomerulus intact was microperfused in vitro at 60 mmHg. When 0.1 microM AII was first applied, luminal diameter decreased by 49 +/- 7.0% (n = 9; P less than 0.0001); however, constriction waned, with the decrease becoming 15 +/- 3.5% at 1 min. After washing the Af-Art, repeated AII caused less constriction (13 +/- 4.0%; P less than 0.0002 vs. first application), showing tachyphylaxis. Pretreatment with Nw-nitro-L-arginine (N-Arg), which inhibits synthesis of nitric oxide (an EDRF), decreased basal diameter by 18 +/- 3.0% (n = 14; P less than 0.0001). N-Arg also augmented AII-induced constriction (86 +/- 6.8%; P less than 0.02 vs. nontreated Af-Art) and rendered it persistent (82 +/- 6.9% at 1 min). Even after pretreatment with N-Arg, repeated AII caused a weaker response, which was restored by washing with kidney homogenate rich in angiotensinase. In conclusion, this study provides evidence that local production of EDRF is an important determinant of the tone of the Af-Art. Our results suggest that the transient nature of AII-induced constriction of the Af-Art may be due to production of EDRF, while tachyphylaxis may be the result of long lasting receptor occupancy.

Endothelium-derived relaxing factor/nitric oxide modulates angiotensin II action in the isolated microperfused rabbit afferent but not efferent arteriole.

It has been reported that sensitivity to angiotensin II (Ang II) is higher in efferent (Ef) than afferent (Af) arterioles (Arts). We tested the hypothesis that this is due to arteriolar differences in the interaction between Ang II and endothelium-derived relaxing factor/nitric oxide (EDNO). Rabbit Af-Arts with glomerulus intact were microperfused in vitro at a constant pressure. Ef-Arts were perfused from the distal end of either the Af-Art (orthograde perfusion) or the Ef-Art (retrograde perfusion) to eliminate influences of the Af-Art or glomerulus, respectively. Ang II did not alter Af-Art luminal diameter until the concentration reached 10(-9) M, which decreased the diameter by 11 +/- 2.6% (n = 11; P < 0.002). In contrast, Ef-Arts became significantly constricted at concentrations as low as 10(-11) M with either perfusion. Surprisingly, the decrease in Ef-Art diameter at 10(-10), 10(-9), and 10(-8) M was significantly greater with retrograde perfusion (44 +/- 6.9%, 70 +/- 5.6%, and 74 +/- 4.1%, respectively; n = 5) than with orthograde perfusion (16 +/- 4.2%, 25 +/- 2.9%, and 35 +/- 3.5%; n = 9). ENDO synthesis inhibition with 10(-4) M nitro-L-arginine methyl ester (L-NAME) decreased the diameter to a greater extent in Af-Arts (22 +/- 3.0%; n = 11) compared to Ef-Arts with either orthograde (9.5 +/- 2.3%; n = 8) or retrograde perfusion (1.2 +/- 2.1%; n = 6). With L-NAME pretreatment, Af-Art constriction induced by 10(-10) M (14 +/- 4.0%, n = 9) and 10(-9) M Ang II (38 +/- 3.9%) was significantly greater compared to nontreated Af-Arts. In contrast, L-NAME pretreatment had no effect on Ang II-induced constriction in Ef-Arts with either perfusion. In conclusion, this study demonstrates higher sensitivity of Ef-Arts to Ang II, particularly with retrograde perfusion. Our results suggest that EDNO significantly modulates the vasoconstrictor action of Ang II in Af-Arts II but not Ef-Arts, contributing to the differential sensitivity to Ang II.

N-acetyl-seryl-aspartyl-lysyl-proline prevents cardiac remodeling and dysfunction induced by galectin-3, a mammalian adhesion/growth-regulatory lectin.

Galectin-3 (Gal-3) is secreted by activated macrophages. In hypertension, Gal-3 is a marker for hypertrophic hearts prone to develop heart failure. Gal-3 infused in pericardial sac leads to cardiac inflammation, remodeling, and dysfunction. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), a naturally occurring tetrapeptide, prevents and reverses inflammation and collagen deposition in the heart in hypertension and heart failure postmyocardial infarction. In the present study, we hypothesize that Ac-SDKP prevents Gal-3-induced cardiac inflammation, remodeling, and dysfunction, and these effects are mediated by the transforming growth factor (TGF)-beta/Smad3 signaling pathway. Adult male rats were divided into four groups and received the following intrapericardial infusion for 4 wk: 1) vehicle (saline, n = 8); 2) Ac-SDKP (800 microg x kg(-1) x day(-1), n = 8); 3) Gal-3 (12 microg/day, n = 7); and 4) Ac-SDKP + Gal-3 (n = 7). Left ventricular ejection fraction, cardiac output, and transmitral velocity were measured by echocardiography; inflammatory cell infiltration, cardiomyocyte hypertrophy, and collagen deposition in the heart by histological and immunohistochemical staining; and TGF-beta expression and Smad3 phosphorylation by Western blot. We found that, in the left ventricle, Gal-3 1) enhanced macrophage and mast cell infiltration, increased cardiac interstitial and perivascular fibrosis, and causes cardiac hypertrophy; 2) increased TGF-beta expression and Smad3 phosphorylation; and 3) decreased negative change in pressure over time response to isoproterenol challenge, ratio of early left ventricular filling phase to atrial contraction phase, and left ventricular ejection fraction. Ac-SDKP partially or completely prevented these effects. We conclude that Ac-SDKP prevents Gal-3-induced cardiac inflammation, fibrosis, hypertrophy, and dysfunction, possibly via inhibition of the TGF-beta/Smad3 signaling pathway.

Effect of High Salt Intake in Mutant Mice Lacking Bradykinin-B2 Receptors

Renal kinins release prostaglandins and nitric oxide via the B2 receptor, promoting diuresis and natriuresis; hence, they may also contribute significantly to blood pressure regulation. We hypothesized that mutant mice lacking the gene encoding for the bradykinin-B2 receptor (B2-KO) become hypertensive when placed on a long-term high-salt diet. To test this, B2-KO and control mice were placed on either a normal (0.2%) or high-Na+ diet (3.15% in food plus 1% saline as drinking water) for 8 weeks. Systolic blood pressure was determined during weeks 6 and 8 by a computerized tail-cuff system. At the end of the 8-week period, mice were anesthetized for determination of mean blood pressure, renal blood flow, and renal vascular resistance. In B2-KO mice maintained on high Na+, systolic blood pressure was 15 mm Hg higher than in knockout animals on normal Na+ (P < .01). In contrast, there was no difference in blood pressure in control mice fed either a normal or a high-Na+ diet. Consistent with the systolic blood pressure data, direct mean arterial pressure revealed that B2-KO mice on high Na+ were hypertensive (115 +/- 6 in B2-KO on high-Na+ diet versus 79 +/- 2.8 in B2-KO on normal Na+, P < .0001); renal blood flow was reduced by 20% (P < .05) and renal vascular resistance was doubled (P < .0001) compared with B2-KO mice on normal Na+. In contrast, control mice on high Na+ were normotensive and tended to have increased renal blood flow and decreased renal vascular resistance compared with control mice on a normal Na+ diet. These findings indicate that kinins play an important role in preventing salt-sensitive hypertension; this may be achieved by maintaining renal blood flow under conditions of high salt intake.

Paracrine Systems in the Cardioprotective Effect of Angiotensin-Converting Enzyme Inhibitors on Myocardial Ischemia/Reperfusion Injury in Rats

After transient episodes of ischemia, benefits of thrombolytic or angioplastic therapy may be limited by reperfusion injury. Angiotensin-converting enzyme inhibitors protect the heart against ischemia/reperfusion injury, an effect mediated by kinins. We examined whether the protective effect of the angiotensin-converting enzyme inhibitor ramiprilat on myocardial ischemia/reperfusion is due to kinin stimulation of prostaglandin and/or nitric oxide release. The left anterior descending coronary artery of Lewis inbred rats was occluded for 30 minutes, followed by 120 minutes of reperfusion. Immediately before reperfusion rats were treated with vehicle, ramiprilat, or the angiotensin II type 1 receptor antagonist losartan. We tested whether pretreatment with the kinin receptor antagonist Hoe 140, the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester, or the cyclooxygenase inhibitor indomethacin blocked the effect of ramiprilat on infarct size and reperfusion arrhythmias. In controls, infarct size as a percentage of the area at risk was 79 +/- 3%; ramiprilat reduced this to 49 +/- 4% (P < .001), but losartan had little effect (74 +/- 6%, P = NS). Pretreatment with Hoe 140, NG-nitro-L-arginine methyl ester, or indomethacin abolished the beneficial effect of ramiprilat. Compared with the 30-minute ischemia/120-minute reperfusion group, nonreperfused hearts with 30 minutes of ischemia had significantly smaller infarct size as a percentage of the area at risk, whereas in the 150-minute ischemia group it was significantly larger. This suggests that reperfusion caused a significant part of the myocardial injury, but it also suggests that compared with prolonged ischemia, reperfusion salvaged some of the myocardium. Ventricular arrhythmias mirrored the changes in infarct size. Thus, angiotensin-converting enzyme inhibitors protect the myocardium against ischemia/reperfusion injury and arrhythmias; these beneficial effects are mediated primarily by a kinin-prostaglandin-nitric oxide pathway, not inhibition of angiotensin II formation.

A local kallikrein-kinin system is present in rat hearts.

It has been reported that kinins mediate part of the beneficial cardiac effects induced by treatment with angiotensin-converting enzyme inhibitors in situations such as ischemia-reperfusion injury, myocardial infarction, and cardiac hypertrophy. However, it is not known whether the heart contains an independent kallikrein-kinin system. We measured kallikrein in tissue and in the incubation medium of heart slices. Heart slices released active and total (trypsin-activatable) kallikrein into the medium (46 +/- 5 and 380 +/- 18 pg bradykinin/mg, respectively, after 1 hour and 78 +/- 6 and 654 +/- 14 pg bradykinin/mg after 2 hours, n = 7). Release was not due to tissue damage because lactate dehydrogenase, a cytosolic marker, decreased from 8.9 +/- 2.9 to 2.9 +/- 1.0 U/mg per hour. Although kallikrein was released, total tissue kallikrein in the slices did not change (423 +/- 25 pg bradykinin/mg in nonincubated slices and 370 +/- 42 pg bradykinin/mg after 2 hours, P = NS), suggesting pool replenishment. Cardiac kallikrein activity was inhibited by incubation with anti-glandular kallikrein antibodies. Pretreatment with the protein synthesis inhibitor puromycin (10 mg IP) lowered release of active kallikrein from 78 +/- 6 to 22 +/- 4 pg bradykinin/mg and total kallikrein from 654 +/- 14 to 113 +/- 9 pg bradykinin/mg (P < .001). By using reverse transcription polymerase chain reaction with kallikrein family oligonucleotide primers and a specific kallikrein probe, we found that mRNA for tissue kallikrein is present in both atrial and ventricular RNA. Kallikrein activity was also detected in primary cultures of neonatal rat atrial and ventricular cardiocytes and their incubation medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelial Nitric Oxide Gene Knockout Mice Cardiac Phenotypes and the Effect of Angiotensin-Converting Enzyme Inhibitor on Myocardial Ischemia/Reperfusion Injury

We tested the hypothesis that nitric oxide (NO) released by endothelial NO synthase (eNOS) is not only important in blood pressure regulation but also involved in cardiac function and remodeling and in the cardioprotective effect of angiotensin-converting enzyme inhibitors (ACEi). With the use of a 2D Doppler echocardiography system equipped with a 15-MHz linear transducer, we evaluated left ventricular (LV) morphology and function in conscious eNOS knockout mice (eNOS(-/-); n=15) and their wild-type littermates (eNOS(+/+); n=16). We also studied whether in eNOS(-/-) mice (1) myocardial ischemia/reperfusion injury is more severe and (2) the cardioprotective effect of ACEi is diminished or absent. In comparison with the wild type, eNOS(-/-) mice had significantly increased systolic blood pressure (128+/-3 versus 108+/-5 mm Hg; P<0.001) and decreased heart rate (531+/-22 versus 629+/-18 bpm; P<0.001) associated with increased LV posterior wall thickness (0.80+/-0.04 versus 0.64+/-0.02 mm; P<0.001) and LV mass (18.3+/-0.9 versus 13.1+/-0.5 mg/10 g body weight; P<0.01). Despite hypertension and LV hypertrophy, LV chamber dimension, shortening fraction and ejection fraction (indicators of LV contractility), and cardiac output did not differ between the 2 strains, which indicates that LV function in eNOS(-/-) mice is well compensated. We also found that in eNOS(+/+) mice, ACEi decreased the ratio of myocardial infarct size to area at risk from 62.7+/-3.9% to 36.3+/-1.6% (P<0. 001), whereas in eNOS(-/-) mice this effect of ACEi was almost abolished: the ratio of myocardial infarct size to area at risk was 67.2+/-2.9% in the vehicle-treated group and 62.7+/-3.9% in mice treated with ACEi. Moreover, infarct size in vehicle-treated eNOS(-/-) mice was not significantly different from eNOS(+/+) mice given the same treatment. We concluded that (1) endothelium-derived NO plays an important role in the regulation of blood pressure homeostasis; (2) NO released under basal conditions has no significant impact on cardiac function; and (3) ACEi protect the heart against ischemia/reperfusion injury in mice and that this effect is mediated in part by endothelium-derived NO.

Novel NAD(P)H Oxidase Inhibitor Suppresses Angioplasty-Induced Superoxide and Neointimal Hyperplasia of Rat Carotid Artery

Abstract— Neointimal proliferation occurring after vascular or endovascular procedures is a major complication leading to end-organ or limb ischemia. In experimental models, balloon injury has been shown to induce NAD(P)H oxidase to produce vascular superoxide anion (O2·−) production, which has been implicated in cell proliferation, but a direct link is still unclear. We postulated that inhibition of arterial NAD(P)H oxidase, resulting in decreased O2·−, would lessen the neointimal hyperplasia caused by balloon injury to the common carotid artery (CCA). Sprague-Dawley rats were implanted with osmotic minipumps containing either vehicle, a cell-permeant peptide that inhibits NAD(P)H oxidase (gp91ds-tat, 10 mg/kg per day), or a scrambled peptide control (scrmb-tat). Two days after pump implantation, the left CCA was injured using an intravascular balloon embolectomy catheter (2F Fogarty). Systolic blood pressure was monitored by tail cuff. Fourteen days after injury, CCAs were harvested and analyzed by digital morphometry. Rats in both groups remained normotensive, with no significant differences in systolic blood pressure. Reactive oxygen species measurements after injury indicated a significant reduction in vascular O2·− in rats infused with gp91ds-tat, and the neointima/media area and thickness ratios were significantly lower in their arteries compared with control. On the contrary, no significant change in overall CCA diameter was observed in any group. Our data indicate that in response to balloon injury of the rat carotid artery, NAD(P)H oxidase activity contributes to neointimal hyperplasia and is involved in vascular cell proliferation and migration during restenosis.