Coexpression of VEGF and angiopoietin-1 promotes angiogenesis and cardiomyocyte proliferation reduces apoptosis in porcine myocardial infarction (MI) heart Zhengxian Taoa, Bo Chena, Xiao Tana, Yingming Zhaoa, Liansheng Wanga, Tiebing Zhua, Kejiang Caoa, Zhijian Yanga,1, Yuet Wai Kanb,1, and Hua Suc a Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, People’s Republic of China; and bDepartment of Medicine and cCenter for Cerebrovascular Research, Department of Anesthesia and Perioperative, University of California, San Francisco, CA 94143-0793 VEGF and angiopoietin-1 (Ang1) are two major angiogenic factors being investigated for the treatment of myocardial infarction (MI). Targeting VEGF and Ang1 expression in the ischemic myocardium can increase their local therapeutic effects and reduce possible adverse effects. Adeno-associated viral vectors (AAVs) expressing cardiac-specific and hypoxia-inducible VEGF [AAV-myosin light chain-2v (MLC)VEGF] and Ang1 (AAV-MLCAng1) were coinjected (VEGF/Ang1 group) into six different sites of the porcine myocardium at the peri-infarct zone immediately after ligating the left descending coronary artery. An identical dose of AAV-Cytomegalovirus (CMV)LacZ or saline was injected into control animals. AAV genomes were detected in the liver in addition to the heart. RT-PCR, Western blotting, and ELISA analyses showed that VEGF and Ang1 were predominantly expressed in the myocardium in the infarct core and border of the infarct heart. Gated single-photon emission computed tomography analyses showed that the VEGF/Ang1 group had better cardiac function and myocardial perfusion at 8 wk than at 2 wk after vector injection. Compared with the saline and LacZ controls, the VEGF/Ang1 group expressed higher phosphorylated Akt and BclxL, less Caspase-3 and Bad, and had higher vascular density, more proliferating cardiomyocytes, and less apoptotic cells in the infarct and peri-infarct zones. Thus, cardiac-specific and hypoxia-induced coexpression of VEGF and Ang1 improves the perfusion and function of porcine MI heart through the induction of angiogenesis and cardiomyocyte proliferation, activation of prosurvival pathways, and reduction of cell apoptosis. coronary disease | gene therapy T ransfer of angiogenic genes to the ischemic myocardium is a promising approach under development for the treatment of myocardial infarction (MI). Animal and clinical data indicated that angiogenic factors such as fibroblast growth factor (1–3), VEGF (4–7), angiopoietins (8), and hepatocyte growth factor (9, 10) can induce angiogenesis in ischemic myocardium and improve cardiac function of MI heart. VEGF and angiopoietin-1 (Ang1) can also activate a prosurvival signaling pathway and attract stem cell homing (11). Obstacles that prevent clinical application of angiogenic gene therapy include short duration of action of the factors, inadequate transduction or expression of the genes, immune reactions against the delivery vector, and unwanted angiogenesis caused by the uncontrolled expression of angiogenic factors. To overcome these obstacles, we used (i) adeno-associated viral vector (AAV) serotype 1 to reduce the immune reaction and increase the duration of transgene expression and (ii) cardiac myosin light chain-2v (MLC) promoter and the hypoxia response element (HRE) to drive VEGF and Ang1 expression in a cardiacspecific and hypoxia-inducible manner (12–15). The notions that mammalian cardiomyocytes irreversibly withdraw from the cell cycle soon after birth and that the mammalian heart is a terminally differentiated organ with no intrinsic capacity to regenerate after myocardial injury have been seriously challenged in recent years (16). Nevertheless, the innate re- www.pnas.org/cgi/doi/10.1073/pnas.1018925108 generative ability of the adult heart is limited and not enough to restore cardiac function adequately after a MI. Several laboratories have shown that some angiogenic factors can induce myocyte proliferation (9, 17–21). Therefore, in addition to analyzing the angiogenic effect of VEGF and Ang1, we have also studied their roles in promoting cell proliferation. In this study, we used a porcine acute MI model to study the benefits of AAV-mediated, cardiac-specific, and hypoxia-inducible coexpression of VEGF and Ang1 and investigated the underlying mechanisms. Similar to what we have shown in the mouse acute MI model (14), the AAV vectors that we have developed could express VEGF and Ang1 in a cardiac-specific and hypoxia-inducible manner in the pig MI model. Coexpression VEGF and Ang1 in porcine MI heart induced angiogenesis, stimulated cardiomyocyte proliferation, and reduced apoptosis. Together, they resulted in the improvement of cardiac functions. Results Cardiac-Specific and Hypoxia-Inducible VEGF and Ang1 Expression. AAV-MLCVEGF and AAV-MLCAng1 were coinjected into six sites of the myocardium around the ischemic area immediately after ligation of the left anterior descending coronary artery (LAD). Eight weeks after the injection, the distribution of AAV1MLCVEGF genome was analyzed by PCR using a pair of human VEGF specific primers with the genomic DNA isolated from the infarct, peri-infarct, and normal tissues of the myocardium, lung, liver, and kidney of all animals (SI Materials and Methods). The vector genome was detected in the DNA isolated from all three myocardial zones (infarct, peri-infarct, and normal) and liver of VEGF/Ang1-injected pigs (Fig. 1A). No vector was detected in the DNA isolated from the lung or kidney. Thus, the AAVMLCVEGF and AAV-MLCAng1 were successfully delivered into the myocardium, although some vectors leaked into liver even when they were injected directly into myocardium. RT-PCR (Fig. 1B) and Western blot (Fig. 1C) showed that human VEGF and Ang1 expression were detected in all three zones of myocardium, with a lower level of expression in the normal zone than in the infarct and peri-infarct zones (P < 0.01). No human VEGF and Ang1 expressions were detected in the liver; even the vector genomes were presented (Fig. 1 B and C). These results indicated that the gene expression mediated by Author contributions: Y.W.K., Z.Y., and H.S. designed research; Z.T., B.C., X.T., Y.Z., L.W., T.Z., and K.C. performed research; Z.T., B.C., X.T., Y.Z., and Z.Y. analyzed data; and Z.T., Z.Y., and H.S. wrote the paper. The authors declare no conflict of interest. Freely available online through the PNAS open access option. 1 To whom correspondence may be addressed. E-mail: [email protected] or yw. [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1018925108/-/DCSupplemental. PNAS Early Edition | 1 of 6 MEDICAL SCIENCES Contributed by Yuet Wai Kan, December 20, 2010 (sent for review July 31, 2010) Fig. 1. Vector distribution and VEGF and Ang1 expressions. (A) Distribution of AAV genomes after intramyocardial injection. Representative pictures of RT-PCR (B) and Western blots (C). Bar graphs are quantitative data of RT-PCR (B) and Western blot (C), respectively. *P < 0.01 vs. normal myocardium. #P < 0.05 vs. normal myocardium. I, infarct zone; P, peri-infarct zone; N, normal zone; K, kidney; Li, liver; L, lung; LacZ, infarct myocardium of LacZ group; saline, infarct myocardium of saline group. AAV-MLCVEGF and AAV-MLCAng1 was cardiac-specific and hypoxia-inducible in the porcine acute MI model. Improvement of Myocardial Perfusion and Cardiac Function. Gated single-photon emission computed tomography (SPECT) was used to compare myocardial perfusion and cardiac functions at 2 and 8 wk after treatment. The VEGF/Ang1 group had better left ventricular ejection fraction (LVEF) at 8 wk than at 2 wk (P < 0.01) (Fig. 2B). Myocardial perfusion score of the VEGF/Ang1 group was significantly improved at 8 wk compared with 2 wk (P < 0.01) (Fig. 2 A and C). Although the left ventricular end diastolic volume (LVEDV) of the VEGF/Ang1 group at 2 and 8 wk was not different (P > 0.05), the left ventricular end systolic volume (LVESV) was significantly reduced at 8 wk compared with 2 wk (P < 0.05) (Fig. 2D). None of these measurements showed any significant changes in the saline- or LacZ-injected controls (P > 0.05) (Fig. 2 B, C, and E). Thus, coexpression of VEGF and Ang1 improves myocardial perfusion and cardiac functions of porcine acute MI heart. No Increase of VEGF and Ang1 Levels in the Serum. To test whether intramyocardial injection of AAV1-MLCVEGF and AAV1MLCAng1 would increase serum levels of VEGF and Ang1, we analyzed human VEGF and Ang1 levels in the sera of treated pigs at 0, 1, 3, and 7 d and 2, 3, 4, and 8 wk after vector injection by ELISA. No significant increases in human VEGF and Ang1 levels in VEGF/Ang1 group were detected compared with the LacZ and saline groups (P > 0.05) (Table 1). Thus, intramyocardial injection of AAV1-MLCVEGF and AAV1-MLCAng1 did not increase the serum VEGF and Ang1 levels. Reduction of Cardiomyocyte Apoptosis in Infarct and Peri-Infarct Myocardium. The hearts of the VEGF/Ang1 group showed sig- nificant decrease in TUNEL-positive nuclei in the myocardium of Fig. 2. Myocardial perfusion and cardiac function. (A) Representative images of SPECT of saline- (Top), LacZ- (Middle), and VEGF/Ang1-treated (Bottom) porcine acute MI hearts. Thirty-two images were acquired, 40 s each, at every 5.6° throughout a 180° arc, beginning 45° right anterior oblique and ending 45° left posterior oblique. There was better myocardial perfusion in VEGF/Ang1-treated heart at 8 wk compared with 2 wk (red arrows), but similar myocardial perfusion in saline- and LacZ-injected heart between 2 and 8 weeks (white arrows). (B) Bar graph showing that LVEF improved at 8 vs. 2 wk after VEGF/Ang1 injection (*P < 0.01). (C) Myocardial perfusion score improved at 8 vs. 2 wk after VEGF/Ang1 injection (*P < 0.01). (D) LVESV improved at 8 vs. 2 wk after VEGF/Ang1 injection (*P < 0.05). (E) LVEDV was not changed at 8 vs. 2 wk after VEGF/Ang1 injection (P > 0.05). infarct and peri-infarct zones compared with the LacZ and salineinjected controls (P < 0.01) (Fig. 3 A and B). The percentage of apoptotic cardiomyocyte at the infarct and peri-infarct zones of VEGF/Ang1 group (33 ± 5.7 and 13 ± 3.3) was significantly lower compared with the saline (53 ± 7.9 and 27 ± 6.7) and LacZ (56 ± 10.1 and 25 ± 6.3) groups (P < 0.01). The percentage of apoptotic cardiomyocyte in the normal myocardial zone was similar among the three groups (P > 0.05) (Fig. 3 C and D). Consistent with the cytological analysis, caspase-3 expression in the infarct and periinfarct zones of VEGF/Ang1 group was the lowest among the three groups (Fig. 3E). To analyze the mechanism of decreased cellular apoptosis mediated by the injections, the apoptotic pathway was analyzed by Western blots. There were more prosurvival proteins, such as phosphorylated Akt and Bcl-xL, and less proapoptotic proteins, such as Bad, in VEGF/Ang1-injected hearts compared with those injected with LacZ and saline. Bcl-2 expression was similar among all three zones of the three groups (Fig. 3E). The results suggest that the overexpression of VEGF and Ang1 activated the prosurvival pathways, which resulted in reduction of cardiomyocyte death. Increase of Vascular Density in Myocardial Infarct and Peri-Infarct Zones. To analyze the angiogenesis mediated by coexpression of VEGF/Ang1, small blood vessels on lectin-stained sections were counted by two investigators who were blinded to the experimental groups. The VEGF/Ang1 group had significantly more capillaries in the myocardial infarct (48 ± 8.3/mm2) and periinfarct (92 ± 15.5/mm2) zones than those injected with saline Table 1. VEGF and Ang1 levels in the sera (μg/μL) VEGF Ang1 0d 1d 3d 7d 2 wk 3 wk 4 wk 8 wk 9 ± 2.1 22 ± 5.0 8 ± 2.8 24 ± 7.4 8 ± 2.2 23 ± 7.0 8 ± 1.8 22 ± 8.6 9 ± 3.1 23 ± 7.9 8 ± 2.5 23 ± 10.3 10 ± 4.2 26 ± 9.7 9 ± 2.6 24 ± 10.6 No significant increase of VEGF and Ang1 levels in the sera of pigs was detected 0, 1, 3, and 7 d and 2, 3, 4, and 8 wk after the vector injection (P > 0.05). 2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1018925108 Tao et al. (22 ± 8.7 and 60 ± 13.9/mm2) and LacZ (23 ± 8.6 and 59 ± 16/mm2; P < 0.01) (Fig. 4 A and B). Similarly, the density of α-smooth muscle actin (α-SMA)-positive vessels in the myocardial infarct (15 ± 4.2/mm3) and peri-infarct (28 ± 10.1/mm2) zones of VEGF/Ang1 group was significantly higher than in saline (8 ± 4.4 and 18 ± 5.4/mm2) and LacZ (9 ± 4.6 and 19 ± 6.7/mm2) groups (P < 0.01) (Fig. 4 C and D). The densities of capillaries and α-SMA–positive vessels in the infarct and peri-infarct zones were not significantly different between the LacZ and saline groups (P > 0.05) (Fig. 4 B and D). To identify newly formed blood vessels, we used an antibody specific to Ki67 to examine proliferating endothelial cells. We detected significantly more Ki67-positive endothelial cells in VEGF/Ang1-injected hearts (12 ± 4.5/mm2) than in saline- (3 ± 2.3/mm2) and LacZ-injected (3 ± 2.3/mm2) controls (P < 0.01) (Fig. 4 E and F). Thus, overexpression of VEGF and Ang1 induced active angiogenesis in the ischemic myocardium. Increased Cardiomyocyte Proliferation. To determine whether overexpression of VEGF and Ang1 increases cardiomyocyte proliferation, we did double labeling using antibodies against Ki67 and troponin T. As shown in Fig. 5 A and B, there were more Ki67-positive cardiomyocytes in the VEGF/Ang1-injected hearts (14 ± 2.2%) than that in saline- (4 ± 1.1%) and LacZ- (3 ± 0.8%) injected controls (P < 0.01). Flow cytometry data were consistent with the immunostaining data. There were more S phase cardiomyocytes (DipS) in the VEGF/Ang1 group (43 ± 9.2%) compared with saline (16 ± 6.0%) and LacZ (16 ± 4.2%) groups (P < 0.01) (Fig. 5 C and D). The expression of cell cycle proteins was also analyzed (Fig. 5E). p27, a cell cycle inhibitor, was 2.6- and 3.5-fold lower in the infarct and peri-infarct zones of VEGF/Ang1 group compared with the saline group, and 2.3- and 4.1-fold lower compared with the LacZ group (Fig. 5F). The expression of cyclin D2 in the infarct and peri-infarct zones of VEGF/Ang1 group was increased 2.4and 2.6-fold compared with the saline group and 2.2- and 2.7fold compared with the LacZ group (Fig. 5F). Similarly, cdk4 levels in the infarct and peri-infarct zones of the VEGF/Ang1 group were increased 3.6- and 1.9-fold compared with the saline group and 4.0- and 2.0-fold compared with the LacZ group (Fig. 5F). The infarct and peri-infarct zones of VEGF/Ang1 group Tao et al. expressed 2.3- and 2.2-fold higher cyclin A protein than in the saline group and 2.4- and 1.9-fold higher cyclin A protein than in the LacZ group (Fig. 5F). The expression of cdk2 in the infarct and peri-infarct zones of the VEGF/Ang1 group increased 3.7and 3.4-fold compared with saline group and 3.8- and 3.2-fold Fig. 4. Vascular density. (A) Representative pictures of lectin-stained sections. (B) Bar graphs showing the quantification of microvessels. (C) Representative pictures showed α-SMA–positive arterioles. (D) Bar graphs showing the quantification of α-SMA–positive vessels (50–100 μm diameter). (E) Immunofluorescence of the Ki67-positive cells in the border zone of the infarct. (F) Bar graph showing the quantification of Ki67-positive vessels. *P < 0.01 vs. LacZ and saline groups. PNAS Early Edition | 3 of 6 MEDICAL SCIENCES Fig. 3. Cardiomyocyte apoptosis. (A) Representative images of TUNEL-stained sections. TUNEL-positive nuclei are green, and DAPI is blue. (B) Bar graph showing the quantification of the apoptotic cells. *P < 0.05 vs. LacZ and saline groups. (C) Representative images. (D) Bar graph showing the percentage of apoptotic cardiomyocytes by flow cytometry. *P < 0.05 vs. LacZ and saline groups. (E) Representative Western blots showing expressions of prosurvival and proapoptotic genes. I, infarct zone; P, peri-infarct zone; N, normal zone. Fig. 5. Cardiomyocyte proliferation. (A) Representative pictures of sections stained with troponin T (green), Ki67 (red), and DAPI (blue). (B) Bar graph showing the quantification of Ki67-positive myocytes. (C) Flow cytometry data. The areas denoted by the diagonal lines are cardiomyocytes in S phase (DipS), and they show significantly more DipS cardiomyocytes in the VEGF/ Ang1 group than in the saline and LacZ groups. (D) Bar graphs showing the quantification of cardiomyocytes in S phase. (E) Representative images of Western blots showing the expression of genes related to cell cyclin. (F) Bar graphs showing the quantification of cyclin protein. *P < 0.01 vs. LacZ and saline groups. N, normal zone; P, peri-infarct zone; I, infarct zone. compared with LacZ group (Fig. 5F). Taken together, these results suggested that VEGF/Ang1 could drive cardiomyocytes into S phase and increase cardiomyocyte proliferation through the Akt/cyclin D2 and cyclin A pathways. Discussion About 6–12% of symptomatic patients with extensive coronary artery disease are not amenable to conventional treatment, such as percutaneous transluminal coronary angioplasty (PTCA) or coronary artery bypass grafting (CABG) (22). Although available treatments for severe coronary insufficiency are often helpful, restenosis of coronary vessels occurs in 30–35% of the patients (23). Therefore, the development of new treatment strategies is still necessary. It has been reported that unregulated and continuous expression of VEGF can lead to angioma formation at the site of injection (24, 25). A high level of circulating VEGF in acute myocardial infarction can induce acute cor pulmonale, resulting in increased mortality (26). Thus, control of angiogenic factors expression both in levels and locations is important to enhance their local therapeutic efficiency and decrease their possible adverse effects. Many vectors can be used for in vivo gene deliveries. The AAV vector has many advantages compared with other vectors, such as it has the potential for mediating long-term transgene expression and inducing less immunogenicity (27). Hoshijima et al. (28) have used AAV2 to express a pseudophosphorylated mutant of human phospholamban (PLN) in BIO14.6 cardiomyopathic hamsters. The AAV2/PLN treatment enhanced myocardial sarcoplasmic reticulum (SR) Ca2+ uptake and suppressed progressive impairment of left ventricular systolic function and contractility for 28– 30 wk, thereby protecting cardiac myocytes from cytopathic plasmamembrane disruption (28). As to acute myocardial infarction, the early gene expression is crucial for the treatment. We compared gene expression and therapeutic effect of AAV1-5 4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1018925108 vectors. Our result shows that the onset of gene expression delivered by AAV serotypes 1 has minimal delay (15). Because the porcine heart resembles the human heart in size and coronary structure, the pig was selected as the animal model in this study. Our results showed that, although the AAV genomes were detected in liver after intramyocardial injection of AAVMLCVEGF and AAV-MLCAng1, no gene expression was found. VEGF and Ang1 expressions were only indentified in the myocardial infarct, peri-infarct zone along with a low level in normal myocardium. Also, these angiogenic factors were not increased in the systemic circulation. These results confirmed that the 250-bp MLC promoter and nine copies of HRE in our AAV vector can control VEGF and Ang1 gene expression specifically in the ischemic myocardium of pigs. The lower level of VEGF and Ang1 expressions detected in the normal myocardium could partially be because of hypoxia related to anesthesia or thoracotomy surgery. We have shown in previous publications that overexpression of VEGF and Ang1 improves cardiac function in mouse and rat MI heart models through their angiogenic and antiapoptotic functions (4, 15). Here, we have showed that VEGF/Ang1 induced neovascular formation and reduced myocardial apoptosis in infarct and peri-infarct zones of porcine acute MI heart. Hemangioma was not detected in the VEGF/Ang1-treated heart. Myocardial perfusion score and LVEF of the VEGF/Ang1-injected pigs were significantly improved. The LVESV was lower at 8 wk compared with 2 wk. No improvements were observed in the saline- and LacZ-injected controls. In the American College of Cardiology (ACC)/American Heart Association (AHA)/American Society for Nuclear Cardiology (ASNC) Guidelines for the Clinical Use of Cardiac Radionuclide Imaging, gated SPECT is an important noninvasive method used for assessment of myocardial perfusion, left ventricular function, and myocardial viability. Increase of LVEF and reduction of LVESV indicate improvement of cardiac function (29). This study showed that coexpression of VEGF and Ang1 resulted in improvement of myocardial perfusion and cardiac function in pigs with acute myocardial infarction. We have previously reported that VEGF/Ang1 delivered by adenovirus vector reduces cardiomyocyte apoptosis in vitro through the Akt/Bcl-2 pathway (4). In this study, Akt was also activated in VEGF- and Ang1-transduced porcine MI heart. Bad expression was lower in the VEGF/Ang1 group than in the LacZ and saline groups, but Bcl-xL expression was significantly upregulated. Therefore, this study in the porcine heart confirmed our previous finding that VEGF and Ang1 provide cardiac protection through activation of the Akt/Bcl-2 pathway. In addition to inducing angiogenesis and reducing apoptosis, we show in this study that coexpression of VEGF and Ang1 in MI heart also increases cell proliferation through activation of Akt kinase, up-regulation of cyclin D2/cdk4 and cyclin A/cdk2 expression, driving of cardiomyocyte into S phase, and promoting proliferation of cardiomyocytes. It has been shown that Akt kinase expands the population of cycling myocytes in the postnatal heart and the number of progenitor cells expressing markers of myocyte lineage in the adult heart (30). It is also possible that VEGF/Ang1 treatment in vivo might increase proliferation of resident stem cells in the heart or recruit peripheral stem cells to the infarcted myocardium. Different vector delivery routes have been tested in animal MI models and clinical trials. It is not yet known which one is the safest and most effective. The effect of i.v. administration of angiogenic growth factors is minimal and is likely because of first-pass uptake by the lungs, resulting in considerably lower concentration in the myocardium compared with that with intracoronary administration (31). Intracoronary injection requires skillful technique of catheterization. Epicardial myocardial injection has been attempted successfully with an adenoviral vector containing the VEGF121 gene that was injected directly into ischemic myocardium of pig hearts during thoracotomy. Both myocardial perfusion and function in this model were significantly improved (32). Thus, this apTao et al. Materials and Methods AAV Vector Construction and Production. AAV-MLCVEGF and AAV-CMVLacZ constructed for previous experiments were used in this study (11–14). AAV-MLCAng1 was constructed by replacing human VEGF gene in the AAV-MLCVEGF vector with human Ang1 cDNA. AAV vectors were packaged in AAV serotype one capsid and prepared by the three plasmid cotransfection system (33). More details are described in SI Materials and Methods. Myocardial Infarction and AAV Vector Injection. Twenty-four Yorkshire pigs (male, 3–4 mo old, 35 ± 2.7 kg body weight) were housed in the animal care facility of Nanjing Medical University. The animals were randomly divided into three groups of eight: AAV1-MLCVEGF and AAV1-MLCAng1 coinjected group (VEGF/Ang1 group), AAV1-CMVLacZ-injected group (LacZ), and Hepes saline-injected group (saline). The detailed procedures for MI creation and AAV vector injection were described in SI Materials and Methods. Myocardial Perfusion and Cardiac Function. Gated SPECT was performed in the pigs with a commercially available system (ECAM+; Siemens). Left ventricular end diastolic volume (LVEDV), left ventricular end systolic volume (LVESV), and left ventricular ejection fraction (LVEF) were determined by QGS software. The pigs were habituated to the experimental environment for 2 h before the SPECT analysis. The imaging was assessed at a dose of 0.3 m Ci/kg 99mTc sestamibi (99mTc-MIBI) at 2 and 8 wk after LAD ligation and vector injection. Images were acquired 60–90 min after 99mTc-MIBI injection using a multihead camera with high-resolution collimators. The camera energy window (20%) was set on the 140 ke V photopeak of 99mTc-MIBI. Particular care was taken to avoid movement of the pig or overlap from extracardiac activity. Thirtytwo images (64 × 64 matrix) were acquired for 40 s each with 180° rotation. Tao et al. The tomograms were reconstructed in the vertical and horizontal long- and short-axis planes. The myocardial perfusion score was calculated as follows. The left ventricular cavity was divided into 17 segments for assessment of the myocardium (34). Seven segments are assigned to the LAD distribution area. The five-point scoring used to grade each segment was as follows: 0, absent perfusion; 1, severe hypoperfusion; 2, moderate hypoperfusion; 3, mild hypoperfusion; 4, normal perfusion. The normal total score of LAD distribution areas is 28 (35). ELISA. Blood samples were collected at 0, 1, 3, and 7 d and 2, 3, 4, and 8 wk after the LAD ligation and vector injection. The samples were centrifuged for 10 min at 1,000 × g, and the serum was collected. The levels of human VEGF and Ang1 in serum were quantified using an ELISA kit for human VEGF and Ang1 (Quantikine; R&D Systems). The samples were measured in duplicates. PCR and RT-PCR Analysis. The TRIzol RNA isolation system (GIBCO/BRL) was used to isolate RNA and genomic DNA from the heart, liver, lung, and kidney. We performed PCR and RT-PCR analysis as described in SI Materials and Methods. Immunoprecipitation and Western Blotting Analysis. The tissues were gently homogenized in lysis buffer (50 mM Tris, pH 7.5, 10 mM sodium phosphate, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 10 mM NaF, 5 mM iodoacetate, 1 mM benzamidine, 5 μg/mL leupeptin, 5 μg/mL aprotinin) and incubated on ice for 30 min. The lysates were centrifuged at 10,000 × g for 20 min to remove insoluble material, and the total protein concentration was determined by modified Bradford assay. All samples were stored at −80 °C before electrophoresis. The detailed procedures for immunoprecipitation and Western blotting analysis are given in SI Materials and Methods. Histological and Immunohistochemical Analysis. Hearts were fixed in 10% formalin, embedded in paraffin, and sectioned. Serial sections were made from the apex of the heart to the site of the ligation. Sections were stained with specific antibodies against α-SMA, Ki67, and troponin T (Thermo Scientific) antibodies. Nuclei were stained with DAPI. Lectin-FITC (Santa Cruz) was used to visualize the blood vessels. The immunohistochemical staining was carried out according to the manufacturer’s instructions, and signals were visualized by incubating the sections with Alexa Fluor-488– or -555– labeled secondary antibody (Molecular Probes). For quantification of vessel densities in the myocardium, four equivalent sections from each group were randomly selected, and six visual fields from each section were observed. As a surrogate of vessel counting, vessel density was determined by lectin optical density measurements using the QWIN100 immunohistochemical imaging analyzer system (Leica). Cardiomyocyte Apoptosis. Cardiomyocyte apoptosis was measured by double immunofluorescence staining of TUNEL using the In Situ Cell Death Detection Kit, Fluorescein (Roche). SI Materials and Methods contains more details. Flow Cytometry Analysis. Cell suspensions were prepared using the Medimachine System (Becton-Dickinson). Cardiomyocyte cells were collected by flow cytometry with the use of an antibody to α-sarcomeric actinin. The cells (1 × 106 per sample) were fixed in 70% ethanol, double stained for FITC– Annexin V binding and propidium iodide, and analyzed by flow cytometry using a FACS-SCAN apparatus (Becton-Dickinson). Cell cycle distribution was analyzed for DNA content using propidium iodide in flow cytometry. Statistical Analysis. Statistical analysis was performed with SPSS 10.0 software. Student t tests and one-way ANOVA testing were used to compare the differences among groups, with statistical significance considered if P < 0.05. The data are presented as mean ± SD. ACKNOWLEDGMENTS. The authors thank Drs. Li Yuan and Ying-Bin Ge for their technical assistance, Dr. Xiao Xiao for providing the plasmid containing AAV rep and AAV serotype 1 cap genes, Dr. Feng Jianlin and Dr. Yang Guoping for their technical assistance, and Grant Chen for editing the manuscript. This work was supported by a project grant of the Key Faculty of Medical Renaissance Program of Jiangsu province, National Nature Science Foundation of China (Grant 30971254), National Basic Research Program (973 program; Grant 2008CB517303), National Institutes of Health Grant HL67969 (to Y.W.K.), and American Heart Association Scientist Development Grant 0535018N (to H.S.). PNAS Early Edition | 5 of 6 MEDICAL SCIENCES proach was selected in this study. One of the limitations of the procedure is that it needs an open surgery and may not applicable to most of the patients. Recently, we developed a set of catheterbased percutaneous endocardial intramyocardial injection system guided by EnSite NavX for delivery of genes or cells to the myocardium. The vectors that we have tested in this paper can be delivered to patients using our newly developed method. The limitations of this study include (i) a lack of groups that were injected with AAV-MLCVEGF or AAV-MLCAng1 individually because of the cost of the pig experiment. Thus, the effect of coexpression of VEGF and Ang1 was not compared with individual expression. However, our previously published paper on the mouse MI model showed that AAV-mediated coexpression of Ang1 and VEGF results in a decreased vascular leakage compared with VEGF expressed alone. That report also showed a smaller infarct size and better cardiac function when AAV-MLCVEGF and AAV-MLCAng1 were coinjected (11). (ii) Because Akt inhibitor was not used in this study, there is no direct evidence that VEGF- and Ang1-induced increase of cell cyclin gene expression and cell proliferation is dependent on Akt signaling pathways. (iii) The functions of VEGF and Ang1 on induction of endogenous stem cell homing, proliferation, and differentiation and endothelial progenitor incorporation into the neovasculature were not studied. (iv) The effects of VEGF and the other angiogenic factors seem to be multifactorial; it is not clear how much of the therapeutic benefit that we identified in this study can be attributed to the increase of perfusion. (v) The therapy was administered immediately after coronary ligation. This study was targeted to acute MI to reduce mortality by avoiding a second operation. Future studies will be performed to address these issues and study the therapeutic effects of this approach in chronic MI. In summary, we have shown that intramyocardial coinjection of AAV1- MLCVEGF and AAV1-MLCAng1 can result in cardiacspecific and hypoxia-inducible gene expression in porcine acute MI heart, induce angiogenesis and cardiomyocyte proliferation, reduce cardiomyocyte apoptosis, and improve myocardial perfusion and cardiac functions. Thus, the usefulness of AAV1MLCVEGF and AAV1-MLCAng1 in protecting patients from cardiac injury warrants further investigation. 1. 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