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Cardiovascular Research 2007 76(2):292-302; doi:10.1016/j.cardiores.2007.07.003
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Copyright © 2007, European Society of Cardiology

LOX-1 deletion alters signals of myocardial remodeling immediately after ischemia–reperfusion

Changping Hua,b,1, Abhijit Dandapata,1, Jiawei Chena, Yoshiko Fujitac, Nobutaka Inouec, Yosuke Kawased, Kou-ichi Jishaged, Hiroshi Suzukie,f, Tatsuya Sawamurac and Jawahar L. Mehtaa,*

aDepartment of Internal Medicine, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, AR, USA
bDepartment of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha, China
cDepartment of Vascular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan
dChugai Research Institute For Medical Science, Inc., Gotenba, Shizuoka, Japan
eResearch Unit for Functional Genomics, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan
fDepartment of Developmental and Medical Technology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan

*Corresponding author. University of Arkansas for Medical Sciences, 4301 West Markham St., Slot 532, Little Rock, AR 72205-7199, USA. Tel.: +1 501 296 1401; fax: +1 501 686 8319. mehtajl{at}uams.edu

Received 10 April 2007; revised 26 May 2007; accepted 11 July 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective Chronic ischemia is associated with alterations in genes that result in myocardial remodeling. An important biochemical basis of cardiac remodeling is generation of reactive oxygen species (ROS). A few studies have suggested that acute ischemia triggers signals for remodeling. We examined the hypothesis that targeted deletion of lectin-like oxidized-LDL receptor (LOX-1) may inhibit signals related to cardiac remodeling.

Methods and results We generated LOX-1 knockout (KO) mice on C57BL/6 (wild-type mice) background, and subjected wild-type and KO mice to ischemia–reperfusion (I–R). The wild-type mice developed a marked reduction in left ventricular systolic pressure and ±dp/dtmax and an increase in left ventricular end-diastolic pressure following I–R, and this change was much less in the LOX-1 KO mice, indicating preservation of left ventricular function with LOX-1 deletion. There was evidence for marked oxidative stress (NADPH oxidase expression, malondialdehyde and 8-isoprostane) following I–R in the wild-type mice, much less so in the LOX-1 KO mice (P<0.01). In concert, collagen deposition (Masson's trichrome and Picro-sirius red staining) increased dramatically in the wild-type mice, but only half as much in the LOX-1 KO mice (P<0.01). Collagen staining data was corroborated with procollagen-I expression. Further, fibronectin and osteopontin expression increased in the wild-type mice, but to a much smaller extent in the LOX-1 KO mice (P<0.01).

Conclusions These findings provide compelling evidence that LOX-1 is a key modulator of cardiac remodeling which starts immediately following I–R.

KEYWORDS Remodeling; Ischemia; Reperfusion; NADPH oxidase; Extracellular matrix


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Heart failure is often the end result of cardiovascular disease states, such as myocardial ischemia [1]. Heart failure is characterized by abundant accumulation of extracellular matrix (ECM) proteins in the extracellular space. Among the ECM proteins, collagens constitute up to 85% [2,3]. Collagen type I is usually present in the form of thick fibres with a high tensile strength, and is considered a major determinant of myocardial stiffness [3,4]. Fibroblasts are the major source of collagen-I in the myocardium [3,5]. Proliferation of cardiac fibroblasts and deposition of collagen are directly associated with both systolic and diastolic, especially the latter, heart failure [6].

Collagen accumulation in the heart depends not only on its production, but also on its degradation by proteinases, such as matrix metalloproteinase (MMP-2, MMP-3 and MMP-9) [3]. Experimental studies suggest that reactive oxygen species (ROS) released in the early stages of ischemia–reperfusion (I–R) play a major role in the activation of MMPs, and that NADPH oxidase activation, a major source of ROS in the ischemic heart, is a key event in this process [7,8]. Release of cytokines and activation of renin–angiotensin system resulting in the formation of angiotensin II (Ang II) during acute ischemia are also associated with release of ROS and changes in collagens and MMPs [9,10].

Many investigators have highlighted the importance of matricellular protein osteopontin as a key mediator in the cardiovascular system, specifically in vascular remodeling, vascular calcification and left ventricular remodeling [11–13]. Recently, it has been shown that an osteopontin-NADPH oxidase signaling cascade promotes MMP-9 activation [14].

LOX-1 is a lectin-like oxidized-LDL receptor [15]. In previous studies, we showed that LOX-1 is involved in the genesis of oxidant stress and inflammation during myocardial I–R [16,17]. LOX-1 can also act as an adhesion molecule for inflammatory cells [18]. In other studies, we showed that insertion of LOX-1 plasmids in cardiac fibroblasts that are naturally low expressers of LOX-1 alters the biology of fibroblasts to pro-inflammatory phenotype [19]. Further oxidized-LDL treatment enhances collagen formation in fibroblasts that can be blocked by a LOX-1 antibody. These observations collectively suggest that LOX-1 may be an important player in myocardial I–R injury not only by inducing oxidative stress, but also by inducing signals for collagen and MMPs in the ischemic tissues.

We hypothesized that LOX-1 deletion would, by altering the major mediator of myocardial I–R injury, i.e. oxidant stress, improve cardiac diastolic function during ischemia–reperfusion. In addition, it would block or reduce the signal for myocardial remodeling process. Our findings in LOX-1 knockout (KO) mice reveal that "taking away" LOX-1 indeed limits early cardiac remodeling signal following I–R, and this effect is mediated by inhibition of NADPH oxidase.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
C57BL/6 mice (also referred to as wild-type mice) were originally obtained from Jackson Laboratories. The homozygous LOX-1 KO mice were developed as described recently [20], and backcrossed 8 times with C57BL/6 strain to replace the genetic background. 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 University of Arkansas for Medical Sciences, Little Rock, Arkansas. This investigation conforms with 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 mice were utilized in the present studies at 8–10 weeks of age. All experimental procedures were performed in accordance with protocols approved by the Institutional Animal Care and Usage Committee.

2.1 PCR analysis of LOX-1 expression
Wild-type and LOX-1 KO mice were killed with CO2 anesthesia, and blood, aorta and heart tissues were collected. Genomic DNA was obtained using DNAzol® Reagents (Invitrogen). PCR analysis of genomic DNA was done with the primer pair for deleted portion of LOX-1 gene: 5'-ggccaaccatggctatgggagaatgg-3' and 5'-cagcgaacacagctccgtcttgaagg-3', and for neomycin resistant gene: 5'-cgtttccgctgt caccgg-3' and 5'-caacgctatgtcctgatagcggtcc-3'. 30 cycles of PCR were performed at 94 °C for 40 s, 60 °C for 1 min and 72 °C for 1 min. PCR-amplified products were visualized by ultraviolet light following electrophoresis in 1.5% agarose gel containing ethidium bromide.

2.2 Immunofluorescence staining
The expression of LOX-1 protein and the uptake of ox-LDL were analyzed using immunofluorescence staining. The cryothin sections of aorta were treated with Cy3-labeled anti-mouse LOX-1 monoclonal antibody (1 µg/ml, TS58). Thoracic aortas were incubated for 12 hours at 37 °C in DMEM/10% FCS containing 10 µg/ml DiI-labeled oxidized LDL which was prepared as described previously [21], then washed with PBS three times and snap frozen. To confirm the presence of endothelium, immunostaining with anti-von Willebrand factor, biotinylated anti-CD31 and avidin-FITC was performed, and subjected to observation under a laser confocal microscope.

2.3 Myocardial ischemia–reperfusion protocol
Animals were anesthetized with sodium pentobarbital (60 mg/kg, IP). Anesthesia was maintained via supplemental doses of sodium pentobarbital (30 mg/kg, IP) as needed. Mice were mechanically ventilated with room air using a Harvard respirator (model 683). The respirator's tidal volume was set at 1.4 ml/min and the rate at 110 strokes/min. Electrocardiographic leads were connected to the chest and limbs for continuous monitoring throughout the experiment.

After equilibration period of 10 min, a left thoracotomy was performed in the fourth intercostal space and the pericardium opened to expose the heart. A 6-0 silk suture was passed around the left coronary artery at a point two thirds of the way between its origin near the pulmonary conus and the cardiac apex and a snare was formed by passing both ends of the suture through a piece of polyethylene tubing. Occlusion of the coronary artery, by clamping the snare against the surface of the heart, caused an area of epicardial cyanosis with regional hypokinesis and ECG changes. Reperfusion was achieved by releasing the snare and was confirmed by conspicuous hyperemic blushing of the previously ischemic myocardium and gradual resolution of the changes in the ECG signal. Another group of animals underwent the same procedure but without ligation of the coronary artery.

The chest wall was approximated and covered with Parafilm wax paper to prevent desiccation. Anesthetized mice were subjected to 60 min of coronary artery occlusion followed by 60 min of reperfusion, except the sham groups.

2.4 Assessment of left ventricular hemodynamics
To assess the hemodynamic status, a 1.4-Fr Millar (SPR-671) pressure transducer catheter was inserted through the right carotid artery into the left ventricle (LV); the position of the catheter was confirmed by typical wave form. Hemodynamic measurements were recorded at baseline and during reperfusion. Analog inputs from the pressure transducer were amplified using a Bridge amplifier and digitized with a PowerLab data-acquisition system (AD Instruments). All parameters were calculated from an average of 30 consecutive beats at each time point. Subsequent off-line evaluations provided LV systolic pressure (LVSP) and LV end-diastolic pressure (LVEDP). The first derivatives of the pressure over time (±dp/dtmax) were calculated from LV pressure tracings as a marker of systolic and diastolic ventricular function, respectively.

2.5 Determination of infarct size
At the end of reperfusion, the left coronary artery was re-occluded in the same location as before, and 1 ml 1% Evans blue (Sigma) was injected into the LV cavity and was allowed to perfuse the non-ischemic portions of the heart. The entire heart was excised, weighed, rinsed of excess blue dye, trimmed of right ventricular and atrial tissue, and sliced into 1-mm-thick sections from the apex to base. The slices were incubated in 1% triphenyl tetrazolium chloride (TTC, Sigma) at 37 °C for 15 min to stain the viable myocardium brick red. The slices were then fixed in a 10% formalin solution for 24 h. Each slice was imaged with computer-assisted planimetry (NIH Image J 1.34s) by an observer blinded to sample identity and following parameters was analyzed: (1) area at risk (AAR) as a percent of the left ventricle (LV) (AAR/LV), (2) the infarct area (IA) as a percent of AAR (IA /AAR).

2.6 Determination of oxidant stress in the left ventricular tissues
At the end of reperfusion, entire LV was homogenized in ice-cold 20 mM phosphate buffer. Malondialdehyde (MDA) was measured in the LV homogenate spectrophotometrically and expressed as nmol MDA/g. We also measured 8-isoprostane, a nonenzymatic metabolite of ROS by enzyme immunoassay (EIA) in the LV homogenates as described previously [22]. The tissue 8-isoprostane level was expressed as ng/g.

2.7 Quantitative analysis of collagen positive area
Paraffin-embedded heart tissues were cut into 5 to 6 sections each 5 µm thick, and the sections were stained with Masson's trichrome and Picro-sirius red. The images were captured by digital imaging system and analyzed with Image pro software (Media Cybernetics). Area positive for collagen was recorded for each section and averaged for each mouse. Data were obtained from at least 3 mice in each group.

2.8 Expression analysis
At the end of reperfusion, entire LV was isolated for the expression analysis of LOX-1, NADPH p22phox, NADPH p47phox, procollagen-I, MMPs, osteopontin and fibronectin using standard methodologies of RT-PCR and Western blot [9]. Total RNA from the entire LV was extracted using Trizol (Invitrogen). The PCR primers and conditions employed are shown in Table 1. Densities of protein and mRNA bands relative to β-actin were analyzed.


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  		Table 1 Primer sequences used for RT-PCR

 
Real-time RT-PCR analysis for the expression of NADPH oxidase (p22phox and p47phox subunits) was performed as described previously [23]. GAPDH was used as a standard control. The primers used are as follows. GAPDH: forward, 5'-AACTTTGGCATTGTGGAAGG-3'; reverse, 5'-ACACATTGGGGGTAGGAACA-3'. p22phox: forward 5'-GCCAACGA GCAGGCGCTGGC-3'; reverse, 5'-CTCGAGGGTATTCCAGCAG-3; p47phox: forward, 5-GTGTACATGTTCCTGGTTAAG-3'; reverse, 5'-ATGGAACTCGTAGATCTCG-3'.

2.9 Statistical analysis
Data are expressed as means±SE. The between-group difference in the infarct size was evaluated by unpaired t-test. All hemodynamic values were analyzed with a two-way ANOVA with repeated measures. All other data were analyzed by a two-way ANOVA with a Bonferroni post-hoc test. A P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Confirmation of LOX-1 deletion
In the genomic DNA from wild-type mice, we amplified a 403 bp LOX-1 fragment by PCR. This fragment was absent in the LOX-1 KO mice tissues. Instead, a neomycin-specific PCR fragment of 564 bp was detected. A representative experiment is shown in the Fig. 1 A. Further, LOX-1 expression was not detected in the vascular tissues of LOX-1 KO mice, although its presence in the endothelium of wild-type mice was confirmed by immunostaining. The endothelial integrity was confirmed by simultaneous staining with von Willebrand factor (Fig. 1 B left). Further, the uptake of ox-LDL in endothelium was undetectable in the LOX-1 KO mice, but clearly seen in the wild-type mice (Fig. 1 B right). These data confirm the genotype of wild-type and LOX-1 KO mice.


Figure 1
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  		Fig. 1 Evidence for the deletion of LOX-1. (A) PCR analysis of LOX-1 in blood, aorta and heart; (B) Immunofluorescence staining of LOX-1 protein and ox-LDL uptake in aorta.

 
3.2 Ischemia–reperfusion induces expression of LOX-1
In keeping with previous studies [16,17], expression of LOX-1 was increased during I–R in the hearts of wild-type mice. The LOX-1 KO mice, as expected, did not show LOX-1 protein at baseline or during I–R (Fig. 2 A).


Figure 2
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  		Fig. 2 (A) Western blot analysis of LOX-1 expression in the myocardium; (B) Infarct area (IA) as percent of area at risk (AAR) in different groups of mice; (C) Levels of MDA in the heart tissue; (D) Levels of 8-isoprostane in the heart tissue. WT: wild-type mice; I–R: ischemia–reperfusion.

 
3.3 Targeted deletion of LOX-1 limits myocardial injury and preserves myocardial function
The basal values of indices of cardiac function (HR, LVSP, LVEDP and ±dP/dtmax) were similar in the wild-type and LOX-1 KO mice. In both groups of mice, sham I–R caused no significant change in cardiac function (Table 2).


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  		Table 2 Effect of 60-min ischemia followed by 60-min reperfusion on hemodynamic parameters in wild-type and LOX-1 KO mice

 
Following I–R, there was a marked decrease in HR, LVSP, and ±dp/dtmax, and a significant increase in LVEDP in the wild-type mice. Reduction in –dp/dtmax and increase in LVEDP indicate diastolic dysfunction. Importantly, despite similar period of I–R in the LOX-1 mice, there was much less deterioration of cardiac function parameters indicating a significant preservation of cardiac function (P<0.01 vs. wild-type mice).

As shown in Fig. 2 B, there were no differences in AAR in the two groups of mice. In the wild-type mice, I–R resulted in extensive infarct (64.1%±10.6% of AAR). In contrast, LOX-1 KO mice had much smaller infarct (21.4%±3.5% of AAR, P<0.01 vs. wild type mice).

3.4 Myocardial ischemia–reperfusion induces oxidative stress
There is release of large amounts of ROS in the early stages of reperfusion, and NADPH oxidase activation is a major source of ROS in this process [7,8]. Release of ROS causes peroxidation of lipids in the heart. We measured MDA, an index of lipid peroxidation, 8-isoprostane, a nonenzymatic metabolite of ROS, and the expression of NADPH oxidase in the mice hearts. As shown in Fig. 2C and D, I–R caused a significant increase in MDA and 8-isoprostane levels in the wild-type mice hearts. In contrast, LOX-1 KO mice hearts had much lower MDA and 8-isoprostane (P<0.01 vs. wild-type mice), indicating much less ROS release. As shown in Fig. 3, both p22phox and p47phox subunits (mRNA and protein) of NADPH oxidase were markedly increased during I–R in both wild-type and LOX-1 KO mice (vs. sham I–R mice), but the LOX-1 KO mice had a much smaller increase (P<0.01 vs. the wild-type mice).


Figure 3
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  		Fig. 3 Expression of NADPH oxidase p22phox and p47phox subunits. (A) Representative protein and mRNA bands determined by Western blot and RT-PCR, respectively; (B) Quantitative protein data; (C) Quantitative mRNA data determined by real time RT-PCR. WT: wild-type mice; I–R: ischemia–reperfusion.

 
3.5 LOX-1 ablation reduces collagen deposition and MMPs expression
In the wild-type mice, I–R resulted in a marked increase in collagen accumulation (vs. sham I–R mice). In contrast, LOX-1 KO mice had much less collagen accumulation (P<0.01 vs. wild-type mice). The results of Masson's trichrome and Picro-sirius red staining were similar. Representative examples are shown in Fig. 4 (upper left panel), and the summary data are shown in Fig. 4 (lower left panel).


Figure 4
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  		Fig. 4 Collagen and procollagen-I changes following ischemia–reperfusion (I–R). Upper left panel: collagen in representative heart sections (original magnification x20); Lower left panel: quantitative data on collagen accumulation in heart sections; Upper right panel: representative procollagen-I bands (mRNA and protein); Lower right panel: quantitative data on procollagen-I protein. WT: wild-type mice; I–R: ischemia–reperfusion.

 
In support of the collagen data, procollagen-I expression (both mRNA and protein) was found to be markedly increased during I–R in the wild-type mice (P<0.01 vs. sham I–R mice), and the increase was much less in the LOX-1 KO despite I–R (Fig. 4, upper and lower right panel). Next, we determined the expression of MMPs in the heart tissues (Fig. 5). Expression (both mRNA and protein) of MMP-2, MMP-3 and MMP-9 in the wild-type mice was also increased following I–R (P<0.01 vs. wild-type mice). It is of note that the basal expression of MMP-2 and MMP-3 was significantly lower in the LOX-1 KO mice than in the wild-type mice. Nonetheless, expression of all three MMPs following I–R remained lower in the LOX-1 KO mice as compared to the wild-type mice.


Figure 5
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  		Fig. 5 Expression of matrix metalloproteinases (MMPs) determined by RT-PCR and Western analysis. WT: wild-type mice; I–R: ischemia–reperfusion.

 
3.6 LOX-1 ablation attenuates expression of osteopontin and fibronectin
Osteopotin has been shown to interact with fibronectin and plays a role in matrix organization, stability and wound healing [13]. As shown as in Fig. 6, expression (both mRNA and protein) of osteopontin as well as fibronectin increased significantly during I–R in both wild-type and LOX-1 KO mice, but the absolute levels of both remained much lower in the LOX-1 KO mice than in the wild-type mice (P<0.01).


Figure 6
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  		Fig. 6 Expression of fibronectin and osteopontin determined by RT-PCR and Western analysis. WT: wild-type mice; I–R: ischemia–reperfusion.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
4.1 Summary of main findings
Using state-of-the-art gene knockout technology, we provide convincing evidence that LOX-1 plays a potent role in the induction of myocardial infarct and dysfunction during I–R since the LOX-1 KO mice exhibited a much smaller infarct size and much less cardiac functional deterioration than the wild-type mice despite similar degree of I–R. Importantly, we provide conclusive evidence of expression of remodeling signals following a brief period of I–R in the wild-type mice and attenuation of these signals in the LOX-1 KO mice. The salutary changes in cardiac function in the LOX-1 KO mice were associated with attenuated NADPH oxidase expression, much less ROS release, and decreased collagen deposition.

4.2 Over-expression of collagen-I and metalloproteinases during ischemia–reperfusion
One of the early changes after the onset of ischemia is LV stiffness which persists during chronic ischemia and results in diastolic dysfunction. Although diastolic dysfunction during chronic ischemia has been ascribed to excessive collagen synthesis, enhanced collagen expression 1–2 h after onset of ischemia described in this study is a relatively novel finding. In earlier in vitro studies, we found that a brief exposure of cardiac fibroblasts to Ang II results in enhanced collagen-I synthesis [9,24]. We also showed that exposure of cardiac fibroblasts to anoxia-reoxygenation stimulates fibroblast growth as well as collagen type I synthesis and protein expression [25]. Collagen synthesis in response to Ang II and anoxia-reoxygenation was thought to be a response to the release of ROS. In this process, activation of MAP kinases and the redox-sensitive transcription factor NF-{kappa}B were noted to play an important role. It is of note that the cytokine TNF{alpha} that is released during I–R can also induce collagen synthesis in myofibroblasts [10].

Generally, collagen expression in the heart has been thought to occur several days or weeks after myocardial infarction [26–28]. It is of note that Takino et al [29] showed release of carboxyterminal propeptide of type I procollagen (PICP) in patients with myocardial infarction soon after deployment of reperfusion strategy. Release of PICP peaked at 2–3 weeks after myocardial infarction and correlated with end-diastolic volume. Our observations of increased collagen signals early after I–R are in concordance with the in vitro studies from our laboratory [9,24,25] and the in vivo study by Takino et al [29].

The marked increase in collagen noted in the present in vivo study was found to be associated with release of MMPs (–2, –3 and –9). The release of MMPs early after I–R has been frequently observed in the in vivo setting [30], and is thought to contribute to cardiac dilation and rupture during the acute stage of myocardial ischemia. Li et al [16] showed MMP release, expression of adhesion molecules and neutrophil accumulation in the I–R myocardium following a brief period of I–R in the Sprague Dawley rats. In vitro studies have also demonstrated that MMP release (by fibroblasts) during exposure of cardiac fibroblasts to anoxia-reoxygenation is triggered by redox-sensitive signals [25].

4.3 Inhibition of collagen-I and MMPs by LOX-1 deletion
Simultaneous release of MMPs and upregulation of collagen expression following I–R suggests that the two processes are inter-related and represent a cellular attempt to regulate the remodeling process. As discussed previously, expression of both MMPs and collagens may be a response to ROS release. Li et al [16] observed that I–R in the rat hearts was associated with over-expression of LOX-1, a finding reproduced in the present study in the wild-type mice. It is of note that LOX-1 activation has been linked to the release of ROS [31]. LOX-1 also acts as an adhesion molecule [18], and this may explain as to why the LOX-1 antibody administration before I–R reduced adhesion molecule expression and neutrophil accumulation in hearts exposed to I–R [16].

We showed that LOX-1 deletion reduces the expression of MMPs and procollagen-I at transcriptional level. We also documented a marked reduction in diastolic function as –dP/dtmax fell and LVEDP rose in the wild-type mice subjected to I–R. These alterations may well relate to the increase in collagen signal and deposition in the I–R myocardium. Importantly LOX-1 KO mice had significantly less decline in –dP/dtmax and much less increase in LVEDP in concert with a reduction in collagen deposition in the myocardium.

Reduction in procollagen-I may represent a decrease in oxidant stress in the LOX-1 KO mice. NADPH oxidase is the major source of ROS in the heart [7]. We measured NADPH oxidase expression and the myocardial MDA and 8-isoprostane levels, and found that NADPH oxidase (both p22phox and p47phox subunits) expression and ROS release and resultant lipid peroxidation increased dramatically in the wild-type mice subjected to I–R. This increase in NADPH oxidase and ROS release and resultant lipid peroxidation were much less in the LOX-1 KO mice. The absence of increase in NADPH oxidase and ROS release and resultant lipid peroxidation during I–R in the LOX-1 KO mice is in keeping with a previous report indicating that LOX-1 activation enhances superoxide anion generation [31].

We do not know the exact source of ROS in the present experiments, but it could be cardiomyocytes, fibroblasts, endothelial cell and/or neutrophils. All these cell types have been shown to generate ROS [32].

4.4 Osteopontin and fibronectin expression soon after myocardial ischemia
We observed that the expression of osteopontin as well as fibronectin increased in wild-type mice exposed to I–R. Osteopontin is an adhesion protein implicated as an important mediator of the profibrotic effects of Ang II in the heart. Osteopontin also acts as an adhesion molecule and has been implicated in chemoattraction of monocytes and in cell-mediated immunity [33]. It is also important in smooth muscle migration [34]. The plasma levels of the secreted glycophosphoprotein osteopontin have been associated with the presence and extent of coronary artery disease, especially with coronary calcification and restenosis after coronary intervention [11,12]. In the myocardium exposed to ischemia, osteopontin has been shown to interact with fibronectin suggesting its possible role in matrix organization, stability and wound healing [13]. Kossmehl et al [13] showed that the expression of MMPs, fibronectin and osteopontin was significantly elevated in the infarct area in porcine hearts subjected to 2 h of ischemia and 4 h of reperfusion. Simultaneously, large amounts of PICP were released in the perfusate. Fibroblast-like cells from the infarct area exhibited an enhanced ostepontin and fibronectin expression compared to fibroblasts derived from the control non-infarcted myocardium. Trueblood et al [27] examined the importance of osteopontin in the osteopontin null mice and found that the LV chamber dilation after myocardial infarction was approximately twice as great in osteopontin null mice as in the wild-type mice. Procollagen-I accumulation was also much less in the osteopontin null mice. Collins et al [35] showed that osteopontin is formed in response to Ang II, and the osteopontin null mice had much less cardiac fibroblasts proliferation and much less ECM accumulation after three weeks of Ang II infusion. Interestingly, osteopontin null mice had reduced MMP-2 and MMP-9 activity [36].

The signal for osteopontin expression seems to involve oxidant stress and related pathways. Xie et al [37] showed that p42/44 MAPK is a critical component of the ROS-sensitive signaling pathway activated by Ang II that regulates osteopontin gene expression. In another study in apoE KO mice [38], vitamin E decreased aortic 8-isoprostane and reduced both aortic macrophage infiltration and osteopontin expression. Lai and coworkers [14] demonstrated that osteopontin, via activation of NADPH oxidase-derived superoxide anion formation, promotes upregulation of MMP-9 in primary aortic myofibroblasts and smooth muscle cells under hyperglycemic conditions in vitro. Thus, there appears to be a strong link between NADPH oxidase-induced oxidant stress, osteopontin expression and MMP expression and activity. Gorin et al [39] have similarly shown a relationship between NADPH oxidase activation and fibronectin generation in both in vitro and in vivo conditions. In keeping with these studies, it was not surprising that the expression of osteopontin, fibronectin and MMPs was lower in the hearts of LOX-1 KO mice that had low levels of NADPH oxidase (both p22phox and p47phox subunits) and reduced myocardial 8-isoprostane and MDA content.

4.5 Signal for cardiac remodeling after ischemia–reperfusion
It is now amply evident that a host of mediators are expressed during I–R, including cytokines and Ang II, which account for oxidative stress mostly by activating NADPH oxidase system. The intense oxidant stress, particularly in the infarct-prone region (area at risk), induces upregulation of genes, such as fibronectin, osteopontin, collagen and MMPs soon after ischemia. Enhanced expression of fibronectin, osteopontin and collagen leads to myocardial diastolic dysfunction. Attenuation of the expression of these signals in LOX-1 KO mice suggests that LOX-1 could be a relevant therapeutic target in the management of ischemia-associated myocardial dysfunction.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This study was supported by funds from the Department of Veterans Affairs (JLM), a grant from the American Heart Association (PLH), grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Ministry of Health, Labor and Welfare of Japan, the National Institute of Biomedical Innovation, Japan Science and Technology Agency, and the New Energy and Industrial Technology Development Organization (T.S).


   Notes
 
1 These two authors contributed equally. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

  1. Wood D.A. Preventing clinical heart failure: the rationale and scientific evidence. Heart (2002) 88:15–22.[Abstract/Free Full Text]
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