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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Circulation. Author manuscript; available in PMC 2012 September 13.
Published in final edited form as:
PMCID: PMC3181087
NIHMSID: NIHMS318651

Double Knockdown of Prolyyl Hydroxylase and Factor Inhibiting HIF with Non-Viral Minicircle Gene Therapy Enhances Stem Cell Mobilization and Angiogenesis After Myocardial Infarction

Mei Huang, PhD,1,2 Patricia Nguyen, MD,1 Fangjun Jia, PhD,2 Shijun Hu, PhD,2 Yongquan Gong, MD, PhD,1,3 Patricia E. de Almeida, PhD,1,2 Li Wang, MD, PhD,1,2 Divya Nag,1 Mark A. Kay, MD, PhD,4 Amato J Giaccia, PhD,5 Robert C. Robbins, MD,3,6 and Joseph C. Wu, MD, PhD1,2,6

Abstract

Background

Under normoxic conditions, hypoxia inducible factor-1 alpha (HIF-1α) is rapidly degraded by two hydroxylases, prolyl hydroxylase (PHD) and factor inhibiting HIF-1 (FIH). Because HIF-1α mediates the cardioprotective response to ischemic injury, its up-regulation 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 sequences for inhibiting PHD isoenzyme 2 (shPHD2) and FIH (shFIH) were inserted into novel non-viral minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit+ cardiac progenitor cells (CPCs) demonstrated higher expression of angiogenesis factors in the double knockdown group compared to the single knockdown and shScramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially following LAD ligation in adult FVB mice (n=60). Functional studies using magnetic resonance imaging (MRI), echocardiography, and pressure-volume (PV) loops showed greater improvement in cardiac function in the double knockdown group. To assess mechanism(s) 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 (FACS) 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 (LCM) analysis confirmed up-regulation of HIF-1α protein and angiogenesis genes, respectively.

Conclusions

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

Keywords: hypoxia inducible factor, prolyl hydroxylases, factor inhibiting HIF, RNA interference, minicircle, ischemic heart disease, laser capture microdissection, “mousenized” mouse model

INTRODUCTION

Coronary artery disease (CAD) 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 (such as 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-1 alpha (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 isoenzymes (PHD1-3) 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 co-activator 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 sequences inserted into a novel, non-viral minicircle vector to inhibit PHD isoenzyme 2 (shPHD2) and FIH (shFIH), which then prevented HIF-1α degradation. We found that up-regulation of HIF-1α enhances stem cell mobilization (via molecular imaging and fluorescence-activated cell sorting) and increases myocardial angiogenesis (via laser capture microdissection and immunohistochemistry), leading to significant improvement in cardiac function following myocardial infarction (MI).

MATERIALS AND 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 PHD2 gene and 8 for FIH gene. The targeting sequences are shown in Supplemental Figure 1. 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% fetal bovine serum. Mouse HL-1 atrial myocytes were cultured in 10% fetal bovine serum (BioWhittaker), 10 μg/ml insulin (Life Technologies), 50 μg/ml endothelial cell growth supplement (Upstate Biotechnology), 1 μM retinoic acid (Sigma), 10 μM norepinephrine (Sigma), 100 units/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 fetal bovine serum (Gibco), Dulbecco's modified Eagle's medium, Ham's F-12 medium, insulin-transferrin-selenium, leukemia inhibitory factor (10 ng/ml), basic fibroblast growth factor (10 ng/ml), epidermal growth factor (20 ng/ml), and penicillin-streptomycin-glutamine. Minicircle vectors with shPHD2 and shFIH driven by H1 promoter were co-transfected into mouse C2C12, HL-1, and CPC using lipofectamine 2000 (Invitrogen) with the plasmid pHRE-SV40-FLuc as control for determining knockdown efficiency (Supplemental Figure 1). After minicircle shRNA transfection, cells were cultured for 1 day before being subjected to hypoxia condition with 5% CO2, 1% O2, and 94% N2 at 37°C for 48 hours.

Quantitative polymerase chain reaction (q-PCR) 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 PCR was used to compare the expression of angiogenic genes (bFGF, VEGF, FLT, KDR, TGF, and 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 Biosystem (ABI). PCR reactions were performed on the ABI 7900HT system.

Angiogenesis cytokine array following hypoxia exposure

At the end of the hypoxic treatment, the supernatant was collected for angiogenesis cytokine array (Panomics). In two 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. Inter array normalization was performed by using positive control spots (eight 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 myocardial infarction (MI)

Ligation of the mid left anterior descending (LAD) artery was performed in adult female FVB mice (Charles River Laboratories, Wilmington, MA) by a single experienced microsurgeon (YG). Survival rate for each surgery group was between 80%-90%. MI was confirmed by myocardial blanching and EKG 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 peri-infarct zone. Study protocols were approved by the Stanford Animal Research Committee.

Activation of endogenous c-kit+ cardiac progenitor cells 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 (Perkin Elmer, Waltham, MA) for 1 hour after which goat anti-mouse c-kit antibody (R&D systems) was added (1:100) overnight at 4°C. Slides were washed in 1M Tris/NaC1 followed by secondary antibody incubation for 2 hours at room temperature.11 For fluorescence activated cell sorting (FACS) of c-kit+ CPCs in the heart (n=5 mice per group), we excised the peri-infarct zone and digested it using 0.1% collagenase IV into single cell suspension. FACS was performed using c-kit (APC-Cy7 conjugated, BD Biosciences, Oxford, UK) antibody as previously described.12

Creation of bone marrow chimeric mouse model

In order 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 from C57BL/6 mice (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, 2-3 day old NOD/SCID IL-2Rg (-/-) pups received myeloablative treatment consisting of 100 cGy gamma irradiation. Sixteen hours later, pups received an intra-hepatic injection of 1×106 lineage negative bone marrow cells isolated from adult C57BL/6 mice. Six to eight weeks later, hematopoietic engraftment was confirmed by detecting engraftment of donor-derived (H2kb+) multi-lineage immune cells in peripheral blood of mousenized mice via FACS. Once donor cell engraftment was confirmed, the “mousenized” mice underwent LAD ligation followed by injection of shPHD2 + shFIH versus shScramble into the peri-infarct zone (n=3 mice per group). Cells harvested from the peri-infarct zone were stained with Allophycocyanin (APC)-conjugated c-kit and R-Phycoerythrin (PE)-conjugated H2kbantibodies. Donor-derived c-kit cells were identified by FACS as c-kit+ and H2kb+.

Analysis of angiogenesis genes using laser capture microdissection (LCM) and RNA extraction

Murine hearts were removed after perfusion with 20 ml phosphate buffer saline (PBS), embedded in OCT, and immediately frozen in liquid nitrogen. Seven thick tissue sections of left ventricle were prepared on a polyethylene napthalate (PEN) membrane coated slides (MicroDissect GmbH, Leica, Germany). For laser microdissection, slides were thawed briefly and air-dried 5 min 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, Leica, Germany). The dissected tissues were placed on the caps of micro-centrifuge 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 one 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 of ANOVA. ANOVA (one tail, equal variance) with post-hoc testing was used to analyze qPCR, angiogenesis cytokine array, and myocardial perfusion. Repeated measures of ANOVA were used to analyze MRI, bioluminescence imaging, and echocardiographic data. Differences were considered significant at P-values of <0.05. Unless specified, data are expressed as mean ± standard deviation.

RESULTS

In vitro characterization of shFIH and shPHD2 double knockdown

We measured activation of HIF-1α and subsequent up-regulation of angiogenesis genes after hydroxylase inhibition using shRNAs under normoxic and hypoxic conditions. Plasmid pHRE-SV40-FLuc is a hypoxia sensing 5xHRE-SV40 promoter driving FLuc cassette. The 5 copies of hypoxia response element (5xHRE) 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 p/sec/cm2/sr) had significantly higher FLuc bioluminescence signals compared to cells transfected with shPHD2 (3.41×105±57,184 p/sec/cm2/sr), shFIH (4.48×104±4,513 p/sec/cm2/sr), and shScramble control (2.86×104±1,934 p/sec/cm2/sr), indicating increased binding of 5xHRE-SV40 promoter by HIF-1α following 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 hypoxia responsive elements (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 Supplemental Figure 2.

Figure 1
In vitro characterization of shPHD2 + shFIH knockdown

To quantify luciferase activity, we lysed the cells and determined the luminescence activity normalized to protein concentration (Supplemental Figure 3). 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 were also transfected by minicircle shRNA and pHRE-SV40-FLuc. Comparable results were also observed in these two cell types (Supplemental Figure 4).

To confirm the pHRE-SV40-FLuc imaging signals, mRNA was isolated and q-PCR was performed for detection of HIF-1α and downstream angiogenesis genes. As shown in Figure 1B, relative expression of six genes related to angiogenesis (e.g., 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 since 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 if up-regulation of HIF-1α protein via shRNA knockdown of PHD2 and FIH can also exert similar effects, supernatant from transfected C2C12 cells was used for angiogenesis assays. Figures 1C-D demonstrated significant up-regulation of several angiogenesis activators (e.g., FGFα, IL-6, Leptin, VEGF, TNFα, and TGFα) following double knockdown.

Interestingly, both IFNγ and TIMP1 were also up-regulated in the double knockdown group (Figure 1D). IFNγ is a soluble cytokine, which has anti-viral, immuno-regulatory, and anti-tumor properties.16 In contrast, TIMP1 (tissue inhibitor metalloproteinases) is a 28 kD 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 TIMP1 after transfection of C2C12 cells with a minicircle carrying the following genes: 1) HIF-1α [positive control], 2) an inhibitor of HIF-1α [siHIF1α], 3) shFIH and shPHD2 [double knockdown], and 4) blank vector [negative control]. We found that siRNA inhibition of HIF-1α led to significantly lower TIMP1 and IFNγ expression levels. In contrast, over-expression of minicircle HIF-1α and minicircle shPHD2 + shFIH led to significantly higher levels of TIMP1 (P<0.001 for both) and IFNγ (P<0.05 for both) compared to control (Supplemental Figure 5). Consistent with these findings, Yang et al. demonstrated that shRNA inhibition of HIF-1α can dramatically decrease the expression of TIMP1 mRNA expression and protein levels.23 Taken together, these data confirm that similar to physiologic 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 week 4 and 8 following MI, significant improvement in LVEF was seen in the double knockdown group compared to other groups (Figure 2). The shPHD2 knockdown group also had improvement in LVEF compared to shScramble group in week 4 and 8. These findings were also confirmed by serial echocardiograms (Supplemental Figure 6). In addition, invasive hemodynamic parameters showed that the increase in LVEF was associated with lower left ventricular end-diastolic volume (LVEDV) and left ventricular endsystolic volume (LVESV) in double shRNA knockdown compared with shScramble (Supplemental Figure 7). Finally, shPHD2 + shFIH treatment improved myocardial perfusion as determined by measuring the distribution of fluorescent microspheres within myocardial tissues after injection into the LV cavity (Supplemental Figure 8). These results also coincide with observed differences in HIF-1α expression at week 4 and 8 in the shPHD2 + shFIH group compared to the other groups (Supplemental Figure 9).

Figure 2
Evaluation of heart function after minicircle shRNA injection

Double knockdown enhances bone marrow cell homing to the injured myocardium

Previous studies have demonstrated 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 GFP and FLuc and intravenously injected into syngeneic FVB mice with MI and shRNA therapy. Double knockdown significantly increased the number of BMC recruitment to the myocardium from days 1 to 28 as assessed by longitudinal bioluminescence imaging (Figure 3A-B). GFP and troponin-T double staining also demonstrated more GFP+ cells in the shPHD2 + shFIH group (Supplemental Figure 10).

Figure 3
Dual shRNA therapy increases recruitment of bone marrow cells after MI

Double knockdown activates c-kit+ cardiac progenitor cells

Next we assessed whether double knockdown can also induce proliferation of c-kit+ CPCs. Immunostaining showed higher presence of c-kit+ CPCs near the peri-infarct area in the shPHD2 + shFIH group (Figure 4A). To validate the histological data, we then performed FACS analysis for c-kit+ CPCs following excision of the peri-infarct tissue and digestion into single cell suspension. FACS confirmed the number of c-kit+ CPCs was significantly greater in the shPHD2 + shFIH group compared to shScramble control (Figure 4B-C). 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 bone marrow cells from C57BL/6 murine (H2kb). Bone marrow cells from C57BL/6 strain were identified by the expression of H2kb MHC Class-I haplotype. Following 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 compared to control (2.46±0.18% vs. 1.05±0.06%; P<0.01). Similarly, there was greater endogenous cell activation (H2kb-/c-kit+) in the treatment group compared to control (1.06±0.06% vs. 0.50±0.09%; P<0.05) (Supplemental Figure 11). Of the number of ckit+ 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% vs. 30.9%±4.3%). Overall, the data suggest both processes (endogenous activation and bone marrow recruitment of ckit+ cells) are involved with double knockdown therapy.

Figure 4
Dual shRNA therapy increases activation of c-kit+ cardiac progenitor cells

Ex vivo histological analysis confirmed in vivo functional data

Following imaging, animals were sacrificed and their hearts explanted. H&E staining showed thicker heart wall size for the shPHD2 + shFIH group compared to shScramble control (Figure 5A), confirming the positive functional data seen by MRI, echocardiography, and PV-loop. Minicircle vector containing shPHD2 + shFIH significantly decreased left ventricular scarring compared to shFIH and shScramble control (Figure 5B). Immunohistochemistry of the peri-infarct region by CD31 staining also showed the highest neovascularization in the shPHD2 + shFIH group (615±57 vessels/mm2) compared to the other two treated groups, shPHD2 (478±36 vessels/mm2) and shFIH (231±18 vessels/mm2), with the lowest neovascularization in the shScramble group (179±23 vessels/mm2) (*P<0.05 compare to shFIH and shScramble; **P<0.01 compared to shPHD2) (Figure 5C). To analyze the in vivo transfection efficiency of the minicircle vector, we injected minicircle GFP into the peri-infarct zone of murine hearts. Cells were harvested 1 week later and were stained with mouse cTnT and CD31 antibody for cardiac and endothelial cells, respectively. FACS analysis indicated that there were 10.1±1.4% GFP+/TnT+ 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 in Figure 1A and Supplemental Figure 4.

Figure 5Figure 5
Histological analysis of minicircle based shRNA therapy

Double knockdown promotes angiogenesis and HIF-1α stabilization in myocardial tissues

To further elucidate the mechanism(s) of shPHD2 and shFIH therapy, we harvested the peri-infarct area tissue by laser capture microdissection (LCM) and performed q-PCR of angiogenesis genes at 2 weeks post MI (Figure 6A). The remote non-ischemic 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 compared to shFIH and shScramble groups (Figure 6B). To confirm the LCM q-PCR results, we also performed Western blots of HIF-1α protein levels. Infarcted hearts were harvested at week 1, 4, and 8 following 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 decays over time, protein levels also decreased progressively from week 1 to week 8 as expected (Supplementary Figure 9). This may explain why there is no significant difference between LVEF measured at week 4 and 8. However, LVEF was significantly higher in both week 4 and 8 in the double knockdown group compared to the shScramble group. At 14 days post-infarction, the double knockdown group also had a significantly lower percentage of TUNEL-positive apoptotic cells in the peri-infarct area compared to shPHD2, shFIH, and shScramble groups (Supplemental Figure 12). Finally, 1 week after minicircle shRNA delivery, the double knockdown group had higher HIF-2α protein expression in the peri-infarct area (Supplemental Figure 13). HIF-2α is another transcription factor, which is stabilized in hypoxic tissue. Similar to HIF-1α, HIF-2α complex binds to hypoxia-response elements (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
Activation of myocardial angiogenesis genes after dual shRNA therapy

DISCUSSION

In this study, we describe a novel minicircle vector mediating double shRNA knockdown of PHD2 and FIH, resulting in up-regulation of HIF-1α protein in a murine model of myocardial infarction. The major findings can be summarized as follows: (1) shRNA targeting PHD2 + FIH increases bone marrow cell homing to the myocardium, (2) activates endogenous c-kit+ cardiac progenitor cells, (3) promotes myocardial neoangiogenesis, (4) decreases cellular apoptosis, and (5) importantly, improves cardiac function following MI. We demonstrated stable and efficient double knockdown of two hydroxylases using dual shRNAs inserted into novel non-viral minicircle vectors, resulting in up-regulation of HIF-1α. Minicircle has several advantages over both viral-based and conventional plasmid vectors. Compared to viral vectors, minicircle has a better safety profile, allows larger expression cassette, and possibly easier clinical translation due to simple good manufacturing practices. Compared to 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 physiologic response to hypoxia. In response to cardiac hypoxia, bone marrow cells in the peripheral blood home to the site of injury and cardiac progenitor cells 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 anti-apoptotic factors, which can improve the recovery of ischemic myocardium and the function of non-ischemic regions.33, 34 Mobilization of these cells is controlled by several genes, including vascular endothelial growth factor (VEGF), granulocyte macrophage-colony stimulating factor (GM-CSF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), erythropoietin, and stromal cell derived factor-1 (SDF-1); all of which are regulated by HIF-1α.3, 35, 36 For example, previous studies have shown that exogenous administration of GM-CSF 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 IGF 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 via 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 following dual shRNA therapy, which to our knowledge has not been previously reported.

Unlike other approaches using single gene therapy (e.g., VEGF, FGF, IGF), HIF-1α up-regulation can harnesses more cardio-protective components of the ischemic reperfusion response. shRNA dual therapy alters the post-translational 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 physiologic 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 following MI. Taken together, these data suggest that double knockdown of PHD2 and FIH using safe non-viral minicircle vectors may provide a promising new therapy for ischemic heart failure.

Supplementary Material

Acknowledgments

Funding sources. This work was supported in part by grants from NIH HL093172, HL095571 (JCW), American Heart Association Beginning Grant in Aid (MH), and American College of Cardiology/General Electric Cardiovascular Young Investigator Grant (PN).

Footnotes

Conflict of Interest Disclosures: None

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