Ischemic Mouse Heart Research Articles

Interferon regulatory factor 9 is an essential mediator of heart dysfunction and cell death following myocardial ischemia/reperfusion injury.

This study aimed to investigate whether interferon regulatory factor 9 (IRF9) is involved in the pathogenesis of myocardial ischemia-reperfusion (I/R) injury and to explore the underlying molecular mechanisms of this process. Cell death plays a major role in myocardial I/R injury. We recently determined the importance of IRF9 in coordinating molecular events in response to hypertrophic stress in cardiomyocytes. However, the roles of IRF9 in lethal myocardial injury remain to be elucidated. The involvement of IRF9 was assessed via functional assays in a mouse myocardial I/R injury model by genetic knockout and cardiomyocyte-specific transgenic overexpression of IRF9, and its effects on cardiomyocyte apoptosis and inflammation were further studied in vivo and in vitro. IRF9 was upregulated in human ischemic heart tissue and mouse hearts after I/R injury. Ablation of IRF9 protected the heart against I/R-induced cardiomyocyte death, development of inflammation, and loss of heart function. In contrast, cardiomyocyte-specific transgenic overexpression of IRF9 aggravated myocardial reperfusion injury and inflammation. IRF9 negatively regulated the Sirt1-p53 axis under I/R conditions in vivo and in vitro. Downregulation of Sirt1 expression and its downstream apoptosis-related signaling cascade, which results from I/R, was ameliorated by loss of IRF9 and exacerbated by overexpression of IRF9. Cardiomyocyte-specific deletion of Sirt1 abolished the protective effect of IRF9 knockout against I/R injury, which further indicated that IRF9 mediated myocardial reperfusion injury by modulating the Sirt1-p53 axis. Thus, IRF9 may be a novel therapeutic target for the prevention of I/R injury resulting from revascularization therapy after acute myocardial infarction (MI).

Cardiac fibroblast glycogen synthase kinase-3β regulates ventricular remodeling and dysfunction in ischemic heart.

Myocardial infarction-induced remodeling includes chamber dilatation, contractile dysfunction, and fibrosis. Of these, fibrosis is the least understood. After myocardial infarction, activated cardiac fibroblasts deposit extracellular matrix. Current therapies to prevent fibrosis are inadequate, and new molecular targets are needed. Herein we report that glycogen synthase kinase-3β (GSK-3β) is phosphorylated (inhibited) in fibrotic tissues from ischemic human and mouse heart. Using 2 fibroblast-specific GSK-3β knockout mouse models, we show that deletion of GSK-3β in cardiac fibroblasts leads to fibrogenesis, left ventricular dysfunction, and excessive scarring in the ischemic heart. Deletion of GSK-3β induces a profibrotic myofibroblast phenotype in isolated cardiac fibroblasts, in post-myocardial infarction hearts, and in mouse embryonic fibroblasts deleted for GSK-3β. Mechanistically, GSK-3β inhibits profibrotic transforming growth factor-β1/SMAD-3 signaling via interactions with SMAD-3. Moreover, deletion of GSK-3β resulted in the significant increase of SMAD-3 transcriptional activity. This pathway is central to the pathology because a small-molecule inhibitor of SMAD-3 largely prevented fibrosis and limited left ventricular remodeling. These studies support targeting GSK-3β in myocardial fibrotic disorders and establish critical roles of cardiac fibroblasts in remodeling and ventricular dysfunction.

Abstract 18289: Cardiac Macrophages Adopt Profibrotic/M2 Phenotype in Ischemic Hearts

Background: Macrophages (macs) that over express urokinase plasminogen activator(uPA) adapt a profibrotic M2 phenotype in the heart in association with cardiac fibrosis. We tested the hypothesis that cardiac macs are M2 polarized in ischemic mouse and human hearts and that polarization is dependent on mac derived uPA. Methods: Mouse cardiac macs were isolated from hearts of infarcted (MI) and uninjured(UI) wild-type (WT) , uPA overexpressing (SR-uPA), or uPA null mice. RNA was extracted after isolation or culture with vehicle, IL-4 or plasmin. M2 markers Arg1, YM1, and Fizz1 measured with qrtPCR. Human cardiac macs were isolated from tissue at time of LVAD placement or transplantation. Peripheral blood was collected at the same time and monocyte-derived macs (PMDM) generated. Expression of CD206 was measured in isolated and IL-4 treated macs. All values were normalized to GAPDH. Values are meanSEM Results: Macs from WT mice had increased expression of Arg1 and Ym1 following MI (41.36.5 and 70.336, fold change vs UI, n=8, p < 0.007). ANOVA analysis of UI vs MI in WT, SR-uPA, and uPA null showed significant upregulation of Arg1 and Ym1 with MI in all genotypes (n=4-9 per genotype and condition). There was no increase in Fizz1 in any group. Treatment with IL-4 increased expression of Arg1 and YM1, to a greater degree in cardiac macs from SR-uPA versus WT mice. Human cardiac macs expressed increased levels of CD206 in comparison to PMDM (4.91.3). Conclusions: Cardiac macs in mouse and human hearts adapt a M2 phenotype in association with cardiac fibrosis. uPA is not necessary for M2 phenotype development. Altering mac phenotype in the heart is a potential target to modify cardiac fibrosis.

C1q/Tumor Necrosis Factor–Related Protein-9, a Novel Adipocyte-Derived Cytokine, Attenuates Adverse Remodeling in the Ischemic Mouse Heart via Protein Kinase A Activation

C1q/tumor necrosis factor-related protein-9 (CTRP9) is a newly identified adiponectin paralog with established metabolic regulatory properties. However, the role of CTRP9 in postmyocardial infarction remodeling remains completely unknown. This study determined whether CTRP9 may regulate cardiac remodeling after acute myocardial infarction (AMI) and elucidated the underlying mechanisms. Male adult mice were subject to AMI by left anterior descending coronary artery ligation or sham surgery and treated with saline (vehicle) or globular CTRP9 via peritoneal implant osmotic pumps for 6 weeks. H9C2 cardiac cell lines were used in vitro for determining underlying mechanisms. Adipocyte CTRP9 expression and plasma CTRP9 levels were both significantly reduced after AMI. Compared with vehicle, CTRP9 treatment improved animal survival rate (P<0.05), restored cardiac function (P<0.05), attenuated adverse remodeling (P<0.01), and ameliorated cardiomyocyte apoptosis and fibrosis after AMI (P<0.01). Among the multiple antiremodeling molecules determined, AMP-activated protein kinase, protein kinase A (PKA), and Akt were significantly activated in CTRP9-treated heart. Surprisingly, CTRP9 remains cardioprotective in mice with cardiomyocyte-specific overexpression of a mutant AMP-activated protein kinase α2 subunit (AMPK-DN). Additional in vitro experiments demonstrated that administration of either PKA inhibitor or PKA-specific small interfering RNA virtually abolished the antiapoptotic effect of CTRP9 (P<0.05), whereas inhibition of Akt is less effective in blocking CTRP9 cardioprotection. Finally, CTRP9 phosphorylates BCL-2-associated agonist of cell death at its multiple antiapoptotic sites, an effect blocked by PKA inhibitor. We demonstrate that adipokine CTRP9 attenuates adverse cardiac remodeling after AMI, largely via a PKA-dependent pathway.

Protein disulfide isomerase-associated 6 is an ATF6-inducible ER stress response protein that protects cardiac myocytes from ischemia/reperfusion-mediated cell death

Proper folding of secreted and transmembrane proteins made in the rough endoplasmic reticulum (ER) requires oxygen for disulfide bond formation. Accordingly, ischemia can impair ER protein folding and initiate the ER stress response, which we previously showed is activated in the ischemic heart and in culture cardiac myocytes subjected to simulated ischemia. ER stress and ischemia activate the transcription factor, activating transcription factor 6 (ATF6), which induces numerous genes, many of which have not been identified, or examined in the heart. Using an ATF6 transgenic mouse model, we previously showed that ATF6 protected the heart from ischemic damage; however, the mechanism of this protection remains to be determined. In this study, we showed that, in the mouse heart, and in cultured cardiac myocytes, ATF6 induced the protein disulfide isomerase associated 6 (PDIA6) gene, which encodes an ER enzyme that catalyzes protein disulfide bond formation. Moreover, in cultured cardiac myocytes, ER stress-mediated PDIA6 promoter activation was ATF6-dependent, and required an ER stress response element (ERSE) and a nearby CCAAT box element. Electromobility shift assays and chromatin immunoprecipitation showed that ATF6 bound to the ERSE in the PDIA6 promoter, in vitro, and in the mouse heart, in vivo. Gain- and loss-of-function studies showed that PDIA6 protected cardiac myocytes against simulated ischemia/reperfusion-induced death in a manner that was dependent on the catalytic activity of PDIA6. Thus, by facilitating disulfide bond formation, and enhanced ER protein folding, PDIA6 may contribute to the protective effects of ATF6 in the ischemic mouse heart.

Tissue Kallikrein Promotes Cardiac Neovascularization by Enhancing Endothelial Progenitor Cell Functional Capacity

Tissue kallikrein (TK) has been demonstrated to improve neovasculogenesis after myocardial infarction (MI). In the present study, we examined the role and underlying mechanisms of TK in peripheral endothelial progenitor cell (EPC) function. Peripheral blood-derived mononuclear cells containing EPCs were isolated from rat. The in vitro effects of TK on EPC differentiation, apoptosis, migration, and vascular tube formation capacity were studied in the presence or absence of TK, kinin B(2) receptor antagonist (icatibant), and phosphatidylinositol-3 kinase inhibitor (LY294002). Apoptosis was evaluated by flow-cytometry analysis using Annexin V-FITC/PI staining, as well as western-blot analysis of Akt phosphorylation and cleaved caspase-3. Using an MI mouse model, we then examined the in vivo effects of human TK gene adenoviral vector (Ad.hTK) administration on the number of CD34(+)Flk-1(+) progenitors in the peripheral circulation, heart tissue, extent of vasculogenesis, and heart function. Administration of TK significantly increased the number of Dil-LDL/UEA-lectin double-positive early EPCs, as well as their migration and tube formation properties in vitro. Transduction of TK in cultured EPCs attenuated apoptosis induced by hypoxia and led to an increase in Akt phosphorylation and a decrease in cleaved caspase-3 levels. The beneficial effects of TK were blocked by pretreatment with icatibant and LY294002. The expression of recombinant human TK in the ischemic mouse heart significantly improved cardiac contractility and reduced infarct size 7 days after gene delivery. Compared with the Ad.Null group, Ad.hTK reduced mortality and preserved left ventricular function by increasing the number of CD34(+)Flk-1(+) EPCs and promoting the growth of capillaries and arterioles in the peri-infarct myocardium. These data provide direct evidence that TK promotes vessel growth by increasing the number of EPCs and enhancing their functional properties through the kinin B(2) receptor-Akt signaling pathway.

Abstract 8965: C1q/TNF-Related Protein-3, a Newly Identified Adipokine, is a Novel Anti-Apoptotic, Pro-Angiogenic, and Cardioprotective Molecule in the Ischemic Mouse Heart

Obesity/diabetes adversely affects ischemic heart remodeling via incompletely understood mechanisms. Adipocytes produce various adipokines. Delineating these adipokines’ cardiovascular profiles is critical in advancing our knowledge regarding how obesity/diabetes may cause cardiovascular damage. C1q/TNF-related protein-3 (CTRP3) is a newly identified adipokine exerting beneficial metabolic regulation. However, the cardiovascular actions of this novel adipokine remain unknown. The current study determined whether CTRP3 may regulate post-ischemic cardiac remodeling and cardiac dysfunction, and, if so, sought to elucidate the involved underlying mechanisms. Male adult mice were subjected to MI, and treated with saline or CTRP3 via peritoneally implanted osmotic-pumps for 2 weeks. Plasma CTRP3 levels were significantly reduced 4 hours after MI, and remained at low level until 1 week after MI. Supplementation of CTRP3 (beginning 4 hours after MI) at a dose (0.25 μg/g/day) restoring physiological CTRP3 levels in MI animals improved survival rate (P&lt;0.05), rescued cardiac function (determined by echocardiograph and left ventricular catheterization, P&lt;0.01), attenuated cardiomyocyte apoptosis within infarct border zones (determined by TUNEL staining and caspase 3 activation, P&lt;0.01), increased revascularization (evidenced by CD31 and α-SMA immunostaining, P&lt;0.01), and dramatically reduced interstitial fibrosis (41% reduction vs. vehicle group, P&lt;0.01). In vivo CTRP3 administration had no significant effect upon AMPK phosphorylation, but significantly increased Akt phosphorylation, and increased HIF-1α and VEGF expression. Surprisingly, treatment of HUVECs in vitro with CTRP3 failed to increase tube formation. However, pre-conditioned medium from CTRP3-treated cardiomyocytes significantly enhanced HUVEC tube formation, an effect that was blocked by either pre-treatment of cardiomyocytes with a PI3K inhibitor, or pre-treatment of HUVECs with a VEGF inhibitor. Our results demonstrate for the first time that CTRP3 is a novel anti-apoptotic, pro-angiogenic, and cardioprotective adipokine in the ischemic mouse heart, via facilitation of cardiomyocyte-endothelium communication.

Additive effect of AAV-mediated angiopoietin-1 and VEGF expression on the therapy of infarcted heart

Vascular endothelial growth factor (VEGF) is a key angiogenic factor and has been used experimentally for induction of neovasculature in ischemic myocardium. However, blood vessels induced by VEGF are immature. Angiopoietin-1 (ang-1) has the ability to recruit and sustain periendothelial support cells and promote vascular maturation. Thus, co-expression of the two may yield a better result than expression of either one alone. Two adeno-associated viral vectors (AAV), CMVVEGF and CMVang-1 with the CMV promoter driving VEGF or ang-1 gene expression, respectively, were injected into ischemic mouse hearts individually or together in different ratios. The results show that co-injected groups had more capillaries than the CMVang-1 group and similar densities of capillaries and α-actin positive vessels as the CMVVEGF group. Neovasculature induced by CMVVEGF was leaky. In contrast, neovasculature in CMVang-1-injected or CMVVEGF and CMVang-1 co-injected hearts was less leaky than that in CMVVEGF-injected hearts. The group that received CMVang-1 and CMVVEGF in a 1:1 ratio had the smallest infarct size and best cardiac function and regional wall movement among all the groups. We conclude that ang-1 and VEGF can compensate for each others' shortcomings and yield a better therapeutic effect by acting together.

Abstract 1568: Loss of Ischemic Postconditioning in the Mouse Hearts Lacking Adenosine A 1 or Bradykinin B 2 Receptors

Background: Ischemic postconditioning (PostC) is a recently described cardioprotective modality against reperfusion injury, through a series of brief episodes of reperfusion/ischemia at the very onset of reperfusion. It has been well recognized that PostC can activate cellular signaling cascade, in which the role of G protein-coupled membrane receptors serving as upstream triggers of PostC remains to be established. Hence the goal of this study was to determine a definitive role of adenosine A 1 receptors (A1) and bradykinin B 1 or B 2 receptors (B1 or B2) in PostC, using gene knockout (KO) mice. Methods &amp; Results: The hearts isolated from adult male C57BL/6J wild-type mice (C57-WT) or A1, B1, or B2 KO mice (n=7–9 per group) were subjected to 20 min of zero-flow global ischemia and 30 min of reperfusion with or without PostC in a Langendorff isolated, buffer-perfused heart model. PostC, consisted of 6 cycles of 10 sec of reperfusion and 10 sec of ischemia, significantly reduced myocardial infarct size (22.8±3.1%, Mean±SEM) as compared with C57-WT controls (35.1±2.8%, P&lt;0.05). As shown in Figure below, the infarct-limiting protection of PostC was absent in A1-KO (34.9±2.7%) or B2-KO (33.3±1.7%) and was partially attenuated in B1-KO (25.6±2.9%) mice, as compared with the corresponding non-PostC controls under same genetic background (P&gt;0.05). However, cardiac contractile function and coronary flow at the end of reperfusion were not significantly altered by PostC. Conclusion: PostC-induced infarct size reduction in globally ischemic mouse hearts is triggered by activation of multiple G protein-coupled membrane receptors, which include A1, B2, and, to a lesser extent, B1 receptors.

ARGINASE EXPRESSION IN THE MOUSE HEART POST-MYOCARDIAL INFARCTION.

Background Following acute myocardial infarction (MI), there are a number of changes in gene expression that take place in all areas of the heart. It has recently been shown that mRNA levels for arginase (specifically the type I isozyme) are significantly elevated following acute MI in a murine model. The expression of this enzyme could have a significant impact on arginine and polyamine metabolism in the post-MI myocardium and vasculature since it would compete with enzymes such as nitric oxide synthase for access to its substrate, arginine. The goal of this project was to confirm that the protein itself was also overexpressed in the post-MI heart, as well as to define that expression temporally and spatially in the ischemic mouse heart. Study Design and Methods Using primary antibodies directed against arginase I, we evaluated the expression of this enzyme in naive liver, brain, kidney, and heart tissue, as well as tissue from experimentally infarcted (LAD ligature) mice at 4, 24, and 48 hours post-MI. Expression was analyzed via Western blot and immunofluorescence microscopy. Results Western blot data indicated that in naive mouse tissue, arginase expression is highest in the liver, with fairly high levels also noted in the kidney. Naive heart showed very low levels of constitutive arginase expression, whereas brain tissue showed no detectable expression. Experiments on infarcted tissue showed very little induction of protein expression at 4 hours post-MI but notable elevations at 24 hours and even higher levels at 48 hours. These data were further confirmed by immunofluorescence microscopy, which indicated very little evident antibody binding in naive heart and increasing levels of fluorescent activity from 4 to 48 hours post-MI. The 48-hour time point clearly showed the highest levels of arginase I. The fluorescence was consistently localized to that area of the left ventricle (LV) corresponding to the infarct area, with minimal expression noted in the right ventricular (RV) free wall and other remote areas. Additionally, the fluorescence was most intense at the epicardial surface of the heart, although the specific cell type expressing the enzyme could not be definitively defined. Possibilities include subepicardial cardiac myocytes, fibroblasts in the epicardial connective tissue, epithelial cells forming the visceral pericardium (epicardium), or peripheral infiltrating neutrophils. Conclusion These results show that arginase I is preferentially expressed in the epicardial infarct region of the post-MI mouse heart. These results may hold promise for further elucidating the changes in arginine and nitric oxide biochemistry that occur following ischemic insult to the heart.

228. AAV Mediated Hypoxia-Inducible VEGF Expression and Neovascular Formation in Ischemic Pig Myocardium

Rapid, efficient overexpression of angiogenic growth factors in the peri-infarct zone is a promising approach for gene therapy of acute myocardium infarction. Patients suffering from an acute myocardial infarction often require invasive treatment, and gene transfer could be performed in combination with intravascular revascularization strategies or with bypass surgery. Many angiogenic factors such as vascular endothelial growth factor (VEGF), angiopoietins, fibroblast growth factor-4 (FGF-4) and placental growth factor (PlGF) have been shown to be potent inducers of arteriogenesis. VEGF is the most potent among these factors as it is capable of increasing proliferation and migration of endothelial and vascular smooth muscle cells, and attracting stem cell homing. In our previous studies, we have induced neovascular formation in ischemic mouse hearts with adeno-associated viral vector (AAV) mediated VEGF gene transfer. To avoid the harmful effect of excessive angiogenesis, we have constructed an AAV vector (MLCVEGF) with 9 copies of the hypoxia-response element (HRE) of the human erythropoietin (EPO) gene and the promoter of the rat myosin light chain 2v (MLC-2v) controlling VEGF gene expression. Since information derived from mouse model may not be directly applicable to human and the pig heart closely resembles the human heart closely, we tested the hypotheses that MLCVEGF can also mediate hypoxia- inducible VEGF expression and induce neovascular formation in the pig heart. We injected 1|[times]|1011 genomes of MLCVEGF or CMVVEGF (AAV vector with CMV promoter driving VEGF expression) into normal and ischemic pig myocardium after 90 minutes of occlusion and reperfusion of the left anterior descending coronary artery (LAD). CMVLacZ vector (AAV vector with CMV promoter driving LacZ gene expression) was used as control. All vectors were packaged in AAV serotype 1 capsid. Myocardiums were collected 2 weeks after the vector injection. Gene expression was analyzed by real-time RT-PCR and immunohistochemical staining. Capillaries and a-actin positive vessels were quantitated on anti PECAM-1 and smooth muscle a-actin antibody stained sections. We observed robust LacZ and VEGF gene expression at CMVLacZ and CMVVEGF injection sites and found more capillaries and a-actin positive vessels at VEGF vector injected sites than CMVLacZ injection sites. VEGF expression mediated by MLCVEGF vector was low in normal myocardium and was induced about 40-fold in ischemic myocardium 2 weeks after myocardial infarction and vector injection. Conclusions: 1) AAV serotype 1 can infect pig myocardium efficiently. 2) AAV vector containing VEGF controlled by MLC-2v promoter and 9 copies of EPO HRE can mediate hypoxia-inducible gene expression and induce neovascular formation in the pig myocardium.

Ethnic Differences in Blood Pressure Control Among Men at Veterans Affairs Clinics and Other Health Care Sites

Endothelia progenitor cell (EPC)-based revascularization therapies have shown promise for the treatment of myocardial ischemic injury. However, applications and efficacy are limited by the relatively inefficient recruitment of endogenous EPCs to the ischemic area, while implantation of exogenous EPCs carries the risk of tumorigenicity. In this study, we developed a therapeutic protocol that relies on the capacity of neutrophils (NEs) to target lesions and release preloaded EPC-binding molecules for high efficiency capture. Neutrophils were loaded with superparamagnetic iron oxide nanoparticles conjugated to an antibody against the EPC surface marker CD34 (SPIO-antiCD34/NEs), and the therapeutic efficacy in ischemic mouse heart following SPIO-antiCD34/NEs injection was monitored by SPIO-enhanced magnetic resonance imaging (MRI). These SPIO-antiCD34/NEs exhibited unimpaired cell viability, superoxide generation, and chemotaxis in vitro as well as satisfactory biocompatibility in vivo. In a mouse model of acute myocardial infarction (MI), SPIO-antiCD34 accumulation could be observed 0.5 h after intravenous injection of SPIO-antiCD34/NEs. Moreover, the degree of CD133+ EPC accumulation at MI sites was three-fold higher than in control MI model mice, while ensuing microvessel density was roughly two-fold higher than controls and left ventricular ejection fraction was > 50%. Therapeutic cell biodistribution, MI site targeting, and treatment effects were confirmed by SPIO-enhanced MRI. This study offers a new strategy to improve the endogenous EPC-based myocardial ischemic injury repair through NEs mediated SPIO nanoparticle conjugated CD34 antibody delivery and imaging.The efficacy of endogenous endothelial progenitor cell (EPC)-based cardiovascular repair therapy for ischemic heart damage is limited by relatively low EPC accumulation at the target site. We have developed a method to improve EPC capture by exploiting the strong targeting ability of neutrophils (NEs) to ischemic inflammatory foci and the capacity of these treated cells to release of preloaded cargo with EPC-binding affinity. Briefly, NEs were loaded with superparamagnetic iron oxide nanoparticles conjugated to an antibody against the EPC surface protein CD34 (SPIO-antiCD34). Thus, we explored sites targeting with nanocomposites cargo for non-invasive EPCs interception and therapy tracking. We demonstrate that SPIO-antiCD34 released from NEs can effectively capture endogenous EPCs and thereby promote heart revascularization and functional recovery in mice. Moreover, the entire process can be monitored by SPIO-enhanced magnetic resonance imaging including therapeutic cell biodistribution, myocardial infarction site targeting, and tissue repair.

Stem cell implantation in ischemic mouse heart: a high‐resolution magnetic resonance imaging investigation

Advances in the biology of stem cells have evoked great interest in cell replacement therapies for the regeneration of heart tissue after myocardial infarction. However, results from human trials are controversial, since the destination of the injected cells, their engraftment and their long-term fate have remained unclear. Here we investigate whether transplanted cells can be identified in the intact and lesioned murine myocardium employing high-resolution MRI. Cardiac progenitor cells, expressing the enhanced green fluorescent protein (EGFP), were labeled with ultra-small paramagnetic iron-oxide (USPIO) nanoparticles and transplanted into the intact or injured myocardium of mice. Their precise location was determined with high-resolution MRI and compared with histological tissue sections, stained with Prussian blue for iron content. These experiments showed that iron nanoparticle-loaded cells could be identified at high resolution in the mouse heart. However, ischemic myocardium (after cryoinjury or left coronary artery ligation) was characterized by a signal attenuation similar to that induced by USPIO-labeled cells in T2*-weighted MR images, making detection of labeled stem cells in this area by T2*-sensitive contrast rather difficult. In animals with myocardial injury only, the signal attenuated areas were of the same size in proton density- and T2*-weighted MR images. In injured animals also receiving labeled cells the lesioned area appeared larger in T2*--than in proton density-weighted MR images. This sequence-dependent lesion size change is due to the increased signal loss caused by the iron oxide nanoparticles, most sensitively detectable in the T2*-sensitive images. Thus, using the novel combination of these two parameter weightings, USPIO-labeled cells can be detected at high resolution in ischemic myocardium.

Adeno-associated viral vector delivers cardiac-specific and hypoxia-inducible VEGF expression in ischemic mouse hearts

It has been shown that the adeno-associated virus (AAV) vector can deliver the VEGF gene efficiently into the ischemic mouse myocardium. However, the AAV genomes can be found in extracardiac organs after intramyocardial injection. To limit unwanted VEGF expression in organs other than the heart, we tested the use of the cardiac myosin light chain 2v (MLC-2v) promoter and the hypoxia-response element to mediate cardiac-specific and hypoxia-inducible VEGF expression. An AAV vector, MLCVEGF, with 250 bp of the MLC-2v promoter and nine copies of the hypoxia-response element driving VEGF expression, was constructed. Gene expression was studied in vitro by infection of rat cardiomyocytes, rat skeletal myocytes, and mouse fibroblasts with the vector and in vivo by direct injection of the vector into normal and ischemic mouse hearts. With MLCVEGF infection, VEGF expression was higher in cardiomyocytes than the other two cell lines and was hypoxiainducible. VEGF expression was also higher in ischemic hearts than in normal hearts. No VEGF expression was detectable in organs with detectable MLCVEGF vectors other than the heart. MLCVEGF-injected ischemic hearts had more capillaries and small vessels around the injection site, smaller infarct size, and better cardiac function than the negative controls. Hence, MLCVEGF can mediate cardiac-specific and hypoxia-inducible VEGF expression, neoangiogenesis, infarct-size reduction, and cardiac functional improvement.

929. Target Adult Stem Cells to Ischemic Hearts by Regulated Stromal Cell-Derived Factor 1 Gene Transfer

Adult bone marrow-derived stem cells (BMSCs) have emerged as a promising source for regeneration of tissues including those traditionally considered terminally differentiated, for instance, heart and brain. Chronic heart failure afflicts 4.8 million Americans, for those at the end stage of this disease heart transplantation remains the only and last resort. Acute myocardial infarction (AMI) is a leading cause of heart failure. Increasing evidence suggests that the body has a natural response for cardiac repair by up-regulating stromal cell-derived factor 1 (SDF-1), a stem cell active chemokine, which leads to recruitment of circulating BMSCs to ischemic hearts. However, up-regulation of endogenous SDF-1 is transient and inadequate to induce functionally meaningful heart regeneration. Therefore, the current study aimed to enhance stem cell-initiated cardiac repair by targeting circulating BMSCs to ischemic myocardium through targeted and regulated SDF-1 gene expression. To accomplish this goal, we have developed a regulatable system that consists of a MLC-2v promoter for cardiac selective expression as well as an oxygen-sensitive gene switch based on the oxygen-dependent degradation domain of hypoxia inducible factor 1α. It delivers genes of interest in a cardiac-selective and hypoxia-regulated manner in vitro and in vivo. AMI was surgically induced in adult BALB/c mice. DNA plasmid encoding hypoxia-regulated human SDF-1 was administered via intra-myocardial injection. PKH26 labeled BMSCs (6 × 106) were systemically injected immediately after induction of AMI. hSDF-1 expression was evaluated by Western blot analysis and the efficiency of hSDF-1 to attract implanted BMSCs was assessed by immunofluorescent staining and confocal microscopy. 1 week post AMI, hSDF-1 expression was markedly increased in the ischemic mouse heart, while less or no expression was detected in spleen, liver, lung, kidney and skeletal muscle nor in the heart of non-MI group. Confocal microscopy showed that hSDF-1 was highly expressed in the peri-infarct zone, with PKH26-labeled BMSCs enriched nearby. Furthermore, infarct size and cardiac fibrosis were significantly reduced in mice treated with hSDF-1 plus BMSCs compared to BMSCs alone. Taken together, our results indicate that cardiac-selective and hypoxia-inducible SDF-1 gene transfer activated mobilization of BMSCs to ischemic hearts and facilitated post-ischemic repair. This combined approach of regulated gene transfer and adult stem cells may provide a novel, safe and effective strategy for regenerative medicine, leading to efficient and timely repair of injured tissues.

192. Cardiac-Specific and Hypoxia-Responsive Vascular Endothelial Growth Factor Expression in Ischemic Hearts

A problem associated with angiogenic gene therapy is that uncontrolled gene expression of angiogenic factors may cause several side effects, including: hemangioma formation, retinopathy, occult tumor growth and atherosclerotic plaque progression. Previously, we used the hypoxia response element (HRE) isolated from the erythropoietin (Epo) gene enhancer to render vascular endothelial growth factor (VEGF) expression responsive to hypoxia induction in vitro and in ischemic myocardium in vivo. We found in our experiment that adeno-associate viral vector (AAV) can be detected in other organs in addition to the heart after intra-myocardial injection. Thus, unwanted effects may still occur even with HRE controlled VEGF expression, since hypoxia in other tissues in patients with heart failure may results in extra-cardiac VEGF gene expression. To ensure the safety of angiogenic therapy for ischemic heart disease, it is necessary that the gene expression is cardiac specific and hypoxia responsive. In this study, we explored the feasibility of using the cardiac myosin light-chain-2v (MLC-2v) promoter and Epo HRE to mediate cardiac-specific and hypoxia-responsive VEGF gene expression. We constructed an AAV vector with MLC-2v promoter and 9 copies of Epo HRE driving VEGF (MLCVEGF) expression. Gene expression mediated by this vector was studied in vitro by transfection of this vector into a rat cardiac muscle cell line (H9c2), a rat skeletal muscle cell line (L6) and a mouse fibroblast cell line (NIH3T3), and in vivo by direct injection of the vector to normal and ischemic mouse hearts. AAV vectors with CMV driving VEGF (CMVVEGF), and 9HRE plus minimum SV40 promoter driving VEGF (H9VEGF) were used as controls. RESULTS: With MLCVEGF infection, VEGF expression is higher in H9c2 than the other two non-cardiac cell lines, and the expression is hypoxia inducible. In vivo studies indicated that MLCVEGF increased VEGF expression by 74% in ischemic heart compared to normal heart. VEGF expression was detected in liver, kidney, spleen and gonad of CMVVEGF inoculated mice by RT-PCR, but no VEGF expression was detected in organs other than heart and liver of MLCVEGF inoculated mice. CONCLUSION: Our result indicates that MLC-2v promoter and 9 copies of HRE can mediate cardiac-specific and hypoxia-inducible gene expression in vitro and in vivo. Induction of angiogenesis and improvement of cardiac function by this vector, studies currently in progress, may provide a safe approach for the treatment of ischemic heart disease.

622. Regulation of AAV Mediated VEGF Gene Expression by Hypoxia Response Element and Tissue Specific Promoter in the Ischemic Mouse Heart

622. Regulation of AAV Mediated VEGF Gene Expression by Hypoxia Response Element and Tissue Specific Promoter in the Ischemic Mouse Heart

1085. AAV Mediated VEGF Gene Transfer Improves the Function of the Ischemic Mouse Heart

1085. AAV Mediated VEGF Gene Transfer Improves the Function of the Ischemic Mouse Heart