Effects of CXCR4 Gene Transfer on Cardiac Function After Ischemia-Reperfusion Injury
2010; Elsevier BV; Volume: 176; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2010.090451
ISSN1525-2191
AutoresJiqiu Chen, Elie R. Chemaly, Lifan Liang, Changwon Kho, Ahyoung Lee, Jaeho Park, Perry Altman, Alison D. Schecter, Roger J. Hajjar, Sima T. Tarzami,
Tópico(s)interferon and immune responses
ResumoAcute coronary occlusion is the leading cause of death in the Western world. There is an unmet need for the development of treatments to limit the extent of myocardial infarction (MI) during the acute phase of occlusion. Recently, investigators have focused on the use of a chemokine, CXCL12, the only identified ligand for CXCR4, as a new therapeutic modality to recruit stem cells to individuals suffering from MI. Here, we examined the effects of overexpression of CXCR4 by gene transfer on MI. Adenoviruses carrying the CXCR4 gene were injected into the rat heart one week before ligation of the left anterior descending coronary artery followed by 24 hours reperfusion. Cardiac function was assessed by echocardiography couple with 2,3,5-Triphenyltetrazolium chloride staining to measure MI size. In comparison with control groups, rats receiving Ad-CXCR4 displayed an increase in infarct area (13.5% ± 4.1%) and decreased fractional shortening (38% ± 5%). Histological analysis revealed a significant increase in CXCL12 and tumor necrosis factor-α expression in ischemic area of CXCR4 overexpressed hearts. CXCR4 overexpression was associated with increased influx of inflammatory cells and enhanced cardiomyocyte apoptosis in the infarcted heart. These data suggest that in our model overexpressing CXCR4 appears to enhance ischemia/reperfusion injury possibly due to enhanced recruitment of inflammatory cells, increased tumor necrosis factor-α production, and activation of cell death/apoptotic pathways. Acute coronary occlusion is the leading cause of death in the Western world. There is an unmet need for the development of treatments to limit the extent of myocardial infarction (MI) during the acute phase of occlusion. Recently, investigators have focused on the use of a chemokine, CXCL12, the only identified ligand for CXCR4, as a new therapeutic modality to recruit stem cells to individuals suffering from MI. Here, we examined the effects of overexpression of CXCR4 by gene transfer on MI. Adenoviruses carrying the CXCR4 gene were injected into the rat heart one week before ligation of the left anterior descending coronary artery followed by 24 hours reperfusion. Cardiac function was assessed by echocardiography couple with 2,3,5-Triphenyltetrazolium chloride staining to measure MI size. In comparison with control groups, rats receiving Ad-CXCR4 displayed an increase in infarct area (13.5% ± 4.1%) and decreased fractional shortening (38% ± 5%). Histological analysis revealed a significant increase in CXCL12 and tumor necrosis factor-α expression in ischemic area of CXCR4 overexpressed hearts. CXCR4 overexpression was associated with increased influx of inflammatory cells and enhanced cardiomyocyte apoptosis in the infarcted heart. These data suggest that in our model overexpressing CXCR4 appears to enhance ischemia/reperfusion injury possibly due to enhanced recruitment of inflammatory cells, increased tumor necrosis factor-α production, and activation of cell death/apoptotic pathways. Coronary artery disease is a leading cause of death in the United States. Acute myocardial infarction (MI) is a catastrophic manifestation of coronary artery disease that strikes nearly one million Americans each year.1Tissier R Berdeaux A Ghaleh B Couvreur N Krieg T Cohen MV Downey JM Making the heart resistant to infarction: how can we further decrease infarct size?.Front Biosci. 2008; 13: 284-301Crossref PubMed Scopus (34) Google Scholar, 2Yellon DM Baxter GF Protecting the ischaemic and reperfused myocardium in acute myocardial infarction: distant dream or near reality?.Heart. 2000; 83: 381-387Crossref PubMed Scopus (127) Google Scholar, 3Carden DL Granger DN Pathophysiology of ischaemia-reperfusion injury.J Pathol. 2000; 190: 255-266Crossref PubMed Scopus (1415) Google Scholar The mainstay of current therapy in acute MI is the restoration of blood flow (reperfusion) to the affected area through thrombolytic therapy or angioplasty. This reperfusion, although key to myocyte survival, is also associated with myocardial injury.2Yellon DM Baxter GF Protecting the ischaemic and reperfused myocardium in acute myocardial infarction: distant dream or near reality?.Heart. 2000; 83: 381-387Crossref PubMed Scopus (127) Google Scholar One component of reperfusion is the influx of inflammatory cells into the heart. These inflammatory cells are believed to cause further damage to the heart by their release of a variety of enzymes and other factors.4Vermeiren GL Claeys MJ Van Bockstaele D Grobben B Slegers H Bossaert L Jorens PG Reperfusion injury after focal myocardial ischaemia: polymorphonuclear leukocyte activation and its clinical implications.Resuscitation. 2000; 45: 35-61Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 5Gao L Yin H Smith S RJ Chao L Chao J Role of kallistatin in prevention of cardiac remodeling after chronic myocardial infarction.Lab Invest. 2008; 88: 1157-1166Crossref PubMed Scopus (58) Google Scholar However, in recent years, there has been an effort to maximize stem cell recruitment to speed repair of injured myocardium. Investigators have focused on the use of a chemokine, CXCL12 (also known as stromal cell–derived factor-1), which is the main functional ligand for CXCR4, as a new therapeutic modality. However, to date, several human clinical trials of stem cell therapy have shown limited cardiac benefit to individuals suffering from MI.6Abbott JD Huang Y Liu D Hickey R Krause DS Giordano FJ Stromal cell-derived factor-1alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury.Circulation. 2004; 110: 3300-3305Crossref PubMed Scopus (735) Google Scholar Chemokines are a super family of low-molecular-weight proteins (8 to 10 kDa) that have been subdivided into families on the basis of the position of their cysteine residues. There are currently 46 ligands that bind to 18 G protein–coupled receptors.7Luster AD Chemokines–chemotactic cytokines that mediate inflammation.N Engl J Med. 1998; 338: 436-445Crossref PubMed Scopus (3252) Google Scholar Although chemokines are key regulators of leukocyte migration and host defense pathways, excessive production of chemokines has been implicated in the inflammatory components of numerous diseases including chronic heart failure and ischemia-induced myocardial injury.8Pease JE Williams TJ The attraction of chemokines as a target for specific anti-inflammatory therapy.Br J Pharmacol. 2006; 147 Suppl 1: S212-S221PubMed Google Scholar, 9Damas JK Eiken HG Oie E Bjerkeli V Yndestad A Ueland T Tonnessen T Geiran OR Aass H Simonsen S Christensen G Froland SS Attramadal H Gullestad L Aukrust P Myocardial expression of CC- and CXC-chemokines and their receptors in human end-stage heart failure.Cardiovasc Res. 2000; 47: 778-787Crossref PubMed Scopus (191) Google Scholar, 10Sasayama S Okada M Matsumori A Chemokines and cardiovascular diseases.Cardiovasc Res. 2000; 45: 267-269Crossref PubMed Scopus (60) Google Scholar CXCR4 is a 37-kDa G protein–coupled receptor, located principally at the plasmalemma of cardiac myocytes. CXCL12, the predominant CXCR4 ligand, is constitutively expressed in myocardium, detectable in the serum, and is increased early post myocardial infarction.11Ma J Ge J Zhang S Sun A Shen J Chen L Wang K Zou Y Time course of myocardial stromal cell-derived factor 1 expression and beneficial effects of intravenously administered bone marrow stem cells in rats with experimental myocardial infarction.Basic Res Cardiol. 2005; 100: 217-223Crossref PubMed Scopus (199) Google Scholar Expression of CXCR4 is increased in the myocardium of patients with heart failure.9Damas JK Eiken HG Oie E Bjerkeli V Yndestad A Ueland T Tonnessen T Geiran OR Aass H Simonsen S Christensen G Froland SS Attramadal H Gullestad L Aukrust P Myocardial expression of CC- and CXC-chemokines and their receptors in human end-stage heart failure.Cardiovasc Res. 2000; 47: 778-787Crossref PubMed Scopus (191) Google Scholar Both CXCR4 and CXCL12 are expressed and functional in cardiomyocyte.12Segret A Rucker-Martin C Pavoine C Flavigny J Deroubaix E Chatel MA Lombet A Renaud JF Structural localization and expression of CXCL12 and CXCR4 in rat heart and isolated cardiac myocytes.J Histochem Cytochem. 2007; 55: 141-150Crossref PubMed Scopus (39) Google Scholar Whereas the significance of CXCR4 and CXCL12 in cardiac development has already been established,13Ma Q Jones D Borghesani PR Segal RA Nagasawa T Kishimoto T Bronson RT Springer TA Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice.Proc Natl Acad Sci USA. 1998; 95: 9448-9453Crossref PubMed Scopus (1422) Google Scholar, 14Zou YR Kottmann AH Kuroda M Taniuchi I Littman DR Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development.Nature. 1998; 393: 595-599Crossref PubMed Scopus (2121) Google Scholar there is a paucity of information regarding chemokine receptor signaling on adult cardiac myocytes. It was recently demonstrated that activation of CXCR4 results in a direct negative inotropic modulation of cardiac myocyte function in vitro implicating an important role for CXCR4 and its ligand, CXCL12, in heart function.15Pyo RT Sui J Dhume A Palomeque J Blaxall BC Diaz G Tunstead J Logothetis DE Hajjar RJ Schecter AD CXCR4 modulates contractility in adult cardiac myocytes.J Mol Cell Cardiol. 2006; 41: 834-844Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar In the present study, we explored the effects of CXCR4 gene transfer in a rat cardiac ischemia-reperfusion (IR) injury model. We hypothesized that overexpression of CXCR4 in the heart will increase the influx of inflammatory cells into ischemic tissue, enhance host response to oxygen free radicals, and impair the cardiac pump function. In support of this hypothesis, we examined the mechanistic role of CXCR4 in the pathophysiology of myocardial ischemic injury in vivo. Our data demonstrated that CXCR4 overexpression exacerbates the hemodynamic dysfunction and structural deterioration in a rat model of ischemic reperfusion injury. CXCR4 gene transfer increased the infarct size and decreased the cardiac pump function. Collectively, we show that overexpression of CXCR4 plays a potentially critical role dictating the outcome of ischemic reperfusion injury. Recombinant adenoviruses were used in these studies. Ad-EGFP-β-gal contains cytomegalovirus promoter, expression cassettes for β-galactosidase (β-gal), and enhanced green fluorescent protein (EGFP).16Hajjar RJ Schmidt U Matsui T Guerrero JL Lee KH Gwathmey JK Dec GW Semigran MJ Rosenzweig A Modulation of ventricular function through gene transfer in vivo.Proc Natl Acad Sci USA. 1998; 95: 5251-5256Crossref PubMed Scopus (266) Google Scholar The backbone vector, which contains most of the adenoviral genome (pAd.EASY1), was used and the recombination performed in Escherichia coli. CXCR4 cDNA was subcloned into the adenoviral shuttle vector (pAd.TRACK), using the cytomegalovirus long-terminal repeat as a promoter. pAd.TRACK also has a concomitantly expressed green fluorescent protein (GFP) under the control of a separate cytomegalovirus promoter.15Pyo RT Sui J Dhume A Palomeque J Blaxall BC Diaz G Tunstead J Logothetis DE Hajjar RJ Schecter AD CXCR4 modulates contractility in adult cardiac myocytes.J Mol Cell Cardiol. 2006; 41: 834-844Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar Ad-β-gal/GFP and Ad-CXCR4 virus were propagated in HEK293 cells. Viral titer was determined by plaque assay. Stock titers were ≈1010 pfu/ml for each vector with a particle/pfu ratio of approximately ≈102. Contamination with wild-type adenovirus was ruled out by the absence of real time PCR-detectable E1 sequences. Male Sprague-Dawley (SD) (Charles River Laboratories, Wilmington, MA) rats were anesthetized with pentobarbital (60 mg/kg) i.p. and placed on a ventilator. The chest was accessed from the right side through the third intercostal space. The pericardium was opened and a 7-0 suture was placed at the apex of the left ventricle. A 22G catheter containing 200 μl of adenovirus was advanced from the apex of the left ventricle (LV) to the aortic root. The aorta and pulmonary artery were clamped distal to the site of the catheter and the solution was injected. The clamp was held for 10s while the heart pumped against a closed system (isovolumically). The solution containing the adenovirus circulated through the coronary arteries without further manipulation, as described previously.16Hajjar RJ Schmidt U Matsui T Guerrero JL Lee KH Gwathmey JK Dec GW Semigran MJ Rosenzweig A Modulation of ventricular function through gene transfer in vivo.Proc Natl Acad Sci USA. 1998; 95: 5251-5256Crossref PubMed Scopus (266) Google Scholar All procedures were approved by and performed in accordance with the Institutional Animal Care and Use Committee of the Mount Sinai School of Medicine. The 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). One week after adenoviruses (Ad-) administration, a left thoracotomy was performed, and the left anterior descending coronary artery (LAD) was ligated with 6-0 silk suture ≈4 mm from its origin with a slipknot. Successful ligation of the LAD was verified by visual inspection of the left ventricular apex. After 30 minutes, the LAD ligature was released and reperfusion was visually confirmed.17Tarzami ST Miao W Mani K Lopez L Factor SM Berman JW Kitsis RN Opposing effects mediated by the chemokine receptor CXCR2 on myocardial ischemia-reperfusion injury: recruitment of potentially damaging neutrophils and direct myocardial protection.Circulation. 2003; 108: 2387-2392Crossref PubMed Scopus (76) Google Scholar The chest was closed with continuous 2-0 silk suture. After 24 hours reperfusion, rats were re-anesthetized with 50 mg/kg of Ketamine and echocardiograms were performed using a VIVID 7 general electric device with a 14-MHZ probe as previously described.18del Monte F Lebeche D Guerrero JL Tsuji T Doye AA Gwathmey JK Hajjar RJ Abrogation of ventricular arrhythmias in a model of ischemia and reperfusion by targeting myocardial calcium cycling.Proc Natl Acad Sci USA. 2004; 101: 5622-5627Crossref PubMed Scopus (183) Google Scholar LV cavity size, wall motion, fractional shortening, and wall thickening were evaluated on M-mode echocardiography. They were measured from a short-axis view at the level of the papillary muscles. In addition to animals receiving Ad-CXCR4, controls included: 1) Blank control with no IR (Blank), 2) Saline-injected control group who had IR (Control), and 3) Ad–β-gal–injected animals who were also exposed to IR (β-gal). Histological analysis of infarct size was performed by 2,3,5-Triphenyltetrazolium chloride (TTC) staining. Briefly, after echocardiogranphic measurements, heparin (1000 u/ml) 0.4 ml was injected intravenously. The hearts were excised and mounted on a Langendorff apparatus and perfused for 3 minutes under constant pressure (100 cmH20) with K-H buffer at room temperature. The LAD was re-occluded. The hearts were perfused with 5% solution of Phthalo blue dye in normal saline over 3 minutes. The right ventricle of each heart was excised. The hearts were then frozen at −20°C for 20 minutes, followed by transverse sectioning into 2-mm slices. Sections were then incubated in 15 ml of 1.5% TTC for 20 minutes at 37°C. The sections were fixed in 10% formaldehyde. Twenty-four hours later, the slices were weighed and photographed. Color digital images of each transverse slice were obtained using a digital camera (Canon 640A). The blue regions represents nonischemic normal tissue, red regions represents risk area (ischemic but noninfarcted), and unstained pale white regions represent infarct tissue. The blue, red, and white areas were outlined on each color image and measured using Image Plus. On each side, the fraction of the LV area representing infarct-related tissue (average of 2 images) was multiplied by the weight of the section to determine the absolute weight of infarct-related tissue.17Tarzami ST Miao W Mani K Lopez L Factor SM Berman JW Kitsis RN Opposing effects mediated by the chemokine receptor CXCR2 on myocardial ischemia-reperfusion injury: recruitment of potentially damaging neutrophils and direct myocardial protection.Circulation. 2003; 108: 2387-2392Crossref PubMed Scopus (76) Google Scholar, 19Zhu BQ Simonis U Cecchini G Zhou HZ Li L Teerlink JR Karliner JS Comparison of pyrroloquinoline quinone and/or metoprolol on myocardial infarct size and mitochondrial damage in a rat model of ischemia/reperfusion injury.J Cardiovasc Pharmacol Ther. 2006; 11: 119-128Crossref PubMed Scopus (54) Google ScholarIschemicarea=infarct area+risk areaInfarct size/LV mass (%)=∑Infarct weight in eachsectionTotal LV weight×100%Risk area/LV mass (%)=∑red weight in eachsectionTotal LV weight×100%. After gene transfer and ischemia/reperfusion, the hearts were harvested, cut across the suture, and embedded in OCT. The tissues were cryosectioned (5- to 10-μm sections). Subsequently, β-gal/lacZ gene expression was determined as previously described.16Hajjar RJ Schmidt U Matsui T Guerrero JL Lee KH Gwathmey JK Dec GW Semigran MJ Rosenzweig A Modulation of ventricular function through gene transfer in vivo.Proc Natl Acad Sci USA. 1998; 95: 5251-5256Crossref PubMed Scopus (266) Google Scholar Frozen tissue blocks from the above hearts were cut into 5- to 10-μm sections, and parallel sections were prepared for hematoxylin and eosin staining and immunohistochemistry. Immunohistochemical analyses included staining for CXCL12, CXCR4, and tumor necrosis factor (TNF)-α, as well as for macrophages and pan leukocytes markers. The following antibodies were used: (1) rabbit anti-mouse/rat CXCL12 (eBiosciences; San Diego, CA); (2) goat anti-rat CXCR4 antibody (Torrey Pines Biolabs; East Orange, NJ); (3) goat anti-mouse/rat TNF- α, (R&D), (4) mouse anti-rat leukocyte antibody (BD Pharmingen; Franklin Lakes, NJ); and (5) mouse anti- rat macrophage marker (VP-M640, Vector Laboratories; Burlingame, CA) Sections from the hearts were fixed and blocked with the normal serum and incubated with primary antibody for 2 hours at room temperature. For visualization, slides were developed using either the Vectastain ABC kit with DAB as a substrate,20Chen J Kuhlencordt P Urano F Ichinose H Astern J Huang PL Effects of chronic treatment with L-arginine on atherosclerosis in apoE knockout and apoE/inducible NO synthase double-knockout mice.Arterioscler Thromb Vasc Biol. 2003; 23: 97-103Crossref PubMed Scopus (90) Google Scholar or with secondary fluorescent conjugated antibodies; with using a BP530/30 filter for FITC and/or a LP590 filter for Texas red. Images were taken with Axioplan2IE or Zeiss LSM510 META confocal microscope. Rats’ hearts were transected along the LAD ligature to separate ischemic tissue and remote myocardium. The ischemic and remote tissues were lysed in lyses buffer. Western blot analysis was performed using procedures established in our laboratory. For these studies the following primary polyclonal antibodies were used: (1) anti–CXCR4 (Torrey Pines Biolabs; East Orange, NJ); (2) anti-CXCL12 (eBiosciences; San Diego, CA); (3) anti-Caspase 3 (Cell Signaling; Danvers, MA); or (4) anti-Glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH, IMgenex; San diego, CA). A secondary antibody conjugated with alkaline phosphatase (Sigma; St. Louis, MO). The bands were visualized using the enhanced chemiluminescence method according to the manufacturer’s instructions (Pierce; Rockford, IL).21Tarzami ST Cheng R Miao W Kitsis RN Berman JW Chemokine expression in myocardial ischemia: mIP-2 dependent MCP-1 expression protects cardiomyocytes from cell death.J Mol Cell Cardiol. 2002; 34: 209-221Abstract Full Text PDF PubMed Scopus (85) Google Scholar, 22Chen J Tung CH Mahmood U Ntziachristos V Gyurko R Fishman MC Huang PL Weissleder R In vivo imaging of proteolytic activity in atherosclerosis.Circulation. 2002; 105: 2766-2771Crossref PubMed Scopus (324) Google Scholar For each of the hearts examined, the number of inflammatory cells was quantified in the infarcted zone (LV anterior wall), in a blinded manner. Briefly, frozen tissue blocks were cut into 5- to 10-μm sections, stained with hematoxylin and eosin, and examined by light microscopy (×10, ×40, and ×63 objective). For each section, the number of inflammatory cells per mm2 was quantified.17Tarzami ST Miao W Mani K Lopez L Factor SM Berman JW Kitsis RN Opposing effects mediated by the chemokine receptor CXCR2 on myocardial ischemia-reperfusion injury: recruitment of potentially damaging neutrophils and direct myocardial protection.Circulation. 2003; 108: 2387-2392Crossref PubMed Scopus (76) Google Scholar Three sections were assessed per heart. Ten fields were randomly chosen per section, four section per animal, a total of 40 fields per heart (n = 3 per treatment group) were counted using a defined rectangular field area (×40 objective). The number of monocytes and macrophages was also counted under high-power field (×40) using monocyte/macrophage specific antibody (VP-M640, Vector Laboratories; Burlingame, CA). For each of the hearts examined, myocardial cell apoptosis was quantified using a commercially available in situ cell death detection kit (Roche Applied Science, Indianapolis, IN) to find DNA strand breaks using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reagent according to the manufacturer’s protocol. Briefly, a double-staining technique was used (ie, TUNEL staining by using an In Situ Cell Death Detection Kit [Roche, USA] for apoptotic cell nuclei and 4′,6-diamidino-2-phenylindole [DAPI] staining for all cell nuclei). An additional staining was performed with a monoclonal antibody against α-sarcomeric actinin (Sigma; St. Louis, MO) for the identification of myocytes. Cardiomyocytes from at least four slides per block were selected and evaluated immunohistochemically to determine the number and percentage of cells exhibiting positive staining for apoptosis. For each slide, 10 fields were randomly chosen, and a total of 100 to 150 myocytes per field were counted using a defined rectangular field area (×20 and ×40 objective). Apoptotic index was determined (number of TUNEL-positive myocytes/total number of myocytes stained with anti–α-actinin × 100) from a total of 40 fields per heart. Assays were performed in a blinded manner. CXCL12 mRNA levels were determined by QRT-PCR using a QuantiTect SYBR Green RT-PCR Kit (Qiagen Ltd; Valencia CA) on a LightCycler (Roche Diagnostics Ltd., Lewes, UK). Total RNA was isolated from samples with Trizol reagent (Gibco BRL; Carlsbad, CA) according to the manufacturer’s instructions. Primers were designed to generate short amplification products. The sequences of the specific primers were: CXCL12 5′-TTGCCAGCACAAAGACACTCC-3′ and 5′-CTCCAAAGCAAACCGAATACAG-3′; TNF-α 5′-CTCTGCTTGGTGGTTTGCTA-3′ and 5′-CAAGGAGGAGAAGTTCCCAA-3′ 18S: 5′-GTTGGTTTTCGGAACTGAGGC-3′ and 5′- GTCGGCATCGTTTATGGTCG-3′. RT-PCR was performed in 20-μl reaction volumes using 10 pmol of primers. Reverse transcription was performed at 50°C for 20 minutes, and cDNA was amplified for 37 cycles: 94°C for 10 s, 57°C for 15 s, and 72°C for 5 seconds. The relative quantity of gene expression was calculated according to the manufacturer’s recommendations. 18S was used as an internal control to calculate the relative abundance of CXCL12 and TNF- α mRNAs. All data are expressed as SD (±SD). Differences between groups were determined with either a paired t test and/or two-tailed Student t test and/or a one-way analysis of variance followed by a Newman–Keuls post hoc test. Probability values of P < 0.05 were considered to be significant. MI in rats was induced by LAD ligation (30 minutes followed by 24 hours reperfusion). Histological analysis of infarct size was performed by TTC staining. Figure 1, A–D shows that the infarct area as a percentage of LV was significantly increased in CXCR4 group (13.5% ± 4.2%) in comparison with β-gal/GFP group (7.6% ± 3.4%) and saline group (control) (8.0% ± 2.0%). Although the risk area/LV was slightly increased in CXCR4 group, there was no significant statistical difference when it was compared with β-gal/GFP and saline groups. But the infarct/ischemia ratio was significantly increased in CXCR4 group (46% ± 7%) as compared with 36% ± 11% in β-gal/GFP group or 39% ± 8% in the saline group. The infarct/risk ratio was also significantly increased in CXCR4 group (89% ± 30%) as compared with β-gal/GFP control (60% ± 28%, P = 0.02). Cardiac function was also measured by echocardiography. Figure 2A illustrates that movement of the LV anterior wall was diminished in CXCR4 group. The left ventricle internal diastolic/systolic diameter and end diastolic/systolic volume were increased in CXCR4 IR group. Factional shortening was also decreased significantly in CXCR4 group as compared with β-gal/GFP and saline groups (control); (Figure 2B; see Supplemental Table S2, at http://ajp.amjpathol.org). Cardiac CXCR4 overexpression did not have any effect on myocardial function of noninfarcted rats (no IR); (Figure 2C; see Supplemental Table S253 at http://ajp.amjpathol.org).Figure 2CXCR4-overexpressed rats exhibit worsening cardiac function after ischemia-reperfusion in vivo. A: Echocardiography was performed on rats’ hearts post 30 minutes ischemia and 24 hours reperfusion. Short axis. Left, blank control without IR; Middle, β-gal gene transfer with IR; Right, CXCR4 gene transfer with IR. B: Percentage of fractional shortening (%) was calculated: *P < 0.05 compared with blank, **P < 0.05 compared with β-gal and control. Mean ± SD. C: Echocardiography was performed on rats’ hearts before Ad-CXCR4 gene transfer and one week after injection in an absence of any ischemic injury.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Gene transfer was assessed by quantifying expressions of β-gal and CXCR4. β-gal and CXCR4 proteins expression were overexpressed in ischemic myocardium after gene transfer (Figure 3, A–D). These results were confirmed by Western blotting (Figure 3E). The CXCR4-overexpressed group exhibited increased CXCR4 protein expression in ischemic myocardium as compared with β-gal and control (saline injected) groups. Next, we assessed whether changes in CXCR4 expression could effect concomitant expression of its ligand, CXCL12. Interestingly, we found a marked increased expression of CXCL12 in hearts injected with Ad-CXCR4 and exposed to IR as compared with β-gal and control groups as were assessed by immunofluoresence staining (Figure 4A), Western blot (Figure 4B), and quantitative real-time PCR (Figure 4C).Figure 4CXCL12 is upregulated in ischemic myocardium of CXCR4-overexpressed rat. A: CXCL12 protein expression was assessed in hearts injected with either CXCR4 or β-gal and/or control (saline-injected; with IR) by immunofluorescence staining. Frozen sections were fixed and stained with anti-CXCL12 (green) and anti–α actinin (red). Primary Abs visualized with FITC or Texas Red conjugate. Nuclei were stained with DAPI. Images are taken with confocal microscopy. B: Protein lysates were prepared from rats’ hearts one week after CXCR4 gene transfer followed by 30 minutes LAD ligation and 24 hours reperfusion. The ischemic and remote tissues were lysed and analyzed by Western blot. Representative gel of three independent experiments is shown. C: CXCL12 mRNA levels were determined by quantitative real-time PCR (QRT-PCR) using a QuantiTect SYBR Green RT-PCR Kit and using specific primers for CXCL12 and 18S. Primers were designed to generate short amplification products. Densitometric analysis of data from three different experiments is shown. *P < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because inflammatory cells are the important modulators of ischemic injury, we assessed the presence of inflammatory cells in rats overexpressing CXCR4 in the heart. Our data demonstrate that hearts injected with CXCR4 and exposed to IR have significantly more inflammatory cellular infiltrate in the ischemic region (Figure 5A). The number of inflammatory cells was quantified per mm2(Figure 5B). Importantly, there were no significant differences in monocytes/macrophages migrating into the ischemic area of CXCR4, β-gal, and saline injected groups (Figure 5, C and D). The number of monocytes and macrophages was counted in high power fields (×40) using cell type–specific mouse anti-rat macrophage marker (VP-M640) antibody. Importantly, CXCR4 overexpression did not seem to mediate inflammatory reactions in the absence of IR (see Supplemental Figure S1, at http://ajp.amjpathol.org). Cardiomyocyte necrosis and apoptosis play important roles in the pathology of ischemic/reperfusion injury. To determine myocardial apoptosis, in situ detection of apoptotic myocytes was performed by using TUNEL assay. Positive apoptotic nuclei were stained green in infarcted myocardial tissue, and healthy nuclei were stained blue (DAPI staining). In Figure 6A we show there are significantly higher number of TUNEL-positive cells in CXCR4 group in comparison with controls (β-gal–injected and/or saline-injected). Representative pictures were taken at ×40 objective. The number of TUNEL-positive
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