Cell Therapy and Tissue Generation

Double Knockdown of Prolyl Hydroxylase and Factor-Inhibiting Hypoxia-Inducible Factor With Nonviral Minicircle Gene Therapy Enhances Stem Cell Mobilization and Angiogenesis After Myocardial Infarction

Mei Huang, Patricia Nguyen, Fangjun Jia, Shijun Hu, Yongquan Gong, Patricia E. de Almeida, Li Wang, Divya Nag, Mark A. Kay, Amato J. Giaccia, Robert C. Robbins, Joseph C. Wu
https://doi.org/10.1161/CIRCULATIONAHA.110.014019
Circulation. 2011;124:S46-S54
Originally published September 12, 2011

Jump to

Abstract

Background—Under normoxic conditions, hypoxia-inducible factor (HIF)-1α is rapidly degraded by 2 hydroxylases: prolyl hydroxylase (PHD) and factor-inhibiting HIF-1 (FIH). Because HIF-1α mediates the cardioprotective response to ischemic injury, its upregulation may be an effective therapeutic option for ischemic heart failure.

Methods and Results—PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin (sh) sequences for inhibiting PHD isoenzyme 2 and FIH were inserted into novel, nonviral, minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit+ cardiac progenitor cells demonstrated higher expression of angiogenesis factors in the double-knockdown group compared with the single-knockdown and short hairpin scramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially after left anterior descending coronary artery ligation in adult FVB mice (n=60). Functional studies using MRI, echocardiography, and pressure-volume loops showed greater improvement in cardiac function in the double-knockdown group. To assess mechanisms of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double-knockdown group. Fluorescence-activated cell sorting showed significantly higher activation of endogenous c-kit+ cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser-capture microdissection analysis confirmed upregulation of HIF-1α protein and angiogenesis genes, respectively.

Conclusions—We demonstrated that HIF-1α upregulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function.

Introduction

Coronary artery disease is the leading cause of morbidity and mortality in the Western world.1 Despite conventional treatments, a significant number of patients still have refractory angina.2 Cytokine agents are a promising therapeutic option, which can be used as adjunctive treatment to revascularization or cell therapy. Cytokine therapy may act directly on the myocardium (eg, inhibiting apoptosis and stimulating proliferation) or indirectly by mobilization of stem cells from the systemic circulation.3 However, randomized clinical trials have shown mixed results, which may be partly due to the limited benefits of single gene therapy.4 A better option may be to administer hypoxia-inducible factor (HIF)-1α, an upstream transcriptional factor that regulates >100 genes and protects the myocardium from ischemic injury.5,6

Unfortunately, HIF-1α has a biological half-life of only ≈5 minutes under normoxic conditions. Its rapid proteosomal degradation results from hydroxylation of HIF-1α at Pro402 or Pro564 by a set of HIF prolyl hydroxylase (PHD) isoenzymes (1 to 3) that mediate its recognition by von Hippel-Lindau ubiquitin ligase complex.7 Concurrently, factor-inhibiting HIF-1 (FIH) also catalyzes the hydroxylation of HIF-1α Asn803, which blocks its interaction with the transcriptional coactivator p300.8 In contrast, during hypoxia, the lack of hydroxylase activities enables HIF-1α to escape proteosomal destruction and allows it to bind with the HIF-1β subunit to become transcriptionally active.

In this study, we used short hairpin (sh) sequences inserted into a novel, nonviral, minicircle vector to inhibit PHD isoenzyme 2 (shPHD2) and FIH (shFIH), which then prevented HIF-1α degradation. We found that upregulation of HIF-1α enhances stem cell mobilization (through molecular imaging and fluorescence-activated cell sorting [FACS]) and increases myocardial angiogenesis (through laser-capture microdissection [LCM] and immunohistochemistry), leading to significant improvement in cardiac function after myocardial infarction (MI).

Methods

RNA Interference of Mouse PHD2 Gene in Culture Cell

Mouse PHD2 and FIH genes were cloned from mouse embryonic stem cells (SV129 line). We used the PHD2 isoenzyme because a previous study showed higher expression of PHD2 in the heart compared to PHD1 or PHD3 isoenzymes.9 We designed 4 sequences of RNA interference sites for the PHD2 gene and 8 for the FIH gene. The targeting sequences are shown in online-only Data Supplement Figure I. The optimal knockdown fragment was inserted after the H1 promoter in the minicircle parental backbone vector.

Cell Culture, shRNA Transfection, and Hypoxia Exposure

Mouse C2C12 myoblasts were cultured in DMEM medium (high glucose)+10% FBS. Mouse HL-1 atrial myocytes were cultured in 10% FBS (BioWhittaker), 10 μg/mL insulin (Life Technologies), 50 μg/mL endothelial cell growth supplement (Upstate Biotechnology), 1 μmol/L retinoic acid (Sigma), 10 μmol/L norepinephrine (Sigma), 100 U/mL penicillin, 100 μg/mL streptomycin (Life Technologies), and an additional 1× nonessential amino acids (Life Technologies) on 0.01% gelatin-coated plate. Mouse c-kit+ cardiac progenitor cells (CPCs) were isolated based on a previously described protocol.10 CPCs were cultured in 10% embryonic stem cell-qualified FBS (Gibco), DMEM, Ham F-12 medium, insulin-transferrin-selenium, 10 ng/mL leukemia inhibitory factor, 10 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL epidermal growth factor, and penicillin-streptomycin-glutamine. Minicircle vectors with shPHD2 and shFIH driven by H1 promoter were cotransfected into mouse C2C12, HL-1, and CPC using lipofectamine 2000 (Invitrogen), with the plasmid pHRE-SV40-FLuc as control for determining knockdown efficiency (online-only Data Supplement Figure I). After minicircle shRNA transfection, cells were cultured for 1 day before being subjected to the hypoxia condition with 5% CO2, 1% O2, and 94% N2 at 37°C for 48 hours.

Quantitative Polymerase Chain Reaction Analysis of Angiogenesis Genes

At the end of the 48-hour hypoxia treatment, cells were harvested for immediate RNA extraction (n=3 biological replicates). Quantitative polymerase chain reaction (qPCR) was used to compare the expression of angiogenic genes (fibroblast growth factor b [bFGF], vascular endothelial growth factor [VEGF], vascular endothelial growth factor receptor 1 [Flt], vascular endothelial growth factor receptor 2 [KDR], transforming growth factor [TGF], and plasminogen activator inhibitor-1 [PAI-1]) in transfected cells under normoxic versus hypoxic conditions. Total RNA was prepared from C2C12 cells with Trizol reagent (Invitrogen) according to the manufacturer's protocol. The probe sets used in the amplification reaction were obtained from Applied Biosystems. PCR reactions were performed on the ABI 7900HT system.

Angiogenesis Cytokine Array After Hypoxia Exposure

At the end of the hypoxic treatment, the supernatant was collected for angiogenesis cytokine array (Panomics). In 2 separate experiments (duplicate spots, 2 biological replicates), the arrays were hybridized and imaged together. Expression intensities were calculated by adding the total pixel intensity for each spot. Interarray normalization was performed by using positive control spots (8 per array) on each array. Protein expression levels were normalized to PBS controls so that changes in protein expression could be easily assessed.

Animal Surgery To Induce MI

Ligation of the mid-left anterior descending coronary artery was performed in adult female FVB mice (Charles River Laboratories; Wilmington, MA) by a single experienced microsurgeon (Y.G.). Survival rate for each surgery group was between 80% to 90%. MI was confirmed by myocardial blanching and ECG changes. Animals were randomized into shFIH (n=15), shPHD2 (n=15), shPHD2+shFIH (n=15), and shScramble control (n=15) groups. Animals were injected intramyocardially with 25 μg of shRNA or shScramble minicircle vector using a 31-gauge Hamilton syringe. In all groups, the volume of injection was 25 μL in 3 different spots near the periinfarct zone. Study protocols were approved by the Stanford Animal Research Committee.

Activation of Endogenous c-kit+ CPCs With shRNA Therapy

For immunostaining, frozen heart sections (n=5 mice per group) were deparaffinized, and antigen retrieval was performed using 10 mmol/L citrate (pH 6.0). Slides were blocked in TNB (PerkinElmer) for 1 hour after which goat antimouse c-kit antibody (R&D Systems) was added (1:100) overnight at 4°C. Slides were washed in 1 mol/L Tris/NaCl followed by secondary antibody incubation for 2 hours at room temperature.11 For FACS of c-kit+ CPCs in the heart (n=5 mice per group), we excised the periinfarct zone and digested it using 0.1% collagenase IV into single-cell suspension. FACS was performed using c-kit (allophycocyanin-Cy7 conjugated; BD Biosciences) antibody as previously described.12

Creation of Bone Marrow Chimeric Mouse Model

To determine whether c-kit+ cells found in the heart were endogenous or donor-derived bone marrow-derived cells, we generated a hematopoietic chimeric mouse model that we termed the mousenized mouse model. Using a strategy described previously, we used lineage-negative bone marrow cells (BMCs) from C57BL/6 mice (major histocompatibility complex [MHC] class I haplotype, H2kb) to reconstitute NOD/SCID IL-2Rg (−/−) mice (MHC class I haplotype, H2g7), a severely immunocompromised strain that lacks T cells, B cells, and NK cells.13 Briefly, 2- to 3-day-old NOD/SCID IL-2Rg (−/−) pups received myeloablative treatment consisting of 100 cGy gamma irradiation. Sixteen hours later, pups received an intrahepatic injection of 1×106 lineage-negative BMCs isolated from adult C57BL/6 mice. Six to 8 weeks later, hematopoietic engraftment was confirmed by detecting engraftment of donor-derived (H2kb+) multilineage immune cells in peripheral blood of mousenized mice through FACS. After donor cell engraftment was confirmed, the mousenized mice underwent left anterior descending coronary artery ligation followed by injection of shPHD2+shFIH versus shScramble into the periinfarct zone (n=3 mice per group). Cells harvested from the periinfarct zone were stained with allophycocyanin-conjugated c-kit and R-phycoerythrin-conjugated H2kb antibodies. Donor-derived c-kit cells were identified by FACS as c-kit+ and H2kb+.

Analysis of Angiogenesis Genes Using LCM and RNA Extraction

Murine hearts were removed after perfusion with 20 mL PBS, embedded in optimal cutting temperature, and immediately frozen in liquid nitrogen. Seven thick tissue sections of left ventricle were prepared on polyethylene naphthalate membrane-coated slides (MicroDissect GmbH). For LCM, slides were thawed briefly and air dried 5 minutes before dissection. Green fluorescence observed under laser microscopy was used as a landmark for microdissection. Specific green fluorescence tissues, as well as normal cardiac tissues without green fluorescence, were independently dissected out by applying Leica LCM Systems (MicroDissect GmbH). The dissected tissues were placed on the caps of microcentrifuge tubes with 5 μL lysis enhanced buffer. After dissection, tissues were collected by centrifugation at 8000g for 5 minutes. Total RNA extraction and reverse transcription of these samples were performed using a commercial 1-step kit (Invitrogen) (n=5 mice per group).

Statistical Analysis

For calculation of relative gene expression, the expression level of each specific gene was divided by the expression level of GADPH. For statistical analyses of treatment groups, a Box-Cox transformation was used to achieve approximate normality for analysis by ANOVA and repeated-measures ANOVA. ANOVA (1 tail, equal variance) with post hoc testing was used to analyze qPCR, angiogenesis cytokine array, and myocardial perfusion. Repeated-measures ANOVA was used to analyze MRI, bioluminescence imaging, and echocardiographic data. Differences were considered significant at P<0.05. Unless otherwise specified, data are expressed as mean±SD.

Results

In Vitro Characterization of shFIH and shPHD2 Double Knockdown

We measured activation of HIF-1α and subsequent upregulation of angiogenesis genes after hydroxylase inhibition using shRNAs under normoxic and hypoxic conditions. Plasmid hypoxia response element pHRE-SV40-FLuc is a hypoxia-sensing 5xHRE-SV40 promoter driving FLuc cassette. The 5 copies of HRE derived from the erythropoietin gene are activated through binding of the HIF-1 complex,14 which allowed us to monitor the efficacy of the upstream shRNA knockdown compared to shScramble control (Figure 1A). In the normoxic condition, cells transfected with shPHD2+shFIH (5.32×105±32 171 photons/s per cm2/steridian [p/s per cm2/sr]) had significantly higher FLuc bioluminescence signals than cells transfected with shPHD2 (3.41×105±57 184 p/s per cm2/sr), shFIH (4.48×104±4513 p/s per cm2/sr), and shScramble control (2.86×104±1934 p/s per cm2/sr), indicating increased binding of 5xHRE-SV40 promoter by HIF-1α after double shRNA knockdown. A similar, but more robust trend was observed when the cells were exposed to hypoxic conditions. This is an expected finding given that HIF-1α acts by binding to the HREs to drive the expression of FLuc under hypoxic conditions. Western blot confirmed that higher levels of HIF-1α expression are present under hypoxic conditions, as shown in online-only Data Supplement Figure II.

Figure 1.
Figure 1.

In vitro characterization of shPHD2+shFIH knockdown. A, In vitro imaging results indicate that FLuc signals responded more strongly to shPHD2+shFIH under normoxic and 48-hour hypoxic conditions than shFIH alone or shScramble. The increased HIF-1α binds to the 5 copies of hypoxia response element binding site, leading to increased FLuc signals. B, Quantitative polymerase chain reaction analysis confirmed significant upregulation among 6 genes involved in angiogenesis because of activation of the HIF-1α protein from single or double knockdown of PHD2 and FIH. GAPDH was used as a control. C, Angiogenesis protein array data after hypoxia exposure and shScramble, shFIH, shPHD2, and shPHD2+shFIH treatment. D, Quantitative analysis indicates significant upregulation in several angiogenic-related proteins. Two biological replicates per group. bFGF indicates basic fibroblast growth factor; FIH, factor-inhibiting HIF-1; HIF-1α, hypoxia-inducible factor-1α; PAI-1, plasminogen activator inhibitor-1; PHD, prolyl hydroxylase; sh, short hairpin; TGFα, transforming growth factor-α; TIMP-1, tissue inhibitor of metalloproteinase-1; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.*P<0.001 vs shScramble. **P<0.05 vs shScramble.

To quantify luciferase activity, we lysed the cells and determined the luminescence activity normalized to protein concentration (online-only Data Supplement Figure III). The luminescence activity was highest in the double-knockdown group under both normoxic (2495±55 luminescence activity/mg protein) and hypoxic conditions (5232±100 luminescence activity/mg protein). To confirm similar effects in different cell types, mouse HL-1 atrial myocytes and mouse c-kit+ CPCs also were transfected by minicircle shRNA and pHRE-SV40-FLuc. Comparable results were observed in these 2 cell types (online-only Data Supplement Figure IV).

To confirm the pHRE-SV40-FLuc imaging signals, mRNA was isolated and qPCR performed for detection of HIF-1α and downstream angiogenesis genes. As shown inFigure 1B, relative expression of 6 genes related to angiogenesis (eg, bFGF, VEGF, Flt, KDR, TGF, PAI-1) were increased by 28.8±5.3% and 54.3±8.6% after treatment with shPHD2 and double knockdown, respectively. HIF-1α mRNA levels were not changed, which is expected because shRNA affects HIF-1α at the protein and not at the mRNA level. HIF-1α protein can activate several downstream genes responsible for stimulation of angiogenesis.15 To examine whether upregulation of HIF-1α protein through shRNA knockdown of PHD2 and FIH also can exert similar effects, supernatant from transfected C2C12 cells was used for angiogenesis assays.Figure 1C and 1D show significant upregulation of several angiogenesis activators (eg, FGFα, IL-6, leptin, VEGF, tumor necrosis factor-α, and TGF-α) after double knockdown.

Interestingly, both IFN-γ and tissue inhibitor of metalloproteinase-1 (TIMP-1) also were upregulated in the double-knockdown group (Figure 1D). IFN-γ is a soluble cytokine, which has antiviral, immunoregulatory, and antitumor properties.16 In contrast, TIMP-1 is a 28-kDa protein that inhibits the function of metalloproteinases and has been associated with cell growth promotion, matrix binding, apoptosis induction, and angiogenesis regulation.17 Previous studies have shown that HIF-1α regulates IFN-γ and TIMP-1.18–22 To confirm this, we measured the relative expression of TIMP-1 after transfection of C2C12 cells with a minicircle carrying the following genes: 1 HIF-1α (positive control), 2 an inhibitor of HIF-1α (siHIF-1α), 3 shFIH and shPHD2 (double knockdown), and 4 blank vector (negative control). We found that siRNA inhibition of HIF-1α led to significantly lower TIMP-1 and IFN-γ expression levels. In contrast, overexpression of minicircle HIF-1α and minicircle shPHD2+shFIH led to significantly higher levels of TIMP-1 (P<0.001 for both) and IFN-γ (P<0.05 for both) compared with control (online-only Data Supplement Figure V). Consistent with these findings, Yang et al23 demonstrated that shRNA inhibition of HIF-1α can dramatically decrease the expression of TIMP-1 mRNA expression and protein levels. Taken together, these data confirm that similar to physiological hypoxia response, PHD2+FIH double knockdown can effectively stabilize HIF-1α protein and induce HIF-1α-dependent gene activation in vitro.

Improvement of Left Ventricular Function and Perfusion After Dual shRNA Therapy

To examine whether double shRNA knockdown therapy can synergistically improve cardiac function, cardiac MRI was performed. At weeks 4 and 8 after MI, significant improvement in left ventricular ejection fraction (LVEF) was seen in the double-knockdown group compared to the other groups (Figure 2). The shPHD2 knockdown group also had improvement in LVEF compared with the shScramble group at weeks 4 and 8. These findings also were confirmed by serial ECGs (online-only Data Supplement Figure VI). In addition, invasive hemodynamic parameters showed that the increase in LVEF was associated with lower left ventricular end-diastolic volume and left ventricular end-systolic volume in double shRNA knockdown compared with shScramble (online-only Data Supplement Figure VII). Finally, shPHD2+shFIH treatment improved myocardial perfusion as determined by measuring the distribution of fluorescent microspheres within myocardial tissues after injection into the left ventricular cavity (online-only Data Supplement Figure VIII). These results also coincide with observed differences in HIF-1α expression at weeks 4 and 8 in the shPHD2+shFIH group compared with the other groups (online-only Data Supplement Figure IX).

Figure 2.
Figure 2.

Evaluation of heart function after minicircle shRNA injection. A, Cardiac MRI of mice with left anterior descending coronary artery ligation after minicircle injections at week 8. B, Animals injected with shPHD2+shFIH showed significant improvement in LVEF compared with shScramble, shFIH, and shPHD2 after 4 and 8 weeks. Five mice per group. LVEF indicates left ventricular ejection fraction. Other abbreviations as inFigure 1. *P<0.05 vs shScramble. **P<0.01 vs shScramble.

Double Knockdown Enhances BMC Homing to the Injured Myocardium

Previous studies have demonstrated that stem cells are activated and enriched in areas of injury.24,25 Moreover, cytokines have been shown to improve the cardiac stem cell mobilization in the infarction area.12 To confirm angiogenesis-related cytokine function for stem cell homing, BMCs were isolated from transgenic mouse that constitutively express green fluorescent protein (GFP) and FLuc and intravenously injected into syngeneic FVB mice with MI and shRNA therapy. Double knockdown significantly increased the number of BMC recruited to the myocardium from days 1 to 28 as assessed by longitudinal bioluminescence imaging (Figure 3A and 3B). GFP and troponin T double staining also demonstrated more GFP+ cells in the shPHD2+shFIH group (online-only Data Supplement Figure X).

Figure 3.
Figure 3.

Dual shRNA therapy increases recruitment of bone marrow cells (BMCs) after myocardial infarction. A, BMCs with green fluorescent protein-FLuc were administered through the tail vein 24 hours after myocardial infarction. BLI demonstrated a greater number of BMCs homed to the periinfarct region in the double-knockdown group, with peak homing at day 7. B, Quantitative analysis of BLI data at different time points. BLI indicates bioluminescence imaging; p/s/cm2/sr, photons/s per cm2/steridian. Other abbreviations as inFigure 1.

Double Knockdown Activates c-kit+ CPCs

Next, we assessed whether double knockdown also can induce proliferation of c-kit+ CPCs. Immunostaining showed higher presence of c-kit+ CPCs near the periinfarct area in the shPHD2+shFIH group (Figure 4A). To validate the histological data, we then performed FACS analysis for c-kit+ CPCs after excision of the periinfarct tissue and digestion into single-cell suspension. FACS confirmed that the number of c-kit+ CPCs was significantly greater in the shPHD2+shFIH group than in shScramble control (Figure 4B and 4C). To analyze the source of c-kit+ cells, a bone marrow chimeric mouse model (mousenized mouse model) was created.13 The hematopoietic system of NOD/SCID IL-2Rg (−/−) mice (H2g7) was reconstituted using lineage-negative BMCs from C57BL/6 murine (H2kb). BMCs from C57BL/6 strain were identified by the expression of H2kb MHC class I haplotype. After injection with shPHD2+shFIH versus shScramble into the mousenized mice, FACs analysis demonstrated a greater percentage of bone marrow recruitment (H2kb+/c-kit+) in the treatment group than in control group (2.46±0.18% versus 1.05±0.06%; P<0.01). Similarly, there was greater endogenous cell activation (H2kb−/c-kit+) in the treatment group than in the control group (1.06±0.06% versus 0.50±0.09%, P<0.05) (online-only Data Supplement Figure XI). Of the number of c-kit+ cells isolated from the treatment group, a greater percentage of isolated cells originated from the recipient (H2kb−) than the donor (H2kb+) (69.3±3.1% versus 30.9±4.3%). Overall, the data suggest that both processes (endogenous activation and bone marrow recruitment of c-kit+ cells) are involved with double-knockdown therapy.

Figure 4.
Figure 4.

Dual shRNA therapy increases activation of c-kit+ cardiac progenitor cells (CPCs). A, Immunostaining for c-kit and troponin markers in heart sections (c-kit, green; troponin, red; nuclei, blue) (DAPI, magnification 200×). Quantification of c-kit+ CPCs by flow cytometry (B) and bar graph analysis (C). The number of c-kit+ CPCs was analyzed from the excised periinfarct regions after enzymatic digestion into single-cell suspension. Data are presented as mean±SEM. Five mice per group. Abbreviations as inFigure 1. *P<0.05. **P<0.01 vs shScramble.

Ex Vivo Histological Analysis Confirmed In Vivo Functional Data

After imaging, animals were euthanized and their hearts explanted. Hematoxylin-eosin staining showed thicker heart wall size for the shPHD2+shFIH group than for the shScramble control (Figure 5A), confirming the positive functional data seen by MRI, echocardiography, and pressure-volume loop. Minicircle vector containing shPHD2+shFIH significantly decreased left ventricular scarring compared with shFIH and shScramble control (Figure 5B). Immunohistochemistry of the periinfarct region by CD31 staining also showed the highest neovascularization in the shPHD2+shFIH group (615±57 vessels/mm2) compared with the other 2 treated groups (shPHD2, 478±36 vessels/mm2; shFIH, 231±18 vessels/mm2), with the lowest neovascularization in the shScramble group (179±23 vessels/mm2) (P<0.05 compared with shFIH and shScramble; P<0.01 compared with shPHD2) (Figure 5C). To analyze the in vivo transfection efficiency of the minicircle vector, we injected minicircle GFP into the periinfarct zone of murine hearts. Cells were harvested 1 week later and stained with mouse cardiac troponin T and CD31 antibody for cardiac and endothelial cells, respectively. FACS analysis indicated that there were 10.1±1.4% GFP+/troponin T+ cells and 2.7±0.4% GFP+/CD31+ cells (data not shown). Overall, these data demonstrate that minicircle can efficiently transfect cardiac cells in vivo, which is consistent with the in vitro transfection data on HL-1, H9c2, and CPC shown inFigure 1A and online-only Data Supplement Figure IV.

Figure 5.
Figure 5.

Histological analysis of minicircle-based shRNA therapy. A, Representative histology of infarcted hearts injected with shScramble, shFIH, shPHD2, and shPHD2+shFIH at week 4 (hematoxylin-eosin, magnification ×5). B, Quantitative analysis of ventricular wall showed significant improvement in the double-knockdown group compared with shScramble control group. *P<0.05. **P<0.01 vs shScramble. C, Immunostaining for CD31 marker indicated significantly increased neovascularization in the double-knockdown group (staining with FITC and PI, magnification 100×). Five mice per group. Abbreviations as inFigure 1.

Double Knockdown Promotes Angiogenesis and HIF-1α Stabilization in Myocardial Tissues

To further elucidate the mechanisms of shPHD2 and shFIH therapy, we harvested the periinfarct area tissue by LCM and performed qPCR of angiogenesis genes at 2 weeks after MI (Figure 6A). The remote nonischemic tissue of the same heart was used as internal controls. FGF2, VEGF, FRT1, KDR, TGF, and PAI-1 genes showed significantly higher levels among shPHD2+shFIH and shPHD2 groups than the shFIH and shScramble groups (Figure 6B). To confirm the LCM qPCR results, we also performed western blots of HIF-1α protein levels. Infarcted hearts were harvested at weeks 1, 4, and 8 after single- and double-shRNA therapy. Quantitative analysis of the western blots demonstrated that HIF-1α protein levels were significantly higher in shPHD2 and shPHD2+shFIH-treated hearts compared to shScramble and shFIH alone starting at week 1 (data not shown). As the minicircle vector decayed over time, protein levels also decreased progressively from week 1 to week 8 as expected (online-only Data Supplement Figure IX). This may explain why there is no significant difference between LVEF measured at weeks 4 and 8. However, LVEF was significantly higher in both week 4 and week 8 in the double-knockdown group than in the shScramble group. At 14 days postinfarction, the double-knockdown group also had a significantly lower percentage of TUNEL-positive apoptotic cells in the periinfarct area than the shPHD2, shFIH, and shScramble groups (online-only Data Supplement Figure XII). Finally, 1 week after minicircle shRNA delivery, the double-knockdown group had higher HIF-2α protein expression in the periinfarct area (online-only Data Supplement Figure XIII). HIF-2α is another transcription factor that is stabilized in hypoxic tissue. Similar to HIF-1α, HIF-2α complex binds to HREs in the promoters of many genes involved in adaptation to the hypoxic environment. In addition, several endothelial cell-specific genes (Tie-2 and Flk-1) are exclusively regulated by HIF-2α.26

Figure 6.
Figure 6.

Activation of myocardial angiogenesis genes after dual shRNA therapy. A, Frozen slides for laser capture microdissection showing the experimental and control areas used to assess levels of angiogenic gene activation after shRNA therapy (no staining, magnification 50×). B, Quantitative polymerase chain reaction showed significant increase in expression of angiogenic genes in the double-knockdown group compared with the single-knockdown and shScramble control groups. Five mice per group. Abbreviations as inFigure 1.

Discussion

In this study, we describe a novel minicircle vector that mediates double shRNA knockdown of PHD2 and FIH, resulting in upregulation of the HIF-1α protein in a murine model of MI. The major findings can be summarized as follows: shRNA targeting PHD2+FIH 1 increases BMC homing to the myocardium; 2 activates endogenous c-kit+ CPCs; 3 promotes myocardial neoangiogenesis; 4 decreases cellular apoptosis; and, importantly, 5 improves cardiac function after MI. We demonstrated stable and efficient double knockdown of 2 hydroxylases using dual shRNAs inserted into novel, nonviral, minicircle vectors, resulting in upregulation of HIF-1α. Minicircle has several advantages over both viral-based and conventional plasmid vectors. Compared with viral vectors, minicircle has a better safety profile and allows larger expression cassette and possibly easier clinical translation because of simple, good-manufacturing practices. Compared with regular plasmid vectors, minicircle has a significantly higher level and longer duration of transgene expression both in vitro and in vivo.27

HIF-1α is a master transcriptional activator that mediates the physiological response to hypoxia. In response to cardiac hypoxia, BMCs in the peripheral blood home to the site of injury, and CPCs that reside in the myocardium are activated.28–31 These recruited cells can regenerate damaged tissue by differentiating into endothelial cells, smooth muscle cells, and cardiac myocytes.32 These cells also secrete angiogenic or antiapoptotic factors, which can improve the recovery of ischemic myocardium and the function of nonischemic regions.33,34 Mobilization of these cells is controlled by several genes, including VEGF, granulocyte macrophage colony-stimulating factor, FGF, insulin-like growth factor, erythropoietin, and stromal cell-derived factor-1; all of which are regulated by HIF-1α.3,35,36 For example, previous studies have shown that exogenous administration of granulocyte macrophage colony-stimulating factor mobilized endothelial progenitor cells from peripheral blood to the areas of ischemic injury in the hindlimb and myocardium, resulting in neovascularization.31,37 Similarly, activation of CPCs by injection of insulin-like growth factor has been shown to regenerate myocytes and induce vessel growth, leading to improvement in cardiac structure and function.25 Consistent with these findings, we demonstrated that higher levels of HIF-1α through double shPHD2 and shFIH knockdown improved BMC homing and survival as well as enhanced activation of endogenous c-kit+ CPCs in the injured myocardium, resulting in increased angiogenesis and improved cardiac function. We also monitored the spatiotemporal kinetics of exogenously administered BMC recruitment to the ischemic heart after dual shRNA therapy, which to our knowledge has not been previously reported.

Unlike other approaches using single-gene therapy (eg, VEGF, FGF, insulin-like growth factor), HIF-1α upregulation can harnesses more cardioprotective components of the ischemic reperfusion response. The shRNA dual therapy alters the posttranslational modification of HIF-1α, which prevents its degradation in normoxic environments and results in enhanced stem cell mobilization and increased angiogenesis. In addition to promoting potential myocyte regeneration and new vessel growth, HIF-1α may alter tissue metabolism and protect the myocardium against ischemic injury.38 These potential benefits have been demonstrated in studies using HIF-1α/VP16 hybrid gene and HIF-1α activator proteins.39,40

In summary, we demonstrate a novel therapeutic approach to preserve the myocardium by harnessing the normal physiological response to hypoxia. Inhibition of HIF-1α degradation by dual shPHD2 and shFIH therapy produced a robust early expression of HIF-1 protein, which in turn resulted in enhanced BMC homing, activation of endogenous CPCs, increased neoangiogenesis, and decreased cellular apoptosis, leading to improvement of cardiac function after MI. Taken together, these data suggest that double knockdown of PHD2 and FIH using safe nonviral minicircle vectors may provide a promising new therapy for ischemic heart failure.

Sources of Funding

This work was supported in part by grants from the National Institutes of Health HL093172, HL095571, HL099117, and EB009689 (to Dr Wu), American Heart Association Beginning Grant in Aid (to Dr Huang), and American College of Cardiology/General Electric Cardiovascular Young Investigator Grant (to Dr Nguyen).

Disclosures

None.

Footnotes

References

  1. 1.↵
    1. Writing Group Members,
    2. Lloyd-Jones D,
    3. Adams RJ,
    4. Brown TM,
    5. Carnethon M,
    6. Dai S,
    7. De Simone G,
    8. Ferguson TB,
    9. Ford E,
    10. Furie K,
    11. Gillespie C,
    12. Go A,
    13. Greenlund K,
    14. Haase N,
    15. Hailpern S,
    16. Ho PM,
    17. Howard V,
    18. Kissela B,
    19. Kittner S,
    20. Lackland D,
    21. Lisabeth L,
    22. Marelli A,
    23. McDermott MM,
    24. Meigs J,
    25. Mozaffarian D,
    26. Mussolino M,
    27. Nichol G,
    28. Roger VL,
    29. Rosamond W,
    30. Sacco R,
    31. Sorlie P,
    32. Stafford R,
    33. Thom T,
    34. Wasserthiel-Smoller S,
    35. Wong ND,
    36. Wylie-Rosett J,
    37. American Heart Association Statistics Committee and Stroke Statistics Subcommittee
    . Heart disease and stroke statistics–2010 update: a report from the American Heart Association. Circulation. 2010;121:e46–e215
    OpenUrlFREE Full Text
  2. 2.↵
    1. Kim M,
    2. Kini A,
    3. Sharma S
    . Refractory angina pectoris: mechanism and therapeutic options. J Am Coll Cardiol. 2002;39:923–934
    OpenUrlCrossRefPubMed
  3. 3.↵
    1. Beohar N,
    2. Rapp J,
    3. Pandya S,
    4. Losordo DW
    . Rebuilding the damaged heart: the potential of cytokines and growth factors in the treatment of ischemic heart disease. J Am Coll Cardiol. 2010;56:1287–1297
    OpenUrlCrossRefPubMed
  4. 4.↵
    1. Hiona A,
    2. Wu JC
    . Noninvasive radionuclide imaging of cardiac gene therapy: progress and potential. Nat Clin Pract Cardiovasc Med. 2008;5:S87–S95
    OpenUrlCrossRefPubMed
  5. 5.↵
    1. Ockaili R,
    2. Natarajan R,
    3. Salloum F,
    4. Fisher BJ,
    5. Jones D,
    6. Fowler AA III.,
    7. Kukreja RC
    . HIF-1 activation attenuates postischemic myocardial injury: role for heme oxygenase-1 in modulating microvascular chemokine generation. Am J Physiol Heart Circ Physiol. 2005;289:H542–H548
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    1. Shyu KG,
    2. Wang MT,
    3. Wang BW,
    4. Chang CC,
    5. Leu JG,
    6. Kuan P,
    7. Chang H
    . Intramyocardial injection of naked DNA encoding HIF-1alpha/VP16 hybrid to enhance angiogenesis in an acute myocardial infarction model in the rat. Cardiovasc Res. 2002;54:576–583
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    1. Lee J-W,
    2. Bae S-H,
    3. Jeong J-W,
    4. Kim S-H,
    5. Kim K-W
    . Hypoxia-inducible factor (HIF-1)alpha: its protein stability and biological functions. Exp Mol Med. 2004;36:1–12
    OpenUrlCrossRefPubMed
  8. 8.↵
    1. Dames S,
    2. Martinez-Yamout M,
    3. De Guzman R,
    4. Dyson HJ,
    5. Wright P
    . Structural basis for Hif-1 alpha /CBP recognition in the cellular hypoxic response. Proc Natl Acad Sci U S A. 2002;99:5271–5276
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    1. Willam C,
    2. Maxwell PH,
    3. Nichols L,
    4. Lygate C,
    5. Tian YM,
    6. Bernhardt W,
    7. Wiesener M,
    8. Ratcliffe PJ,
    9. Eckardt K-U,
    10. Pugh CW
    . HIF prolyl hydroxylases in the rat; organ distribution and changes in expression following hypoxia and coronary artery ligation. J Mol Cell Cardiol. 2006;41:68–77
    OpenUrlCrossRefPubMed
  10. 10.↵
    1. Fischer KM,
    2. Cottage CT,
    3. Wu W,
    4. Din S,
    5. Gude NA,
    6. Avitabile D,
    7. Quijada P,
    8. Collins BL,
    9. Fransioli J,
    10. Sussman MA
    . Enhancement of myocardial regeneration through genetic engineering of cardiac progenitor cells expressing Pim-1 kinase. Circulation. 2009;120:2077–2087
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    1. Fransioli J,
    2. Bailey B,
    3. Gude NA,
    4. Cottage CT,
    5. Muraski JA,
    6. Emmanuel G,
    7. Wu W,
    8. Alvarez R,
    9. Rubio M,
    10. Ottolenghi S,
    11. Schaefer E,
    12. Sussman MA
    . Evolution of the c-kit-positive cell response to pathological challenge in the myocardium. Stem Cells. 2008;26:1315–1324
    OpenUrlCrossRefPubMed
  12. 12.↵
    1. Meloni M,
    2. Caporali A,
    3. Graiani G,
    4. Lagrasta C,
    5. Katare R,
    6. Van Linthout S,
    7. Spillmann F,
    8. Campesi I,
    9. Madeddu P,
    10. Quaini F,
    11. Emanueli C
    . Nerve growth factor promotes cardiac repair following myocardial infarction. Circ Res. 2010;106:1275–1284
    OpenUrlAbstract/FREE Full Text
  13. 13.↵
    1. Ishikawa F,
    2. Yasukawa M,
    3. Lyons B,
    4. Yoshida S,
    5. Miyamoto T,
    6. Yoshimoto G,
    7. Watanabe T,
    8. Akashi K,
    9. Shultz LD,
    10. Harada M
    . Development of functional human blood and immune systems in NOD/SCID/IL2 receptor γ chain(null) mice. Blood. 2005;106:1565–1573
    OpenUrlAbstract/FREE Full Text
  14. 14.↵
    1. Ruan H,
    2. Su H,
    3. Hu L,
    4. Lamborn KR,
    5. Kan YW,
    6. Deen DF
    . A hypoxia-regulated adeno-associated virus vector for cancer-specific gene therapy. Neoplasia. 2001;3:255–263
    OpenUrlCrossRefPubMed
  15. 15.↵
    1. Brahimi-Horn C,
    2. Berra E,
    3. Pouyssegur J
    . Hypoxia: the tumor's gateway to progression along the angiogenic pathway. Trends Cell Biol. 2001;11:S32–S36
    OpenUrlCrossRefPubMed
  16. 16.↵
    1. Schroder K,
    2. Hertzog PJ,
    3. Ravasi T,
    4. Hume DA
    . Interferon-gamma: an overview of signals, mechanisms and functions. J Leukoc Biol. 2004;75:163–189
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    1. Brew K,
    2. Dinakarpandian D,
    3. Nagase H
    . Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys Acta. 2000;1477:267–283
    OpenUrlCrossRefPubMed
  18. 18.↵
    1. Acosta-Iborra B,
    2. Elorza A,
    3. Olazabal IM,
    4. Martin-Cofreces NB,
    5. Martin-Puig S,
    6. Miro M,
    7. Calzada MJ,
    8. Aragones J,
    9. Sanchez-Madrid F,
    10. Landazuri MO
    . Macrophage oxygen sensing modulates antigen presentation and phagocytic functions involving IFN-gamma production through the HIF-1 alpha transcription factor. J Immunol. 2009;182:3155–3164
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    1. Gerber SA,
    2. Pober JS
    . IFN-alpha induces transcription of hypoxia-inducible factor-1alpha to inhibit proliferation of human endothelial cells. J Immnol. 2008;181:1052–1062
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    1. Misra S,
    2. Fu AA,
    3. Rajan DK,
    4. Juncos LA,
    5. McKusick MA,
    6. Bjarnason H,
    7. Mukhopadhyay D
    . Expression of hypoxia inducible factor-1 alpha, macrophage migration inhibition factor, matrix metalloproteinase-2 and -9, and their inhibitors in hemodialysis grafts and arteriovenous fistulas. J Vasc Interv Radiol. 2008;19:252–259
    OpenUrlCrossRefPubMed
  21. 21.↵
    1. Peters CL,
    2. Morris CJ,
    3. Mapp PI,
    4. Blake DR,
    5. Lewis CE,
    6. Winrow VR
    . The transcription factors hypoxia-inducible factor 1alpha and Ets-1 colocalize in the hypoxic synovium of inflamed joints in adjuvant-induced arthritis. Arthritis Rheum. 2004;50:291–296
    OpenUrlCrossRefPubMed
  22. 22.↵
    1. Higgins DF,
    2. Kimura K,
    3. Bernhardt WM,
    4. Shrimanker N,
    5. Akai Y,
    6. Hohenstein B,
    7. Saito Y,
    8. Johnson RS,
    9. Kretzler M,
    10. Cohen CD,
    11. Eckardt KU,
    12. Iwano M,
    13. Haase VH
    . Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition. J Clin Invest. 2007;117:3810–3820
    OpenUrlCrossRefPubMed
  23. 23.↵
    1. Yang C,
    2. Shuiqing Z,
    3. Mingliang L
    . Effect of shRNA inhibiting HIF1alpha gene on TIMP1 expression in RPE cells. J Huazhong Univ Sci Technolog Med Sci. 2006;26:133–136
    OpenUrlCrossRefPubMed
  24. 24.↵
    1. Oh H,
    2. Bradfute SB,
    3. Gallardo TD,
    4. Nakamura T,
    5. Gaussin V,
    6. Mishina Y,
    7. Pocius J,
    8. Michael LH,
    9. Behringer RR,
    10. Garry DJ,
    11. Entman ML,
    12. Schneider MD
    . Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci U S A. 2003;100:12313–12318
    OpenUrlAbstract/FREE Full Text
  25. 25.↵
    1. Rota M,
    2. Padin-Iruegas ME,
    3. Misao Y,
    4. De Angelis A,
    5. Maestroni S,
    6. Ferreira-Martins J,
    7. Fiumana E,
    8. Rastaldo R,
    9. Arcarese ML,
    10. Mitchell TS,
    11. Boni A,
    12. Bolli R,
    13. Urbanek K,
    14. Hosoda T,
    15. Anversa P,
    16. Leri A,
    17. Kajstura J
    . Local activation or implantation of cardiac progenitor cells rescues scarred infarcted myocardium improving cardiac function. Circ Res. 2008;103:107–116
    OpenUrlAbstract/FREE Full Text
  26. 26.↵
    1. Hu C-J,
    2. Wang L-Y,
    3. Chodosh LA,
    4. Keith B,
    5. Simon MC
    . Differential Roles of hypoxia-inducible factor 1α (HIF-1α and HIF-2α) in hypoxic gene regulation. Mol Cell Biol. 2003;23:9361–9374
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    1. Chen Z,
    2. He C,
    3. Kay MA
    . Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo. Human Gene Therapy. 2005;16:126–131
    OpenUrlCrossRefPubMed
  28. 28.↵
    1. Asahara T,
    2. Masuda H,
    3. Takahashi T,
    4. Kalka C,
    5. Pastore C,
    6. Silver M,
    7. Kearne M,
    8. Magner M,
    9. Isner JM
    . Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res. 1999;85:221–228
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    1. Hattori K,
    2. Heissig B,
    3. Wu Y,
    4. Dias S,
    5. Tejada R,
    6. Ferris B,
    7. Hicklin DJ,
    8. Zhu Z,
    9. Bohlen P,
    10. Witte L,
    11. Hendrikx J,
    12. Hackett NR,
    13. Crystal RG,
    14. Moore MA,
    15. Werb Z,
    16. Lyden D,
    17. Rafii S
    . Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1(+) stem cells from bone-marrow microenvironment. Nat Med. 2002;8:841–849
    OpenUrlCrossRefPubMed
  30. 30.↵
    1. Kalka C,
    2. Masuda H,
    3. Takahashi T,
    4. Gordon R,
    5. Tepper O,
    6. Gravereaux E,
    7. Pieczek A,
    8. Iwaguro H,
    9. Hayashi SI,
    10. Isner JM,
    11. Asahara T
    . Vascular endothelial growth factor(165) gene transfer augments circulating endothelial progenitor cells in human subjects. Circ Res. 2000;86:1198–1202
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    1. Orlic D,
    2. Kajstura J,
    3. Chimenti S,
    4. Limana F,
    5. Jakoniuk I,
    6. Quaini F,
    7. Nadal-Ginard B,
    8. Bodine DM,
    9. Leri A,
    10. Anversa P
    . Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A. 2001;98:10344–10349
    OpenUrlAbstract/FREE Full Text
  32. 32.↵
    1. Jackson KA,
    2. Majka SM,
    3. Wang H,
    4. Pocius J,
    5. Hartley CJ,
    6. Majesky MW,
    7. Entman ML,
    8. Michael LH,
    9. Hirschi KK,
    10. Goodell MA
    . Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest. 2001;107:1395–1402
    OpenUrlCrossRefPubMed
  33. 33.↵
    1. Gnecchi M,
    2. He H,
    3. Liang OD,
    4. Melo LG,
    5. Morello F,
    6. Mu H,
    7. Noiseux N,
    8. Zhang L,
    9. Pratt RE,
    10. Ingwall JS,
    11. Dzau VJ
    . Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nat Med. 2005;11:367–368
    OpenUrlCrossRefPubMed
  34. 34.↵
    1. Kamihata H,
    2. Matsubara H,
    3. Nishiue T,
    4. Fujiyama S,
    5. Tsutsumi Y,
    6. Ozono R,
    7. Masaki H,
    8. Mori Y,
    9. Iba O,
    10. Tateishi E,
    11. Kosaki A,
    12. Shintani S,
    13. Murohara T,
    14. Imaizumi T,
    15. Iwasaka T
    . Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation. 2001;104:1046–1052
    OpenUrlAbstract/FREE Full Text
  35. 35.↵
    1. Cheng M,
    2. Zhou J,
    3. Wu M,
    4. Boriboun C,
    5. Thorne T,
    6. Liu T,
    7. Xiang Z,
    8. Zeng Q,
    9. Tanaka T,
    10. Tang YL,
    11. Kishore R,
    12. Tomasson MH,
    13. Miller RJ,
    14. Losordo DW,
    15. Qin G
    . CXCR4-mediated bone marrow progenitor cell maintenance and mobilization are modulated by c-kit activity. Circ Res. 2010;107:1083–1093
    OpenUrlAbstract/FREE Full Text
  36. 36.↵
    1. Arany Z,
    2. Foo SY,
    3. Ma Y,
    4. Ruas JL,
    5. Bommi-Reddy A,
    6. Girnun G,
    7. Cooper M,
    8. Laznik D,
    9. Chinsomboon J,
    10. Rangwala SM,
    11. Baek KH,
    12. Rosenzweig A,
    13. Spiegelman BM
    . HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1alpha. Nature. 2008;451:1008–1012
    OpenUrlCrossRefPubMed
  37. 37.↵
    1. Takahashi T,
    2. Kalka C,
    3. Masuda H,
    4. Chen D,
    5. Silver M,
    6. Kearney M,
    7. Magner M,
    8. Isner JM,
    9. Asahara T
    . Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med. 1999;5:434–438
    OpenUrlCrossRefPubMed
  38. 38.↵
    1. Kim JW,
    2. Tchernyshyov I,
    3. Semenza GL,
    4. Dang CV
    . HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab. 2006;3:177–185
    OpenUrlCrossRefPubMed
  39. 39.↵
    1. Li J,
    2. Post M,
    3. Volk R,
    4. Gao Y,
    5. Li M,
    6. Metais C,
    7. Sato K,
    8. Tsai J,
    9. Aird W,
    10. Rosenberg RD,
    11. Hampton TG,
    12. Sellke F,
    13. Carmeliet P,
    14. Simons M
    . PR39, a peptide regulator of angiogenesis. Nat Med. 2000;6:49–55
    OpenUrlCrossRefPubMed
  40. 40.↵
    1. Vincent KA,
    2. Shyu K-G,
    3. Luo Y,
    4. Magner M,
    5. Tio RA,
    6. Jiang C,
    7. Goldberg MA,
    8. Akita GY,
    9. Gregory RJ,
    10. Isner JM
    . Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF-1α/VP16 hybrid transcription factor. Circulation. 2000;102:2255–2261
    OpenUrlAbstract/FREE Full Text
View Abstract

This Issue

Circulation
September 13, 2011, Volume 124, Issue 11 suppl 1

Jump to

Article Tools

Share this Article

  • Email
  • Double Knockdown of Prolyl Hydroxylase and Factor-Inhibiting Hypoxia-Inducible Factor With Nonviral Minicircle Gene Therapy Enhances Stem Cell Mobilization and Angiogenesis After Myocardial Infarction
    Mei Huang, Patricia Nguyen, Fangjun Jia, Shijun Hu, Yongquan Gong, Patricia E. de Almeida, Li Wang, Divya Nag, Mark A. Kay, Amato J. Giaccia, Robert C. Robbins and Joseph C. Wu
    Circulation. 2011;124:S46-S54, originally published September 12, 2011
    https://doi.org/10.1161/CIRCULATIONAHA.110.014019
    del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Related Articles

Cited By...

Subjects