Sage Journals: Discover world-class research

Abstract

Background:

Cardiomyocyte apoptosis by coronary microembolization (CME) contributes to myocardial dysfunction, in which mitochondrial pathway and death receptor pathway are activated. Lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) is a membrane protein involved in apoptosis. The study aimed to explore the role of LOX-1 in the activation of these 2 major apoptotic pathways.

Methods:

Twenty Bama miniature swine were randomized into 4 groups (n = 5 per group). The groups were Sham, CME, LOX-1 small-interfering RNA (siRNA), and control siRNA. Microspheres were injected into the left anterior descending artery of swine to establish CME model. Twelve hours after operation, cardiac function, serum c-troponin I level, microinfarct, and apoptotic index were examined. The levels of LOX-1, Bcl-2, Bax, cytochrome c as well as cleaved caspase 9, -8, and -3 were detected.

Results:

Myocardial dysfunction, enhanced serum c-troponin I, microinfarct, and apoptosis were induced following CME. Moreover, CME induced increased expression of LOX-1, Bax, cytochrome c, cleaved caspase 9, -8, and -3 as well as decreased Bcl-2 expression levels. The LOX-1 siRNA reversed these effects by CME except cleaved caspase 8 expression, while the control siRNA had no effect.

Conclusion:

Coronary microembolization induces cardiomyocyte apoptosis via the LOX-1-dependent mitochondrial pathway and caspase 8-dependent pathway.

Keywords

coronary microembolization lectin-like oxidized low-density lipoprotein receptor 1 cardiomyocyte apoptosis caspase

Introduction

Coronary microembolization (CME) resulting from the embolization of atherosclerotic and thrombotic debris into the microvasculature is a severe and frequent complication during percutaneous coronary intervention (PCI), thrombosis, and surgical revascularization.¹ It could induce malignant arrhythmias, contractile dysfunction, reduced coronary reserve, and irreversible myocardial damage including apoptosis and necrosis.² Furthermore, CME is the most likely culprit of absence of reflow and represents a strong independent predictor of poor prognosis. Apoptosis occurs following CME and partially contributes to CME-induced myocardial injury.³ Apoptosis inhibition may attenuate the progress of chronic cardiac dysfunction following CME and improve the prognosis. Activation of mitochondrial apoptotic pathway and death receptor pathway has been considered as 2 major pathways in apoptosis.^4,5 Bcl-2, Bax, cytochrome c, and caspase 9 are markers of the mitochondrial pathway. Caspase 8 activation is a key process of the death receptor pathway. Lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) is a newly identified lectin-like oxidized LOX for several ligands including tumor necrosis factor α (TNF-α).⁶ It expresses itself in atherosclerotic plaques and has been demonstrated to be critical in several cardiovascular diseases.^7
–9 However, whether or not it is involved in the activation of mitochondrial pathway and death receptor pathway following CME is currently unknown. Therefore, we sought to explore the role of LOX-1 in CME-induced apoptosis in a swine model of CME established by injecting microspheres into the left anterior descending (LAD) artery.

Materials and Methods

Animal Modeling and Grouping

The protocol was approved by the Animal Care and Use Committee of Guangxi Medical University, China. The investigation adhered to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication NO. 85-23, revised 1996). Twenty Bama miniature swine (25-30 kg) of either sex were purchased from the Animal Center, Agricultural College of Guangxi University (Nanning, China). Swine were randomized into 4 groups (n = 5 per group): Sham, CME, LOX-1 small interfering RNA (siRNA), and control siRNA. The CME model was induced by injecting plastic microspheres (diameter 42 μm) into the LAD artery. The initial anesthesia was induced gradually by intramuscular injection of the combination of ketamine (20 mg/kg) and atropine (2 mg) and maintained with diazepam injected via left or right ear vein. A 6F radial arterial sheath (Cordis, Fremont, CA) was placed in the right femoral artery. Swine were anticoagulated with 200 IU/kg heparin injected intravenously after a sheath placement, then 100 IU/kg/h to maintain heparinization. A 6F JL 4.0 guiding catheter was utilized for coronary angiography. A 1.8F infusion catheter (Cordis Inc) was then placed in the LAD artery with the tip located between the second and third diagonal branches. The swine in the CME group received a total dosage of 100 000 microspheres with the diameter of 42 μm (Dynospheres; Dyno Particles; Lillestrom, Norway) suspended in a 10 mL normal saline within 40 minutes, while animals in the sham-operated group received the same dosage of normal saline instead. Swine in the LOX-1 siRNA and control siRNA groups were subjected to LOX-1 siRNA and control siRNA, respectively, via intracoronary artery, 24 hours prior to CME.

The Transfection of LOX-1 siRNA

The LOX-1 messenger RNA sequence was found in GenBank (accession: NM_ 213805) and synthesized by GenePharma company (Shanghai, China). The sense strand and the antisense strand of the LOX-1 siRNA were 5′-GGGAACUCACAGAAAUGAUTT-3′ and 5′-AUCAUUUCUGUGAGUUCCCTT-3′, respectively. The control siRNA was purchased from the same company. A total mass of 300 μg LOX-1 siRNA or control siRNA was diluted with 600 μL transfection reagent (Entranster—in vivo; Engreen, Beijing, China) mixed thoroughly by pipetting the solution up and down. Then the mixture was injected into the LAD artery via the 1.8F infusion catheter, followed by 2 mL normal saline injection.

Cardiac Function Monitoring

Twelve hours following operation, swine were anesthetized and positioned in a right lateral decubitus position. This process was performed by an experienced specialist using a GE VIVID 7 system with a 1.5 to 4.3 MHZ transducer. In brief, the transducer was placed on the left anterior chest wall to measure the left ventricular ejection fraction (LVEF), left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), fractional shortening (FS), and cardiac output (CO).

Serum c-Troponin I Measurement

EDTA-anticoagulated blood sample (1.0 mL) was obtained from the femoral artery of each swine 12 hours after operation, prior to euthanasia, and centrifuged at 4000 rpm for 15 minutes. Serum samples were stored at −80°C for detection by electrochemistry method according to manufacturer’s instruction (Roche Inc, Switzerland).

Tissue Sampling

After cardiac function detection, 10% potassium chloride (10 mL) was used to euthanize the swine via ear vein injection. Hearts were isolated and cleaned with normal saline immediately. The anterior wall of the left ventricle dominated by LAD artery was isolated. Parts of myocardial tissue were immediately frozen in liquid nitrogen and stored at −80°C for Western blot analysis. The others were fixed with 4% paraformaldehyde for 12 hours, embedded in paraffin, and serially sectioned into slices of 4 μm thickness for hematoxylin and eosin (HE) staining, hematoxylin basic fuchsin picric acid (HBFP) staining, and terminal deoxynucleotidyl transferase-mediated dUTP Nick End-Labeling (TUNEL) assay.

Microinfarct Size Measurement

The HBFP staining is an important method used to detect early myocardial ischemia or infarct region. The normal myocardial tissue was stained yellow or brown while the ischemia or necrotic myocardial tissue red. A DMR-Q550 pathological image analyzer (Leica, Germany) was used to observe HBFP-stained sections. Microinfarct area was counted in 5 randomly selected visual fields (magnification, × 100) in each section using QWin analysis software (Leica, Germany), and 5 sections per swine were examined. The area of microinfarction was determined by planimetry and expressed as percentage of the total analyzed area.

TUNEL Assay

Apoptotic cardiomyocytes were detected using TUNEL assay kit (Roche, USA). The TUNEL-positive signal was located in nuclei, and apoptotic nuclei were stained yellow–brown while the normal light blue. Meanwhile, morphological features of apoptosis (small, condensed nuclei, cell shrinkage, and nuclear fragmentation) were taken into consideration. In each section, 10 random high-powered fields (magnification, ×400) were observed to count TUNEL-positive nuclei, and apoptotic index (%) was calculated as the percentage ratio of TUNEL-positive cell nuclei to the total nuclei.

Western Blot Analysis

Total protein was extracted using lysis buffer (containing Phenylmethanesulfonyl fluoride [PMSF]). The bicinchoninic acid method was used to determine protein concentration, and 30 μg protein sample was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, Massachusetts), which were blocked with 5% nonfat milk dissolved in 0.05% Tris Buffered Saline with Tween (TBST) at room temperature for 1 hour followed by incubation overnight at 4°C with following primary antibodies: LOX-1 (1:1000, abcam, Shanghai), Bcl-2 (1:500, BIOSS, USA), Bax (1:1000, abcam), cytochrome c (1:1000, Cell Signaling Technology, USA), caspase 9 (1:1000, abcam), caspase 8 (1:1000, Cell Signaling Technology), and caspase 3 antibody (1:1000, abcam). The membranes were washed and then incubated with the fluorescent secondary antibody (1:5000, LICOR, USA) at room temperature for 2 hours. The Odyssey 2-color infrared fluorescence imaging system (LICOR) was utilized to scan the membranes. Band density was determined by Quantity One software (Version 4.6.2, Bio-Rad, USA).

Statistical Analysis

Statistical analyses were performed using SPSS 19.0 statistical software (IBM Corporation, Chicago, IL). Quantitative data were expressed as mean ± standard deviation (SD). Analysis of variance was used for comparison among multiple groups, followed by Student-Newman-Keuls test for the further conduction of post hoc analysis. Pearson correlation analysis was performed for linear correlation. Statistical significance was presented as P < .05.

Results

In this study, there was no statistically significant difference (P > .05) among groups in regard to heart rate or body weight (see Table 1)

Table 1.

Physiological data of Swine among Groups.

Group	Heart Rate, bpm		Gender		Body Weight, kg
Group	Pre	Post	Male	Female	Pre
Sham	84.2 ± 5.93	91.2 ± 3.77	4	1	27.38 ± 2.49
CME	88.2 ± 2.17	83.6 ± 3.58	2	3	27.56 ± 1.47
LOX-1 siRNA	91.8 ± 6.02	87.4 ± 3.36	4	1	27.34 ± 1.56
Control siRNA	83.4 ± 4.83	85.2 ± 5.63	3	2	28.34 ± 0.6

Abbreviations: CME, coronary microembolization; Pre, preprocedure; Post, postprocedure; LOX, low-density lipoprotein receptor; siRNA, small interfering RNA.

Cardiac Dysfunction Following CME

The myocardial dysfunction was induced following CME, characterized by increased LVEDD and LVESD as well as decreased LVEF, FS, and CO (P < .05) as shown in Table 2. The LOX-1 siRNA improved myocardial function following CME (P < .05), while the control siRNA had no significant effect (P > .05).

Table 2.

Effects of LOX-1 siRNA on Cardiac function Following CME.

Group	LVEF, %	FS, %	CO, L/min	LVEDD, mm	LVESD, mm
Sham	68.70 ± 2.73	41.05 ± 2.08	4.19 ± 0.29	32.32 ± 1.53	23.71 ± 1.98
CME	48.81 ± 4.00^a	23.44 ± 3.54^a	2.50 ± 0.23^a	39.28 ± 1.23^a	27.31 ± 2.13^a
LOX-1 siRNA	56.37 ± 4.96^ab	32.74 ± 2.98^ab	3.74 ± 0.32^ab	34.26 ± 2.24^ab	25.53 ± 1.75^ab
Control siRNA	46.23 ± 3.74^a	25.72 ± 3.27^a	2.67 ± 0.31^a	42.39 ± 2.57^a	28.99 ± 2.11^a

Abbreviations: CME, coronary microembolization; LVEF, left ventricular ejection fraction; FS, fractional shortening; CO, cardiac output; LVEDD, left ventricular end-diastolic diameter; LVESD, left ventricular end-systolic diameter; LOX, low-density lipoprotein receptor; siRNA, small interfering RNA.

^aData are presented as mean ± standard deviation; n = 5 for each group.

^aP < .05 versus the sham group.

^bP < .05 versus the CME group.

Elevated Serum c-troponin I Level Following CME

The serum c-troponin I level was significantly enhanced following CME (P < .05) as shown in Table 3. Pretreatment with LOX-1 siRNA attenuated significant serum c-troponin I level (P < .05). The control siRNA had no significant effect on serum c-troponin I level (P > .05).

Table 3.

Effect of LOX-1 siRNA on Serum c-Troponin I Level After CME.^a

Groups	Serum c-troponin I level, ng/mL
Sham	0.038 ± 0.005
CME	0.191 ± 0.009^b
LOX-1 siRNA	0.093 ± 0.008^b,c
Control siRNA	0.215 ± 0.012^b

Abbreviations: CME, coronary microembolization; SD, standard deviation; LOX, low-density lipoprotein receptor; siRNA, small interfering RNA.

^aData are mean ± SD (n = 5 per group).

^bP < .05 versus the sham group.

^cP < .05 versus the CME group.

Microinfarct Following CME

No infarct was observed in the sham group. As shown in Figure 1, HE staining showed multiple focal microinfarct around microspheres. Additionally, cardiomyocytes surrounding microinfarct were edematous with inflammatory cell infiltrates. As shown in Figure 2, HBFP staining revealed nontransmural multiple focal microinfarct in hearts of swine subjected to CME, mainly in the subendocardium, and the anterior wall of the left ventricle. No microinfarct was found in the sham group, while it was significantly evident in the CME group (P < .05). LOX-1 siRNA significantly reduced the microinfarct area (P < .05). The control siRNA had no significant effect (P > .05).

Figure 1.

Pathohistological examination by HE staining at 12 hours following coronary microembolization (magnification, ×200; bar = 50 μm). The HE staining showed multiple focal microinfarct around microspheres with inflammatory cells infiltration. The arrow points to microspheres. HE indicates hematoxylin and eosin.

Figure 2.

Microinfarct measurement with HBFP staining (magnification, ×100; bar = 25 μm). The normal tissue was stained yellow, while the infarct red. A, HBFP staining showed no microinfarct in the sham group (a). Infarct was evident following coronary microembolization (b). The increase was inhibited by LOX-1 siRNA (c), while the control siRNA showed no effect (d). B, The bar graph shows infarct size. Data are mean ± SD (n = 5 per group). ^aP < .05 versus the sham group; ^bP < .05 versus the CME group. HBFP indicates hematoxylin basic fuchsin picric acid; LOX, low-density lipoprotein receptor; siRNA, small interfering RNA; SD, standard deviation.

Cardiomyocyte Apoptosis Following CME

The apoptotic index was significantly enhanced in the CME group (P < .05) as shown in Figure 3. The LOX-1 siRNA pretreatment significantly decreased apoptotic index (P < .05). The control siRNA had no significant effect (P > .05).

Figure 3.

Apoptosis detected by TUNEL staining (magnification, × 400; bar = 100 μm). A, TUNEL-positive signal was located in nuclei, and apoptotic nuclei were stained yellow-brown while the normal cell nuclei light blue. The arrow indicates TUNEL-positive nucleus. a, sham; b, CME; c, LOX-1 siRNA; d, Control siRNA. B, The bar graph shows apoptotic index. Apoptosis was evident in the CME group. LOX-1 siRNA decreased apoptotic index. The control siRNA showed no effect. Data are mean ± SD (n = 5 per group). ^aP < .05 versus the sham group; ^bP < .05 versus the CME group. TUNEL indicates terminal deoxynucleotidyl transferase-mediated dUTP Nick End-Labeling; CME, coronary microembolization; LOX, low-density lipoprotein receptor; siRNA, small interfering RNA; SD, standard deviation.

Expression of LOX-1 protein Following CME

The LOX-1 protein expression detected by Western blot was significantly increased in the CME group (P < .05) as shown in Figure 4. Pretreatment with LOX-1 siRNA significantly inhibited its upregulation (P < .05). The control siRNA had no effect on LOX-1 expression (P > .05).

Figure 4.

LOX-1 upregulation. A, Western blot showed that LOX-1 protein expression was increased by coronary microembolization (CME). LOX-1 siRNA inhibited its increase despite CME. The control siRNA had no effect. Representative blots shown. B, Semiquantitation of bands of LOX-1. Data are mean ± SD (n = 5 per group). ^aP < .05 versus the sham group; ^bP < .05 versus the CME group. LOX indicates low-density lipoprotein receptor; siRNA, small interfering RNA; SD, standard deviation.

Expression of Apoptotic Related Proteins Following CME

The Bcl-2 expression was reduced, and the expression of other proteins (Bax, cytochrome c and cleaved caspase-9, cleaved caspase-8, and cleaved caspase-3) was increased in the CME group compared with those in the sham group as shown in Figures 5 to 7. LOX-1 siRNA reversed the expression of Bcl-2, Bax, cytochrome c, cleaved caspase-9, and cleaved caspase 3 following CME but not cleaved caspase-8. The control siRNA showed no effect.

Figure 5.

Mitochondria apoptosis-related protein activation. A, Bcl-2 expression was decreased, and the expression of Bax, cytochrome c, and cleaved caspase-9 was increased following coronary microembolization (CME). LOX-1 siRNA reversed the expression of CME on the expression of these proteins. The control siRNA had no effect. Representative blots are shown. B, Semiquantitation of bands of Bcl-2, Bax, cytochrome c, and cleaved caspase-9. Data are mean ± SD (n = 5 per group). ^aP < .05 versus the sham group; ^bP < .05 versus the CME group. LOX indicates low-density lipoprotein receptor; siRNA, small interfering RNA; SD, standard deviation.

Figure 6.

Caspase-8 activation. A, Cleaved caspase-8 expression was enhanced following coronary microembolization (CME). Both LOX-1 siRNA and control siRNA showed no effect on cleaved caspase-8 expression. Representative blots are shown. B, Semiquantitation of bands of cleaved caspase-8. Data are mean ± SD (n = 5 per group). ^aP < .05 versus the sham group; ^bP < .05 versus the CME group. LOX indicates low-density lipoprotein receptor; siRNA, small interfering RNA; SD, standard deviation.

Figure 7.

Caspase-3 activation. A, Cleaved caspase-3 expression was enhanced following coronary microembolization (CME). LOX-1 siRNA inhibited its increase, while the control siRNA showed no effect. Representative blots shown. B, Semiquantitation of bands of cleaved caspase-3. Data are mean ± SD (n = 5 per group). ^aP < .05 versus the sham group; ^bP < .05 versus the CME group. LOX indicates low-density lipoprotein receptor; siRNA, small interfering RNA; SD, standard deviation.

Linear Correlation Analysis

The level of LOX-1 positively correlated with apoptotic index (r = .864; P < .01), whereas it negatively correlated with LVEF (r = −.871; P < .01). This is shown in Figure 8.

Figure 8.

Linear correlation analysis. The level of LOX-1 protein was positively correlated with apoptotic index (A) and negatively correlated with LVEF (B). LOX indicates low-density lipoprotein receptor; LVEF, left ventricular ejection fraction.

Discussion

The present study was designed to explore the role of LOX-1 in CME-induced apoptosis in a swine model of CME showing that (1) myocardial dysfunction, microinfarct, enhanced serum c-troponin I level, and apoptosis were induced following CME. (2) Mitochondrial apoptosis-related proteins (Bcl-2, Bax, cytochrome c, and caspase-9), caspase-8, and caspase-3 were all activated in hearts following CME. In addition to that LOX-1 protein expression was markedly enhanced. (3) LOX-1 siRNA reversed these effects by CME but not caspase 8, while the control siRNA showed no effect.

Coronary microembolization and its sequelae are frequent clinical events in patients undergoing PCI. Furthermore, CME seems to be inevitable during PCI but differs in its severity. The mild CME does not cause clinical symptom, while the serious CME may induce no reflow and even sudden death. Bose et al concluded that patients with large necrotic core may be more susceptible.¹⁰ Periprocedural myocardial injury is closely related to microembolization. A model resembling those patients who had embolism in their coronary microvasculature during PCI is critical for understanding the pathophysiology of CME. An ideal animal model that could totally simulate the CME phenomenon seen in clinics does not exist; however, the cardiovascular system of the swine closely resembles that in humans. Some researchers caused CME by injecting 3000 microspheres (42 μm) per mL/min showing perfusion–contraction mismatch with reduced contractile function and unchanged or even elevated blood flow.^11,12 Chen et al induced CME in mini-pigs by injecting a mean dosage of 120 000 microspheres into LAD artery, finding myocardial dysfunction, microinfarcts, elevated inflammatory cytokines, and apoptosis.³ Li et al established CME model in rats by injecting homologous thrombotic material finding inflammation and left ventricular dysfunction.¹³ These results are partially consistent with those in clinical scenarios. These models could partially simulate the CME phenomenon in clinics but differ in its severity. Our experimental approach simulated the occurrence of CME during PCI through injecting a dosage of 100 000 microspheres of 42 μm diameter into the LAD artery finding that myocardial dysfunction, microinfarct with inflammation, elevated serum c-troponin I, and apoptosis were induced in a swine model. These events are consistent with sequelae of patients subjected to CME in clinical settings. Therefore, this swine model of CME could partially simulate the status of patients subjected to CME in clinical scenarios.

LOX-1, Apoptosis-Related Proteins, and Apoptosis Following CME

Cardiomyocytes loss by apoptosis is involved in several cardiovascular diseases including heart failure, ischemia–reperfusion injury, and acute myocardial infarction (MI).^14,15 Decreased apoptosis by caspase inhibition has been determined to improve myocardial function and therefore might improve clinical outcomes.^16
–18 Recent reports determined that apoptosis plays an important role in CME-induced myocardial impairment, despite earlier reports of almost negligible effect.^3,19 This was confirmed in the present study by showing that CME induced increased apoptotic index and cardiac dysfunction. The mitochondria has been the primary concern since it was described as the powerhouse of cells to generate adenosine triphosphate. However, emerging studies have proposed its role in the process of apoptosis.^20,21 It is well known that the involvement of mitochondria is dependent on actions of Bcl-2 and Bax to promote the release of apoptotic proteins such as cytochrome c. Then the released cytochrome c induces the activation of caspase 9 and -3, and therefore apoptosis occurs.⁴ Our previous research revealed that caspase 9, caspase 8, and caspase 3 were activated in the process of apoptosis following CME, which means mitochondria pathway and death receptor pathway were activated following CME.^22,23 Moreover, in the present study, we showed that the expression of Bax and cytochrome c was enhanced, and Bcl-2 expression was decreased following CME.

The LOX-1 is a newly determined membrane receptor for several ligands. It was reported to be highly expressed in atherosclerotic lesion and has been proposed in several cardiovascular diseases including acute coronary syndrome. Li et al showed that LOX-1 upregulation during myocardial ischemia–reperfusion injury was associated with apoptosis and cardiac dysfunction.²⁴ Inhibition of LOX-1 by LOX-1 antibody improved myocardial function and reduced apoptotic index. It means that LOX-1 contributes to apoptosis during ischemia–reperfusion injury. Recently, Hong et al showed that LOX-1-mediated endoplasmic reticulum stress pathway contributes to ox-LDL-induced apoptosis on endothelial cells.²⁵ However, its role in the regulation of apoptosis by CME remains to be elucidated. In the present study, we showed that effects of CME on mitochondria apoptotic proteins were markedly blocked by an LOX-1 siRNA, however it did not lead to any significant changes on cleaved caspase-8 expression. Moreover, we showed that the relative expression of LOX-1 was positively correlated with apoptotic index. These results indicate that CME induces apoptosis by activating the LOX-1-mediated mitochondria pathway and caspase-8-dependent pathway. And signal transduction of cardiomyocyte apoptosis resulting from CME is shown in Figure 9.

Figure 9.

Signal transduction of CME-induced cardiomyocyte apoptosis. CME indicates coronary microembolization; LOX-1, lectin-like oxidized low-density lipoprotein receptor 1.

LOX-1 and Myocardial Injury Following CME

Coronary microembolization resulting from the washout of atherothrombotic debris into coronary microcirculation could induce microinfarct and therefore enhanced serum markers of myocardial impairment, such as troponin I. Final infarct size is proposed as a strong predictor of myocardial function recovery and long-term clinical outcome following infarction. Markers of myocardial necrosis in serum samples including troponin I is generally measured to estimate infarct area.²⁶ In this study, we showed that CME induced cardiac dysfunction, increased microinfarct size, and serum c-troponin I level. Therapeutic strategies targeting to reducing infarct area might therefore improve myocardial function in return.²⁷ Thus, LOX-1 siRNA reduced microinfarct area and serum c-troponin I level and improved myocardial function. These effects may partially be involved in apoptosis inhibition attributed to LOX-1 siRNA. Tumor necrosis factor α has been determined as a casual factor for myocardial dysfunction following CME.²⁸ Furthermore, TNF-α is in vitro stimuli for LOX-1 upregulation.²⁹ This means TNF-α may induce cardiac dysfunction following CME via the LOX-1 signaling pathway, which needs further research.

The level of soluble form of LOX-1 (sLOX-1) is believed to reflect LOX-1 expression of atherosclerotic plaque. Elevated sLOX-1 level of circulating blood is a strongly predictor of atherosclerotic progression. Recently, Kobayashi et al revealed that plasma sLOX-1 could diagnose the early stage of ST-elevation MI.³⁰ Coronary microembolization is thought to be a major cause of PCI-related periprocedural MI. Troponin and CK-MB are well-known biomarker of myocardial injury. Balin et al revealed sLOX-1 levels were significantly in positive correlation with those of troponin and CK-MB.³¹ This means sLOX-1 may be an early and novel biomarker of MI. This will be further confirmed in our future research.

Conclusion

Our results indicate that CME induces cardiomyocyte apoptosis in a swine model via the activation of LOX-1-dependent mitochondrial pathway and caspase-8-dependent pathway. LOX-1 might be a potential target for cardioprotection.

Footnotes

Author Contributions

Lang, L contributed to conception, gave final approval, and agrees to be accountable for all aspects of work ensuring integrity and accuracy; Tao, L contributed to design, contributed to acquisition, analysis, and interpretation, drafted the manuscript, and critically revised the manuscript; You, Z contributed to acquisition, analysis, and interpretation; You, W contributed to acquisition, analysis, and interpretation; Qiang, S contributed to analysis; LI, T contributed to acquisition; Chun, L contributed to analysis and critically revised the manuscript.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by the National Nature Science Foundation of China (NO. 81260042).

References

Heusch

Kleinbongard

Bose

. Coronary microembolization: From bedside to bench and back to bedside. Circulation. 2009;120(18):1822–1836.

De Caterina

Porto

Luigi De Maria

Banning

. Prevention and treatment of coronary distal embolization in the setting of acute myocardial infarction: Pharmacologic approach. Curr Vasc Pharmacol. 2012;10(4):463–467.

Chen

Qian

. Tnf-alpha-induced cardiomyocyte apoptosis contributes to cardiac dysfunction after coronary microembolization in mini-pigs. J Cell Mol Med. 2014;18(10):1953–1963.

Crow

Mani

Nam

Kitsis

. The mitochondrial death pathway and cardiac myocyte apoptosis. Circ Res. 2004;95(10):957–970.

Ashkenazi

Targeting the extrinsic apoptotic pathway in cancer: Lessons learned and future directions. J Clin Invest. 2015;125(2):487–489.

Mehta

. Intracellular signaling of lox-1 in endothelial cell apoptosis. Circ Res. 2009;104(5):566–568.

Kataoka

Kume

Miyamoto

. Expression of lectinlike oxidized low-density lipoprotein receptor-1 in human atherosclerotic lesions. Circulation. 1999;99(24):3110–3117.

Akhmedov

Rozenberg

Paneni

. Endothelial overexpression of lox-1 increases plaque formation and promotes atherosclerosis in vivo. Eur Heart J. 2014;35(40):2839–2848.

Sawamura

Wakabayashi

Okamura

. Lox-1 in atherosclerotic disease. Clin Chim Acta. 2015;440:157–163.

10.

Bose

Von Birgelen

Zhou

. Impact of atherosclerotic plaque composition on coronary micro embolization during percutaneous coronary interventions. Basic Res Cardiol. 2008;103(6):587–597.

11.

Skyschally

Schulz

Erbel

Heusch

. Reduced coronary and inotropic reserves with coronary microembolization. Am J Physiol Heart Circ Physiol. 2002;282(2):H611–H614.

12.

Dorge

Neumann

Behrends

. Perfusion-contraction mismatch with coronary microvascular obstruction: role of inflammation. Am J Physiol Heart Circ Physiol. 2000;279(6):H2587–H2592.

13.

Zhong

Zeng

. Blockade of NF-kappaB by pyrrolidine dithiocarbamate attenuates myocardial inflammatory response and ventricular dysfunction following coronary microembolization induced by homologous micro thrombi in rats. Basic Res Cardiol. 2010;105(1):139–150.

14.

Singh

Kang

. Mechanisms and inhibitors of apoptosis in cardiovascular diseases. Curr Pharm Des. 2011;17(18):1783–1793.

15.

Lee

Gustafsson

. Role of apoptosis in cardiovascular disease. Apoptosis. 2009;14(4):536–548.

16.

Park

Vatner

Yan

. Novel mechanisms for caspase inhibition protecting cardiac function with chronic pressure overload. Basic Res Cardiol. 2013;108(1):324.

17.

Hayakawa

Chandra

Miao

. Inhibition of cardiac myocyte apoptosis improves cardiac function and abolishes mortality in the peripartum cardiomyopathy of galpha(q) transgenic mice. Circulation. 2003;108(24):3036–3041.

18.

Kovacs

Bak

Szendrei

. Non-specific caspase inhibition reduces infarct size and improves post-ischaemic recovery in isolated ischaemic/reperfused rat hearts. Naunyn Schmiedebergs Arch Pharmacol. 2001;364(6):501–507.

19.

Dorge

Schulz

Belosjorow

. Coronary microembolization: The role of tnf-alpha in contractile dysfunction. J Mol Cell Cardiol. 2002;34(1):51–62.

20.

Sreedhar

Arumugam

Thandavarayan

. Myocardial 14-3-3eta protein protects against mitochondria mediated apoptosis. Cell Signal. 2015;27(4):770–776.

21.

Dai

Shi

Gupta

Sabbah

Hale

Kloner

. Bendavia, a mitochondria-targeting peptide, improves postinfarction cardiac function, prevents adverse left ventricular remodeling, and restores mitochondria-related gene expression in rats. J Cardiovasc Pharmacol. 2014;64(6):543–553.

22.

Liu

Zhou

Wen

. Effect of metoprolol on myocardial apoptosis and caspase-9 activation after coronary microembolization in rats. Exp Clin Cardiol. 2013;18(2):161–165.

23.

Wang

. Effect of atorvastatin (lipitor) on myocardial apoptosis and caspase-8 activation following coronary microembolization. Cell Biochem Biophys. 2011;61(2):399–406.

24.

Williams

Liu

. Expression of lectin-like oxidized low-density lipoprotein receptors during ischemia-reperfusion and its role in determination of apoptosis and left ventricular dysfunction. J Am Coll Cardiol. 2003;41(6):1048–1055.

25.

Hong

Bai

Gao

. Ox-ldl induces endothelial cell apoptosis via the lox-1-dependent endoplasmic reticulum stress pathway. Atherosclerosis. 2014;235(2):310–317.

26.

Mueller

. Biomarkers and acute coronary syndromes: an update. Eur Heart J. 2014;35(9):552–556.

27.

Timmers

Henriques

de Kleijn

. Exenatide reduces infarct size and improves cardiac function in a porcine model of ischemia and reperfusion injury. J Am Coll Cardiol. 2009;53(6):501–510.

28.

Thielmann

Dorge

Martin

. Myocardial dysfunction with coronary microembolization: signal transduction through a sequence of nitric oxide, tumor necrosis factor-alpha, and sphingosine. Circ Res. 2002;90(7):807–813.

29.

Kume

Murase

Moriwaki

. Inducible expression of lectin-like oxidized LDL receptor-1 in vascular endothelial cells. Circ Res. 1998;83(3):322–327.

30.

Kobayashi

Hata

Kume

. Soluble lectin-like oxidized low-density lipoprotein receptor-1 as an early biomarker for ST elevation myocardial infarction: time-dependent comparison with other biomarkers: time-dependent comparison with other biomarkers. Circ J. 2011;75(6):1433–1439.

31.

Balin

Celik

Kobat

Baydas

. Circulating soluble lectin-like oxidized low-density lipoprotein receptor-1 levels predict percutaneous coronary intervention-related periprocedural myocardial infarction in stable patients undergoing elective native single-vessel PCI. J Thromb Thrombolysis. 2012;34(4):483–490.

Coronary Microembolization Induces Cardiomyocyte Apoptosis in Swine by Activating the LOX-1-Dependent Mitochondrial Pathway and Caspase-8-Dependent Pathway

Abstract

Background:

Methods:

Results:

Conclusion:

Keywords

Introduction

Materials and Methods

Animal Modeling and Grouping

The Transfection of LOX-1 siRNA

Cardiac Function Monitoring

Serum c-Troponin I Measurement

Tissue Sampling

Microinfarct Size Measurement

TUNEL Assay

Western Blot Analysis

Statistical Analysis

Results

Cardiac Dysfunction Following CME

Elevated Serum c-troponin I Level Following CME

Microinfarct Following CME

Cardiomyocyte Apoptosis Following CME

Expression of LOX-1 protein Following CME

Expression of Apoptotic Related Proteins Following CME

Linear Correlation Analysis

Discussion

LOX-1, Apoptosis-Related Proteins, and Apoptosis Following CME

LOX-1 and Myocardial Injury Following CME

Conclusion

Footnotes

Author Contributions

Declaration of Conflicting Interests

Funding

References