Siragy, Helmy M. MD*; Xue, Chun MD*; Webb, Randy L. PhD†
Central to the pathogenesis of cardiovascular disease and its clinical sequelae in the current paradigm are oxidative stress and endothelial dysfunction with subsequent neurohormonal imbalances causing inflammation, remodeling, vasoconstriction, thrombosis, and plaque rupture.1,2 Among the important mediators whose balance is disrupted by endothelial dysfunction are nitric oxide (NO) and angiotensin II (Ang II). NO is anti-inflammatory and reduces circulating free radicals, fibrosis, and matrix deposition. Further, NO increases blood flow and cyclic guanosine 3′,5′-monophosphate (cGMP) production. The NO-cGMP pathway is antihypertrophic.3 The Ang II type 2 receptor is up-regulated in left ventricular hypertrophy and is coupled to NO-cGMP signaling. The NO-cGMP pathway may, therefore, be an important protective mechanism to prevent left ventricular hypertrophy, ventricular dilatation, and heart failure.
Ang II is a potent vasoconstrictor that magnifies endothelial dysfunction, decreases NO, and induces inflammatory states by enhancing release of interleukin (IL)-6 and other inflammatory markers. Nuclear factor-κB (NF-κB) is central to the inflammatory response, stimulating expression of tumor necrosis factor-α (TNF-α), IL-1, IL-6, and, via a feedback pathway, further NF-κB production.4,5 NF-κB is found in many cell types and is a target for anti-inflammatory treatments.
Several classes of drugs used to treat cardiovascular diseases have the potential to improve this pathologic neurohormonal imbalance. The angiotensin-converting enzyme (ACE) inhibitor benazepril reduces levels of Ang II, fibrosis, and collagen deposition.6 Treatment with an ACE inhibitor attenuates infarct expansion and decreases the rates of reinfarction and heart failure. ACE inhibitors are recognized as first-line therapy in patients with myocardial infarction on the basis of findings from several large clinical trials that demonstrated significant reductions in the risk of fatal and nonfatal cardiovascular events.7–9
The calcium channel blocker (CCB) amlodipine has demonstrated beneficial effects on NO and cGMP and also decreasing fibrosis in a rat model of hypertension.10,11 Results from the Anglo-Scandinavian Cardiac Outcomes Trial-Blood Pressure Lowering Arm (ASCOT-BPLA), which was designed as a comparison of combination treatment regimens, showed that amlodipine with or without an ACE inhibitor reduced cardiovascular and total mortality compared with a β-blocker with or without a diuretic.12
Diuretics have long been the mainstay of antihypertensive drug regimens. A meta-analysis demonstrated reductions in stroke, heart failure, coronary disease, and mortality with diuretics.13 More recently, results of the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) suggested that diuretics may have cardioprotective effects.14
The exact mechanisms by which these drugs confer cardiac protection are not known; nor is it known whether combination treatment with an ACE inhibitor and a CCB provides additive benefit in myocardial ischemia. In this study, we hypothesized that ACE inhibitors, CCBs, or diuretics may reduce some mediators of myocardial inflammation that may relate to cardiac remodeling. We evaluated changes in cardiac interstitial fluid (CIF) levels of nitrate/nitrite (NOX), cGMP, Ang II, and TNF-α during treatment with the ACE inhibitor benazepril, the CCB amlodipine, the combination, or hydrochlorothiazide (HCTZ) after induction of myocardial ischemia in a rat model.
All study procedures were performed in accordance with National Institutes of Health guidelines for the care and use of laboratory animals, and the study was approved by the University of Virginia Animal Care and Use Committee.
Cardiac Microdialysis Technique
Using a microdialysis technique, CIF levels of NOX, cGMP, Ang II, and TNF-α were monitored 5 weeks after sham or left anterior descending coronary artery (LAD) occlusion. The methodology for the microdialysis probe procedure has been described previously.15–17 In brief, each end of a single hollow fiber dialysis tube (5.0 mm long, 0.1 mm internal diameter) with a molecular mass cutoff of 40,000 d (Hospal, Meyzieu, France) was inserted into a manually dilated end of 2 hollow polyethylene tubes 100 mm in length (inflow and outflow) with internal diameter of 0.12 mm and outer diameter of 0.65 mm. The respective in vitro recoveries by dialysis probes of cold and radiolabeled Ang II are 48% and 51%; for cGMP, the respective values are 59% and 70%. Recovery of these peptides in the perfusate is >99.8%, indicating that negligible amounts adhere to the polyethylene tube of the dialysis probes.15
Levels of CIF NOX, cGMP, Ang II, and TNF-α were measured in conscious animals on day 35 after myocardial ischemia or sham surgery. All CIF measurements were performed at the same time on collection days.
Sixty male Lewis inbred rats (Charles River Laboratories, Wilmington, MA) were housed in an air-conditioned room with a 12-hour light/dark cycle and were fed standard laboratory rat chow with free access to tap water. Three days after surgery, ischemic animals began treatment orally by gavage with benazepril 40 mg/kg/d, amlodipine 10 mg/kg/d, combined benazepril-amlodipine at those same dosages, HCTZ 3 mg/kg/d, or 5% dextrose in water vehicle; sham-operated animals also received 5% dextrose in water vehicle. There were 10 animals in each group, and treatment lasted for 5 weeks.
The animals were intubated and ventilated with room air using a positive-pressure respirator (680; Harvard Apparatus, Inc, South Natick, MA). A left thoracotomy was performed via the fourth intercostal space, and the lungs were retracted to expose the heart. With the pericardium open, the left ventricular wall was penetrated with a 31-gauge needle that was tunneled into the distribution area of the LAD approximately 1 mm from the outer surface for 5 mm before exiting the wall. The tip of the needle was inserted into one end of the dialysis probe and pulled until the dialysis fiber was situated in the wall of the left ventricle. Inflow and outflow tubes of the dialysis probe were tunneled subcutaneously through a bevel-tipped stainless-steel tube, emerging near the interscapular region. Coronary artery ligation was performed as described previously,18 and the LAD was ligated for 30 minutes near its origin by using a 7-0 silk suture. The lungs were inflated by increasing positive end-expiratory pressure, and the thoracotomy site was closed in layers. Animals undergoing sham ligation were subjected to a similar surgical procedure, except that the suture was not tightened around the coronary artery. Animals remained on a heating pad until they were awake. Cardiac examination at the study end confirmed the location of the dialysis catheter and presence of an infarction in the groups of animals with LAD ligation.
Blood pressure and heart rate were monitored using a Natsumi BP Pulse Monitor (Peninsula Laboratories, Belmont, CA).
NOX was monitored indirectly because the study was conducted in fully conscious animals at week 5. CIF NOX and cGMP levels in dialysate samples, measured by enzyme immunoassay kit (Cayman Chemical Co Inc, Ann Arbor, MI), had a sensitivity of 2.5 μmol/L for NOX and 0.11 pmol/mL for cGMP and a specificity of 100% for both assays. The intra-assay and interassay cross-reactivity of the assays with other cyclic nucleotides was <0.01%. Cardiac interstitial samples of Ang II were collected in tubes containing a mixed inhibitor solution of 5 mmol/L ethylenediaminetetraacetic acid, 10 nmol/L pepstatin, 20 nmol/L enalaprilat, and 0.125 mmol/L 1,10-phenanthroline and were kept on ice. Levels of Ang II, measured by enzyme-linked immunosorbent assay (SPI bio, F-78180 Montigny le Bretonneux, distributed by Cayman Chemical), had a sensitivity of 0.5 pg/mL for Ang II. Cardiac interstitial samples of TNF-α were measured by enzyme-linked immunosorbent assay using OptEIA Sets (sensitivity: <4 pg/mL with assay range: 15.6 to 1000 pg/mL; Pharmingen, San Diego, CA).
Quantitative Real-time Reverse Transcription-Polymerase Chain Reaction
Quantitative real-time reverse transcription-polymerase chain reaction was conducted for inflammatory markers IL-6 and NF-κB (n=8 in each group). After removal, cardiac tissue was weighed promptly and homogenized on ice, and total cardiac RNA was extracted using RNeasy Kit (Qiagen, GmbH, Hilden, Germany). RNA quality was confirmed by ethidium bromide staining in 2% agarose gel. Single-stranded cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Exon-intron boundaries were determined using the University of California Santa Cruz Genome Bioinformatics Site (http://www.genome.ucsc.edu). Corresponding cDNA primers were selected from M26744, AF079314, and BC063166, the gene codes for rat IL-6 (forward sequence: gcccttcaggaacagctatg; reverse sequence: tgaagtagggaaggcagtgg; length 101 bp), NF-κB p65 (forward sequence: tctgggccatatgtggagat; reverse sequence: tgcttctctccccaggaata; length 106 bp), and β-actin (forward sequence: agccatgtacgtagccatcc; reverse sequence: accctcatagatgggcacag; length 115 bp) sequences. The specificity of the primers was verified by melting curves. Quantitative reverse transcription-polymerase chain reaction was performed using iCycler software (Bio-Rad, Hercules, CA). Nontemplate control was used as a negative control. Samples were calculated with normalization to β-actin. Fold downexpression or upexpression was calculated according to the formula 2(Rt−Et)/2(Rn−En), where Rt is the threshold cycle number for the reference gene observed in the test sample, Et is the threshold cycle number for the experimental gene observed in the test sample, Rn is the threshold cycle number for the reference gene observed in the control sample, and En is the threshold cycle number for the experimental gene observed in the control sample.
The animals were sacrificed at the end of the study, and the hearts were fixed with 4% paraformaldehyde in phosphate-buffered saline. Cardiac tissues were then embedded in paraffin, sectioned as 4 to 5 μm thick, and stained with eosin-hematoxylin. The histology evaluation was observed under light microscopy.
The CIF levels were expressed as mean±standard error. Comparisons between pharmacologic agents in LAD-ligated and sham-treated animals were made by 1-way analysis of variance. Comparisons between treatment groups were examined using the t test. Statistical significance was declared at P<0.05.
There was no change in blood pressure or heart rate in any of the treatment groups.
Cardiac NOX: Responses to Ischemia, Benazepril, Amlodipine, HCTZ, or Combined Benazepril-Amlodipine
Five weeks after surgery, mean CIF NOX levels in sham animals (Fig. 1) were 1.8±0.37 μmol/L. In contrast, mean CIF NOX levels were 0.44±0.14 μmol/L in untreated animals with LAD occlusion (P<0.001). In animals with ischemia, treatment with either amlodipine or benazepril alone caused a significant increase in mean CIF NOX levels to 1.29±0.28 and 1.78±0.37 μmol/L, respectively (P<0.05 vs. untreated ischemia). Coadministration of benazepril and amlodipine caused a further increase in mean CIF NOX levels, compared with either drug alone, to 2.42±0.25 μmol/L (P<0.001 vs. untreated ischemia). HCTZ caused no significant change in mean CIF NOX levels (0.44±0.07 μmol/L) in animals with ischemia.
The response of NOX to combination benazepril-amlodipine was greater than to either drug administered as monotherapy. In animals treated with benazepril or benazepril-amlodipine, NOX concentrations were similar to sham at week 5.
Cardiac cGMP: Responses to Ischemia, Benazepril, Amlodipine, HCTZ, or Combined Benazepril-Amlodipine
Five weeks after surgery, mean CIF cGMP levels in sham animals (Fig. 2) were 1.32±0.44 pmol/mL. In untreated animals with LAD occlusion, mean CIF cGMP levels were 0.68±0.08 pmol/mL (P<0.001). In animals with ischemia, treatment with benazepril alone, but not amlodipine alone (0.80±0.12 pmol/mL), caused a significant increase in mean CIF cGMP levels to 1.16±0.17 pmol/mL (P<0.05). Coadministration of benazepril and amlodipine caused a further increase in mean CIF cGMP levels to 1.36±0.34 pmol/mL (P<0.001) when compared with either drug alone. HCTZ caused no significant change in mean CIF cGMP levels (0.74±0.31 pmol/mL) in animals with ischemia.
Cardiac TNF-α: Responses to Ischemia, Benazepril, Amlodipine, HCTZ, or Combined Benazepril-Amlodipine
At week 5, the respective mean CIF Ang II and TNF-α levels were 8.44±1.36 fmol/mL and 26.3±2.1 pg/mL in sham animals. After LAD occlusion, Ang II reached a mean level of 36.54±4.28 fmol/mL and TNF-α a mean level of 130±22 pg/mL at week 5 (Fig. 3). Treatment with amlodipine, benazepril, or combined benazepril-amlodipine caused a significant reduction in CIF TNF-α in ischemic animals at week 5. The respective mean levels of CIF TNF-α for amlodipine, benazepril, and combined benazepril-amlodipine were 76±20 pg/mL (P<0.01), 53.7±32 pg/mL (P<0.01), and 39.5±8 pg/mL (P<0.001). The increase in CIF TNF-α levels observed after ischemia was not reduced by HCTZ (104±40 pg/mL).
Cardiac Inflammatory Markers: Responses to Ischemia, Benazepril, Amlodipine, HCTZ, or Combined Benazepril-Amlodipine
After LAD occlusion, mean CIF IL-6 (Fig. 4) and NF-κB (Fig. 5) levels were increased significantly (P<0.001) at week 5 as compared with levels in sham animals. Treatment with benazepril or amlodipine alone caused significant reductions in mean CIF IL-6 (P<0.001) and NF-κB (P<0.001 and P<0.05, respectively) levels in ischemic animals at week 5. Combination treatment with benazepril-amlodipine caused a further decrease in mean CIF IL-6 and NF-κB levels compared with either drug alone. The increases in CIF IL-6 and NF-κB levels observed after ischemia were not significantly reduced by HCTZ.
Qualitative Cardiac Structural Changes in Response to Ischemia, Benazepril, Amlodipine, HCTZ, or Combined Benazepril-Amlodipine
Compared with normal hearts, ligation of LAD caused cardiac wall necrosis and cell infiltration (Fig. 6). Neither amlodipine nor HCTZ treatment was beneficial in limiting infarct expansion. However, benazepril clearly attenuated infarct expansion. Combination treatment with benazepril-amlodipine attenuated infarct expansion and inflammatory cellular infiltration, and preserved cardiac structure to a greater extent than treatment with either drug alone or HCTZ.
The results of this study confirm that postmyocardial ischemia, there is a reduction in CIF levels of NOX and cGMP and increases in Ang II and the inflammatory mediator TNF-α. HCTZ had no effect on postischemia CIF levels for any of the parameters measured. Both benazepril and amlodipine significantly increased CIF levels of NOX after ischemia, with benazepril and combination of benazepril-amlodipine having more pronounced effects. Benazepril also increased cGMP significantly, as did combination of benazepril-amlodipine. In contrast, amlodipine did not increase cGMP postischemia. Combined benazepril-amlodipine caused a significant reduction in TNF-α levels when compared with either drug alone. Similar findings for other inflammatory mediators (IL-6 and NF-κB) reinforce the results for TNF-α. Histologic results showed that combined benazepril-amlodipine preserved cardiac structure in this model of myocardial ischemia.
In the present study, we used a unique microdialysis technique to monitor changes in cardiac interstitial levels of Ang II, NOX, cGMP, and TNF-α. This technique has several advantages over traditional methods conducted in blood or urine.17 It allows measurement of CIF in vivo at the site of ischemia, close to target receptors, and over time in conscious animals without causing undesirable hemodynamic changes that may occur with repeated blood sampling in small animals. Acute studies allow only a single measurement of tissue or circulating factors and may not reflect local tissue changes in the organ or concentrations in the interstitial space. In addition, plasma measurements of NOX and other autocoids may not accurately reflect target organ concentrations because they can be formed and degraded quickly. Finally, the molecular weight cutoff of the dialysis membrane functions as a barrier excluding undesirable substances such as carrier proteins and degrading enzymes. The isolation of unbound materials facilitates their bioanalytical measurement in a small volume without the need for additional extraction procedures.
The central role of the renin-angiotensin system in the pathogenesis of cardiovascular disease is well established. Elevated levels of Ang II, a potent vasoconstrictor, combined with a reduction in NO levels, enhances vasoconstriction, inflammation, and the atherogenic process.1,19,20 Blocking the formation of Ang II with an ACE inhibitor increases NO availability, improving flow-mediated vasodilation, blood flow, and cGMP production as well as reducing oxidant stress and inflammation in the endothelium.3,19,21–23 Ang II may activate phosphodiesterases, which degrade intracellular cGMP, and may be associated with the finding in the present study that monotherapy with amlodipine or benazepril increased NOX, whereas only benazepril increased cGMP.24 Results of the present study support the importance of ACE inhibition as a mechanism for restoring the balance in the NO-cGMP pathway to prevent inflammation, ischemia, and cardiac hypertrophy. Benazepril alone significantly improved CIF levels of NOX, cGMP, and inflammatory markers, and attenuated infarct expansion.
CCBs also have beneficial effects on the NO-cGMP pathway and fibrosis in cardiovascular disease.10,11 Improvements in blood flow and endothelial function with amlodipine are linked to antioxidant effects and improved kinin-mediated NO production, suggesting that CCBs may modulate myocardial oxygen consumption.21,22,25,26 Inhibition of proinflammatory cytokines by CCBs is also related to enhanced NO levels and reduction in superoxide production, independent of their calcium channel-blocking activity.27,28 Similar to the findings in the present study, ex vivo studies have shown reductions in the production of TNF-α, IL-6, and NF-κB with CCBs.5,29 Significant reductions in IL-6 have been reported in patients with heart failure treated for 26 weeks with amlodipine.30 Although the present study did not show that amlodipine attenuated infarct expansion, a study in dogs reported that nifedipine limited infarct size by an NO-dependent mechanism.31
Most patients with hypertension require combination therapy to achieve blood pressure goals. Greater blood pressure-lowering effects with benazepril-amlodipine have been demonstrated than with a number of monotherapies,32 and reductions in cardiovascular and total mortality were shown with amlodipine plus the ACE inhibitor perindopril in the hypertension arm of the ASCOT trial.12 The ACE inhibitor-CCB combination examined in the present study is being evaluated in the Avoiding Cardiovascular events through COMbination therapy in Patients Living with Systolic Hyperension (ACCOMPLISH) trial, an ongoing outcome trial comparing benazepril-amlodipine with benazepril-HCTZ.33,34
The rationale for combination therapy with an ACE inhibitor-CCB is based on their synergistic effect on endothelial function and also pathogenic mechanisms of cardiovascular disease, including modulating myocardial oxygen consumption.35–37 This regimen, however, has not been studied in postmyocardial infarction patients.
The present study provides insight into possible mechanisms of clinical benefit from combined ACE inhibitor-CCB treatment in patients with cardiac ischemia. Combined benazepril-amlodipine had a greater effect on all parameters measured than either drug alone. Moreover, attenuation of infarct expansion and preservation of cardiac structure was greater with combined benazepril-amlodipine than with benazepril alone.
In conclusion, the present study shows that combination therapy with benazepril-amlodipine may be beneficial for the management of patients with cardiac ischemia.
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