We hypothesized that lectin-like oxidized LDL receptor-1 (LOX-1) deletion may inhibit oxidative stress signals, reduce collagen accumulation and attenuate cardiac remodeling after chronic ischemia. Activation of LOX-1 plays a significant role in the development of inflammation, apoptosis and collagen signals during acute ischemia. Wild-type and LOX-1 knockout (KO) mice were subjected to occlusion of left coronary artery for 3 weeks. Markers of cardiac hypertrophy, fibrosis-related signals (collagen IV, collagen-1 and fibronectin) and oxidant load (nicotinamide adenine dinucleotide phosphate oxidase expression, activity of mitogen-activated protein kinases and left ventricular (LV) tissue thiobarbituric acid reactive substances) were analyzed. In in vitro experiments, HL-1 cardiomyocytes were transfected with angiotensin II (Ang II) type 1 receptor (AT1R) or type 2 receptor (AT2R) genes to determine their role in the cardiomyocyte hypertrophy. LOX-1 KO mice had 25% improvement in survival over the 3-week period of chronic ischemia. LOX-1 deletion reduced collagen deposition and cardiomyocyte hypertrophy (∼75%) in association with a decrease in oxidant load and AT1R upregulation (all P<0.05). The LOX-1 KO mice hearts exhibited a disintegrin and metalloproteinase 10 (ADAM10) and a disintegrin and metalloproteinase 17 (ADAM17) expression and matrix metalloproteinase 2 activity, and increased AT2R expression (P<0.05). Attenuation of cardiac remodeling was associated with improved cardiac hemodynamics (LV ±dp/dt and cardiac ejection fraction). In vitro studies showed that it is AT1R, and not AT2R overexpression that induces cardiomyocyte hypertrophy. We demonstrate for the first time that LOX-1 deletion reduces oxidative stress and related intracellular signaling, which leads to attenuation of the positive feedback loop involving AT1R and LOX-1. This results in reduced chronic cardiac remodeling.
Heart failure is the leading cause of morbidity and mortality in the United States, and a large component of health-care expenditure goes towards management of patients suffering from heart failure.1, 2 Among the multiple causes of heart failure, chronic myocardial ischemia is perhaps the most common. Post-myocardial infarct remodeling has been divided into an early phase (within 72 h) and a late phase (after 72 h). The early phase involves expansion of the infarct zone. The non-infarcted zone undergoes progressive pathological remodeling 1–2-months after the acute ischemic injury, when myocyte hypertrophy and interstitial fibrosis ensue, resulting in progressive left ventricular (LV) enlargement.
The process of cardiac remodeling is initiated and amplified by a host of biochemical alterations, such as activation of renin–angiotensin system.1, 2 Accordingly, renin–angiotensin system modulators are frequently used in the treatment of patients with heart failure. Release of reactive oxygen species (ROS), perhaps in response to renin–angiotensin system activation, results in oxidant load in the ischemic myocardium.3, 4, 5 Oxidant load may be the basis of cardiomyocyte hypertrophy and interstitial fibrosis, and heart failure.3, 4, 5
Release of ROS activates specific lectin-like oxidized LDL-1 (LOX-1) receptors.6 Besides ROS, LOX-1 is transcriptionally upregulated by activation of angiotensin II type 1 receptor (AT1R), but not by type 2 receptor (AT2R).7 LOX-1 activation is a potent stimulus for endothelial7 and cardiomyocyte apoptosis.8 Li et al.9 showed that inhibition of LOX-1 with a specific binding antibody can reduce cardiomyocyte apoptosis and infarct size in rats subjected to a short period of ischemia. Hu et al.10 with the use of gene knockout (KO) technology showed that LOX-1 deletion can reduce signals of oxidant stress and inflammation, and modify infarct size and left ventricle (LV) hemodynamics in animals subjected to brief period of myocardial ischemia. Thus, it appears that LOX-1 inhibition or deletion can reduce acute ischemic injury to the heart.
Whether LOX-1 abrogation would reduce cardiac functional impairment during the state of chronic ischemia is not known. Further, the differential role of AT1R and AT2R in cardiomyocyte hypertrophy during chronic hypoxia is not known, although it is traditionally believed that AT1R and AT2R exert opposing effects.11, 12 The present study was conducted to address these issues.
Confirmation of LOX-1 deletion and LOX-1 upregulation during chronic ischemia
We amplified a 403 bp LOX-1 fragment in the genomic DNA from wild-type mice by polymerase chain reaction (PCR). This fragment was absent in the LOX-1 KO mice (Figure 1a). LOX-1 protein was not detected in the LOX-1 KO mice, but its presence in the wild-type mice was confirmed by western blot. LOX-1 signal increased by ∼200% in the wild-type mice hearts, but not in the LOX-1 KO mice hearts, subjected to 3 weeks of total ischemia (Figure 1b).
Survival after chronic ischemia
Early survival (within 6 h) after left coronary artery (LCA) ligation was much better in the LOX-1 KO mice compared to wild-type mice (17/20 (85%) vs 25/35 (71.4%), P<0.05). Six wild-type mice died of ventricular fibrillation, three of bradycardia and three of respiratory distress. Among the LOX-1 KO mice, two died of bradycardia and one of respiratory distress. The survival rates in wild-type and LOX-1 KO mice that underwent sham LCA occlusion were comparable (14/16 (87.5%) vs 15/16 (93.8%)). Three mice died with sham LCA occlusion from the absence of spontaneous breathing after closing the chest cavity. There were no additional deaths in the mice (wild-type group n=14; LOX-1 KO group n=15) with sham ischemia (Figure 1c). Over 3 weeks of observation, the survival rate was higher in the LOX-1 KO mice as compared with wild-type mice (14/20, 70% vs 14/35, 40%, P<0.05) subjected to total LCA occlusion. A total of 26 mice (12 in the wild-type group and 14 in the LOX-1 KO group) completed the chronic ischemia protocol. Two wild-type mice and none of the LOX-1 KO mice died during echocardiographic study.
LOX-1 deletion improves LV function after total coronary occlusion
There were no differences with respect to LV wall thickness or ejection fraction between wild-type and LOX-1 KO sham ischemia groups. At 3 weeks after LCA occlusion, LV anterior wall systolic thickness and ejection fraction were markedly decreased in the chronic ischemia groups compared to corresponding sham ischemia groups. Of note, there was much less deterioration of cardiac function in the LOX-1 KO mice. Representative echocardiograms and summary data are shown in Figure 2 and Table 1, respectively. Overall, LV cavity diameter was smaller in the LOX-1 KO mice, and LV anterior wall thickness, ejection fraction and fractional shortening were greater as compared to the wild-type mice (all P<0.05). However, posterior wall thickness was similar in LOX-1 KO and wild-type chronic ischemia groups. Direct hemodynamic measurement also showed much less deterioration of LV function in the LOX-1 KO mice (P<0.05 vs wild-type mice) (Figure 3).
LOX-1 deletion reduces infarct size after total LCA occlusion
There were no differences in the area at risk (AAR)/LV between wild-type and LOX-1 KO mice groups. However, the LOX-1 KO mice had much smaller infarct area than the wild-type mice (Figure 4a), despite similar degree and duration of ischemia.
LOX-1 deletion reduces cardiac hypertrophy after chronic ischemia
The heart weight/tibia length as well as heart weight/body weight and cardiomyocyte cross-sectional area were increased in both groups of mice subjected to chronic ischemia. However, changes in the heart weight as well as cardiomyocyte size were less marked in the LOX-1 KO group (P=0.001; Table 2 and Figure 4c).
Expression of atrial natriuretic peptide was higher in the hearts of wild-type and LOX-1 KO mice subjected to chronic ischemia (P=0.001 vs sham ischemia groups), and the increase was much less in the LOX-1 KO mice. This is in keeping with the data on heart weight and cardiomyocyte size. Concurrently, the expression of AT1R and AT2R was increased in both wild-type and LOX-1 KO mice groups subjected to chronic ischemia, but AT1R expression was less in the LOX-1 KO mice (1.33±0.14 vs 1.78±0.16 a.u. in the wild-type mice, P<0.05), whereas the AT2R expression was more pronounced in the LOX-1 KO mice (2.12±0.12 vs 1.33±0.15 a.u. in the wild-type mice, P=0.001; Figure 4b and Supplementary Figure 1).
Expression of both gp91phox and p47phox subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was increased at 3 weeks of chronic ischemia in both wild-type and LOX-1 KO mice hearts (P<0.05 vs sham ischemia groups), but the LOX-1 KO mice exhibited only a modest increase (P=0.001 vs wild-type chronic ischemia mice; Figure 4b and Supplementary Figure 1).
LOX-1 deletion reduces collagen deposition and related proteins after chronic ischemia
Chronic ischemia induced interstitial fibrosis in LV in all mice, but the collagen accumulation was less in the LOX-1 KO mice hearts (vs wild-type mice hearts). Figure 5a shows representative examples of Masson's trichrome-stained sections, and Figure 5b shows summary data on collagen accumulation. Concurrently, expression of fibronectin, collagen IV-α2 and collagen-1 was found to be markedly increased at 3 weeks of chronic ischemia in both groups of mice, but the increase was less in the LOX-1 KO mice (P<0.05; Figure 5c and Supplementary Figure 2).
We also measured the expression of a disintegrin and metalloproteinase 10 (ADAM10) and a disintegrin and metalloproteinase 17 (ADAM17) (western blotting) and matrix metalloproteinase 2 (MMP2) activity (zymography) in the mice hearts. Both ADAM10 and ADAM17 increased in all mice hearts subjected to chronic ischemia (P<0.01 vs sham ischemia groups), but much less so in the LOX-1 KO mice (P<0.05 vs wild-type mice). Notably, ADAM10 expression did not change significantly in the chronically ischemic LOX-1 KO hearts, but the expression of ADAM17 was enhanced (P<0.05 vs corresponding sham ischemia group). MMP2 activity followed the same pattern as collagen IV-α2 and collagen-1 (Figure 5c and Supplementary Figure 2).
LOX-1 deletion reduces myocardial oxidative stress
Change in AT1R, LOX-1 and NADPH oxidases was associated with enhanced phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-κB) p65, but the expression of their proteins was unchanged (P<0.05 vs sham ischemia groups). The activation of p38 MAPK, stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) and NF-κB p65 proteins was less pronounced in the LOX-1 KO mice hearts (P<0.05 vs wild-type mice hearts; Figure 6a; Supplementary Figure 3). In keeping with the LOX-1 and NADPH oxidase data, the ischemia-related rise in serum malondialdehyde (MDA) and LV tissue thiobarbituric acid reactive substances (TBARS) (AAR) was less pronounced in the LOX-1 KO mice (P<0.05; Figures 6b and c).
AT1R, but not AT2R, overexpression causes cardiomyocyte hypertrophy
The transfection efficiency with AT1R and AT2R cDNA was more than 75%. Transfection of HL-1 cardiomyocytes with AT1R and AT2R genes was confirmed by reverse transcription (RT)-RCR (Figure 7a). Overexpression of AT1R increased the expression (mRNA and protein) of NADPH oxidase (p67phox and p47phox subunits) and cardiomyocyte size by 50% (both P<0.05 vs control); in contrast, AT2R transfection decreased NADPH oxidase (p67phox subunits, P<0.05 vs control), but had no significant effect on cardiomyocyte size (Figure 7b). Importantly, AT1R upregulation resulted in a marked increase in LOX-1 expression (mRNA and protein), whereas AT2R upregulation mildly inhibited LOX-1 protein expression (P=0.059 vs control; Figures 7c and 8).
LOX-1 was first identified in vascular endothelial cells, but later studies showed that it is also expressed in cardiomyocytes, platelets and inflammatory cells.13 Its expression is upregulated after a brief period of myocardial ischemia9, 10 in response to ROS,8 cytokines14 and angiotensin II release.15 LOX-1 activation stimulates migration of inflammatory cells.6, 13, 16 LOX-1 activation itself causes ROS release, AT1R expression and cardiomyocyte hypertrophy17 and collagen formation by fibroblasts.18
Because LOX-1 activation influences many of the processes involved in chronic ischemia-related signals, we postulated that LOX-1 abrogation would attenuate the cardiac remodeling process following total LCA occlusion. This study indeed confirms our principal postulate.
We observed a marked improvement in the survival rate in LOX-1 KO mice (vs the wild-type mice) over the first 6 h period following total LCA occlusion. This probably reflects a marked reduction in ROS release in the LOX-1 KO mice during the early phase of myocardial ischemia. Honjo et al.19 showed that rodents pretreated with anti-LOX-1 antibody had improved survival after lipopolysaccharide administration, and implicated a markedly reduced inflammatory activity as the basis. Importantly, mice missing LOX-1 had smaller infarcts after LCA occlusion, despite similar AAR as the wild-type mice in the present study.10 Thus, it is safe to assume that smaller infarct, less ROS release and limited inflammatory response account for the low mortality in the LOX-1 KO mice in the early stages of myocardial ischemia.
Previous studies showed upregulation of LOX-1 after a brief period of ischemia.10 The present study shows that LOX-1 continued to be overexpressed over 3 weeks of sustained ischemia in the wild-type mice hearts. Importantly, cardiomyocyte hypertrophy and interstitial fibrosis were less marked in the LOX-1 KO mice, suggesting a role for LOX-1 in cardiac remodeling process. The reduction in cardiomyocyte hypertrophy and interstitial fibrosis resulted in improved cardiac function, as assessed by echocardiographic and direct hemodynamic measurements.
We were enthused by the modulation of the cardiac remodeling process by LOX-1 deletion in this model of chronic ischemia. The cardiomyocyte hypertrophy as well as collagen deposition both were markedly reduced. The major stimulus for cardiomyocyte hypertrophy appears to be AT1R expression and activation.17, 20 Based on the analysis of microarray data, Kang et al.21 showed that AT1R is upregulated in hypertrophied hearts following sustained hypertension, and LOX-1 deletion reduces AT1R expression. Several experimental and clinical studies22 have shown that AT1R blockade reduces cardiac hypertrophy, unrelated to change in blood pressure. In accordance with these studies, we observed marked cardiomyocyte hypertrophy in association with enhanced AT1R expression in the hearts of wild-type mice subjected to chronic ischemia, and a significant reduction in AT1R expression and cardiomyocyte hypertrophy in the LOX-1 KO mice hearts, despite the same degree and duration of ischemia. The expression of atrial natriuretic peptide, a marker of cardiomyocyte hypertrophy, paralleled the results of cardiomyocyte size measurement in both wild-type and LOX-1 KO mice.
The role of AT1R and AT2R in cardiomyocyte hypertrophy continues to be debated with many investigators suggesting opposite effects of AT1R and AT2R expression.11, 12, 22, 23, 24 We found that the expression of both AT1R and AT2R was increased in the chronically ischemic wild-type mice hearts. The increase of AT1R, but not AT2R, expression was blunted in the LOX-1 KO mice hearts. To further examine the role of AT1R and AT2R in cardiomyocyte hypertrophy, we transduced HL-1 cardiomyocytes with AT1R and AT2R genes. Forced overexpression of AT1R, but not AT2R, resulted in cardiomyocyte hypertrophy as well as LOX-1 expression. Of note, overexpression of AT2R resulted in the downregulation of p67phox subunits of NADPH oxidase in HL-1 cardiomyocytes. These observations from transduction of cardiomyocytes in vitro suggest inhibiting NADPH oxidase expression of AT2R overexpression in chronically ischemic hearts in vivo.
Chen et al.25, 26, 27 examined the role of LOX-1 in collagen generation by cardiac fibroblasts, and established that both hypoxia and AT1R activation induce fibroblast growth and synthesis of pro-collagen. In keeping with these studies, chronic ischemia-associated collagen accumulation in the LV (peri-infarct zone) was significantly less in the hearts of LOX-1 KO (vs wild-type) mice. The expression of collagen-forming signals, such as collagen IV-α2, pro-collagen-1 and fibronectin, was also less marked in the chronically ischemic LOX-1 KO mice.
ADAM10 and ADAM17, members of the ADAM family (sheddases), are cell surface proteins with a unique structure possessing both potential adhesion and protease domains. They primarily function to cleave membrane proteins at the cellular surface. Once cleaved, the sheddases release soluble ectodomains with an altered location and function. Activation of ADAM10 and ADAM17 has been related to the shedding of collagen.28, 29, 30 We observed increased expression of both ADAM10 and ADAM17 in the chronically ischemic mice hearts (vs sham ischemia mice). The expression of both ADAM10 and ADAM17 was lower in the LOX-1 KO mice hearts than in the wild-type mice hearts, both subjected to the same degree of ischemia. These first observations indirectly link LOX-1 abrogation to the attenuation of ADAMs signaling in collagen formation in the heart during chronic ischemia.
We also measured NADPH oxidases (p47phox and p67phox subunits), serum MDA and LV tissue TBARS as surrogates for ROS release in the ischemic hearts. There was evidence of release of ROS in the chronically ischemic wild-type mice hearts, and LOX-1 abrogation reduced the expression of NADPH oxidases as well as levels of serum MDA and LV TBARS. Although the increased NADPH oxidase expression (and ROS release) has been amply documented following a short period of ischemia in the mice hearts,10 its sustained increase during chronic ischemia has not been established until now. These data are also consistent with the results of in vitro studies,18 which showed that LOX-1 inhibition or abrogation can reduce collagen formation by cardiac fibroblasts via inhibition of NADPH oxidase activity. NADPH oxidase activates MAPKs (p38, p44/42 and SAPK/JNK) and, thereafter, induces translocation of the redox-sensitive transcription factor NF-κB.31, 32 In keeping with this well-defined signaling pathway, we identified a marked increase in the expression of phos-p38MAPK, phos-SAPK/JNK and phos-NF-κB p65 in the wild-type mice ischemic hearts. Further, there was a significant downregulation of MAPK-NF-κB signaling in the LOX-1 KO mice hearts (vs wild-type mice). We believe that the present study is the first to document persistent activation of this pathway in the LV during sustained ischemia induced by total LCA occlusion and its attenuation in the LOX-1 KO mice resulting in improved cardiac function.
Materials and methods
C57BL/6 mice (also referred to as wild-type mice) were obtained from Jackson Laboratories (Bar Harbor, ME, USA). The homozygous LOX-1 KO mice were developed and backcrossed eight times with C57BL/6 strain to replace the genetic background.33 C57BL/6 and homozygous LOX-1 KO (on C57BL/6 background) mice were bred by brother–sister mating and housed in the breeding colony at the University of Arkansas for Medical Sciences (Little Rock, AR, USA). This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Male C57BL/6 and LOX-1 KO mice weighing about 25 g and ∼12 weeks of age were utilized. All experimental procedures were performed in accordance with protocols approved by the Institutional Animal Care and Usage Committee.
The LOX-1 genotypes were verified by PCR analysis of genomic DNA extracted from the tail with the primer pair for deleted portion of LOX-1 gene: 5′-IndexTermGGCCAACCATGGCTTGGGAGAATGG-3′ (Tm=63.5 °C) and 5′-IndexTermCAGCGAACACAGCTCCGTCTTGAAGG-3′ (Tm=63.3 °C); and for neomycin-resistant gene: 5′-IndexTermAGGATCTCGTCGTGACCCATGGCGA-3′ (Tm=65.3 °C) and 5′-IndexTermGAGCGGCGATACCGTAAGCACGAGG-3′ (Tm=64.9 °C). Thermal cycler conditions were denaturation for 3 min at 95 °C, followed by 36 cycles of amplification (30 s at 94 °C, 30 s at 60 °C for annealing, 1 min at 72 °C) and a final incubation of 5 min at 72 °C. PCR products were separated by 1.2% agarose gel electrophoresis and visualized by ethidium bromide staining on Quantity on UV transilluminator (Bio-Rad, Hercules, CA, USA).
Myocardial ischemia protocol
Animals were anesthetized with ketamine hydrochloride (60 mg kg−1) and xylazine hydrochloride (8 mg kg−1) intraperitoneally. After endotracheal intubation, mice were mechanically ventilated (tidal volume, 1.2 ml min−1 and respiration rate, 110 min). Body temperature was maintained between 37.0 °C and 37.5 °C with a heating pad. Electrocardiogram was recorded throughout the experiment. After equilibration period of 5 min, under aseptic conditions, left thoracotomy was performed in the 4th intercostal space and the pericardium opened. An 8-0 silk suture was passed around the LCA at a point 2/3rd of the way between its origin near the pulmonary conus. Occlusion of the LCA caused epicardial cyanosis with regional hypokinesia and electrocardiogram changes typical of acute myocardial infarction. Another group of animals underwent the same procedure, but without LCA ligation (sham ischemia). The thoracic cavity was closed in layers, using 6-0 sutures, and drained to prevent pneumothorax. Endotracheal tube was then removed and animals allowed to recover.
Echocardiographic and hemodynamic assessment of LV function
Echocardiograms were obtained at 3 weeks after the induction of myocardial ischemia using Vevo 770 ultrasound system (Visual-Sonics Inc., Toronto, ON, Canada) with a 30-MHz transducer to visualize the LV. Before echocardiography, mice were sedated using isoflurane (2%, vol vol−1). The anterior chest was shaved and the mice were secured with a tape in the supine position. Two-dimensional directed M-mode images of the LV short axis were taken just below the level of the papillary muscles to analyze wall thickness and chamber diameter. LV ejection fraction and fractional shortening were quantified. All measurements represent the mean of at least 10 consecutive cardiac cycles.
Cardiac hemodynamic status was obtained just before euthanasia. Under anesthesia, 1.4-Fr Millar (SPR-671) pressure transducer catheter (AD Instruments, Colorado Springs, CO, USA) was inserted through the right carotid artery into the LV; the position of the catheter was confirmed by typical waveform. Analog inputs from the LV pressure transducer were amplified using a Bridge amplifier and digitized with a PowerLab data-acquisition system (AD Instruments). Subsequent off-line evaluations provided LV systolic pressure and end-diastolic pressure. The first derivatives of the pressure over time (±dp/dtmax) were calculated from LV pressure tracings as a marker of systolic and diastolic LV function.
Assessment of AAR and area of infarct
The LV AAR and area of infarct were measured as described previously.10
Morphological and histological evaluation
Heart was harvested and cross-sectional areas of cardiomyocytes were analyzed with a Scion imaging system as described previously.34 At least 200 cardiomyocytes were examined in each slice and at least four slices were examined in each heart, and the data averaged. Cardiac hypertrophy was evaluated as the ratio of heart weight to tibia length (mg mm−1). Some heart sections were stained with hematoxylin–eosin staining and Masson's trichrome, and interstitial fibrosis area were quantified.
Determination of oxidative stress
The degree of lipid peroxidation was determined in the tissue taken from the AAR by measuring TBARS.34, 35 The TBARS were expressed as micromoles per gram wet weight. MDA was measured spectrophotometrically in serum samples and expressed as mmol l−1.
Cell culture and transfection
To study the role of AT1R and AT2R in cardiomyocyte hypertrophy, AT1R and AT2R genes were transfected into cultured HL-1 mouse cardiomyocytes. The cardiomyocytes were seeded in T25 flasks or multi-well plates pre-coated with 0.02% gelatin (Becton-Dickinson, Sparks, MD, USA) and 5 μg ml−1 fibronectin (Sigma-Aldrich, St Louis, MO, USA), and cultured in Claycomb medium (SAFC Biosciences, Erie, PA, USA) supplemented with 10% fetal bovine serum, 2 mM L-glutamine (Invitrogen, Carlsbad, CA, USA) and 0.1 mM norepinephrine (Sigma) at 37 °C under 5% CO2. When cells reached 70% confluence, they were transfected with PCMV-SPORT6 plasmid with GFPs, AT1R or AT2R cDNA. The transfection efficiency was evaluated by PCMV-SPORT6-GFP expression using fluorescent microscopy. Cells transfected with empty PCMV-SPORT6 plasmid served as control. Cell size was quantified using an image-analyzing system. At least 50 cardiomyocytes were examined in each slice and at least three slices in each experiment, and the data averaged.
Real-time quantitative PCR
Total RNA was isolated from triplicate control and experimental cultures (three sets of experiment) using AllPrep RNA/DNA Extraction Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. cDNA synthesis was performed using first-strand cDNA synthesis kit (Roche, Indianapolis, IN, USA). Quantitative PCR was performed as described,21 using the Applied Biosystems Fast 7500HT real-time PCR system. Quantitative PCR-specific primers (Table 3) were designed using the Probe-Finder (http://www.roche-applied-science.com) web-based software. The results were analyzed using the SDS 2.3 relative quantification manager software (Applied Biosystems, Carlsbad, CA, USA). The comparative threshold cycles' values were normalized for GAPDH reference genes. Quantitative PCR was performed in triplicate to ensure quantitative accuracy.
Western blot analysis
Expression of LOX-1, AT1R, AT2R, cardiomyocyte-specific protein atrial natriuretic peptide, phosphorylated NF-κB p65 (Ser276), NADPH oxidase (p47phox, p67phox and gp91phox subunits), MAPKs (p38 MAPK and SAPK/JNK) and their activity, and β-actin was assessed by western blot using standard methodologies. Several collagen-related signals were also analyzed by western blotting. Primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Cell Signaling Technology (Danvers, MA, USA) or Abcam Inc. (Cambridge, MA, USA). Anti-LOX-1 antibody was obtained from Dr T Sawamura. The densities of protein bands were quantified by Quantity One Image Analyzer (Bio-Rad), and the band was normalized to that of β-actin.
MMP-2 activity assay
Activity of MMP-2 in heart tissues was determined by gelatin zymography as described.10
Data are presented as mean±s.d. Mice survival was analyzed by the Kaplan–Meier method, and between-group differences were tested by the log-rank test. Between-group means were compared by one-way analysis of variance, followed by Student–Newman–Keuls test. A P<0.05 was considered significant.
This study provides convincing evidence from multiple approaches of the important role of LOX-1 expression in the cardiac remodeling process that takes place after chronic myocardial ischemia. LOX-1 deletion reduces oxidative stress and its intracellular signaling and leads to the interruption of the concept of ‘ischemia begets ischemia’ via attenuation of the positive feedback between AT1R and LOX-1 (Figure 9). LOX-1 abrogation results in amelioration of cardiomyocyte hypertrophy and collagen accumulation, resulting in improved cardiac hemodynamic function and survival of the animals. We suggest that LOX-1 blockade be considered a potential target of therapy for both acute and long-term myocardial ischemia.
a disintegrin and metalloproteinase
- Ang II:
angiotensin II type 1 receptor
angiotensin II type 2 receptor
left coronary artery
lectin-like oxidized LDL receptor-1
left ventricular internal diameter in diastole
left ventricular internal diameter in systole
mitogen-activated protein kinase
nicotinamide adenine dinucleotide phosphate
reactive oxygen species
stress-activated protein kinase/c-Jun NH2-terminal kinase
thiobarbituric acid-reactive substances
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This study was supported in part by funds from the Department of Veterans Affairs.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on Gene Therapy website
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Lu, J., Wang, X., Wang, W. et al. LOX-1 abrogation reduces cardiac hypertrophy and collagen accumulation following chronic ischemia in the mouse. Gene Ther 19, 522–531 (2012). https://doi.org/10.1038/gt.2011.133
- cardiac remodeling
- chronic ischemia
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