Autologous Transplantation of Endothelial Progenitor Cells Genetically Modified by Adeno-Associated Viral Vector Delivering Insulin-Like Growth Factor-1 Gene After Myocardial Infarction

Abstract

The regenerative potential of bone marrow-derived endothelial progenitor cells (EPCs) has been adapted for the treatment of myocardial and limb ischemia via ex vivo expansion. We sought to enhance EPC function by the efficient genetic modification of EPCs in a rat model of myocardial infarction. Peripheral blood EPCs were expanded and transduced, using adeno-associated virus (AAV). AAV-mediated EPC transduction efficacy was 23 ± 1.2%, which was improved by 4.0- to 7.2-fold after pretreatment with the tyrosine kinase inhibitor genistein. Adult rats (n = 7 in each group) underwent myocardial infarction by left anterior descending coronary artery occlusion, and received autologous EPCs transduced by AAV-IGF-1 or AAV-lacZ into the periinfarct area. Echocardiography demonstrated that cardiac function in the IGF-1-EPC group was significantly improved compared with the lacZ-EPC control group 12 weeks after myocardial infarction. In addition, IGF-1-expressing EPCs led to reduced cardiac apoptosis, increased cardiomyocyte proliferation, and increased numbers of capillaries in the periinfarct area. AAV expression was limited to the targeted heart region only. Pretreatment with genistein markedly improved AAV transduction of EPCs. IGF-1-expressing EPCs exhibit favorable cell-protective effects with tissue-limited expression in rat heart postinfarction.

Introduction

T he plasticity of stem and progenitor cells has attracted great interest for regenerative medicine to treat inherited and acquired diseases. The regenerative potential of bone marrow-derived endothelial progenitor cells (EPCs) has been demonstrated to improve cardiac function not only in animal studies (Kawamoto et al., 2001, 2003) but also in clinical trials (Losordo et al., 2007). In these animal models investigators have often performed ex vivo expansion of EPCs followed by incorporation into foci of neovascularization. Physiological evidence of neovascular function (Kawamoto et al., 2001, 2003) was noted, which resulted in improved myocardial function or a higher rate of limb salvage. A further refinement of this approach would involve enhancement of EPC functions after efficient genetic modification. Although EPCs have been genetically modified with naked DNA, liposomes, and adenoviral vectors, none of these approaches has achieved long-term gene expression.

Adeno-associated viral (AAV) vectors, which are derived from a nonpathogenic human parvovirus, have been widely applied as vectors for systemic and local gene delivery (Manno et al., 2006; Aiuti et al., 2007; Van Vliet et al., 2008). The physical stability of AAV makes it advantageous for in vivo use, and transgene expression can persist long term in a wide range of tissues including heart muscle (Aikawa et al., 2002; Inagaki et al., 2006; Liu et al., 2006; Van Vliet et al., 2008). In addition, we have demonstrated that AAV vectors are capable of achieving safe and efficient transduction of bone marrow cells without abolishing cell function when used in in vitro and in vivo models (Iwakura et al., 2005; Chng et al., 2007).

Insulin-like growth factor (IGF)-1 is a 7.6-kDa polypeptide growth factor expressed by many cells and tissues during embryonic and postnatal development (Liu and LeRoith, 1999; Khan et al., 2002; Delafontaine et al., 2004). IGF-1 influences various biological processes through binding to a membrane–anchored receptor (IGF-1R), although at higher concentrations, IGF-1 can also activate the insulin receptor (Khan et al., 2002). The in vivo action of IGF-1 is modulated by a family of circulating IGF-binding proteins (Delafontaine et al., 2004). IGF-1 is secreted from the liver in response to growth hormone (GH) and promotes cellular proliferation in bone, muscle, fat, and other tissues (Liu and LeRoith, 1999; Khan et al., 2002; Delafontaine et al., 2004). Human and murine IGF-1 deficiency causes severe intrauterine growth retardation, perinatal lethality, postnatal growth retardation, and delayed development in a variety of organs (Liu and LeRoith, 1999), indicating that IGF-1 is a key regulator of cell development. Overexpression of IGF-1 attenuates myocyte necrosis and apoptosis after infarction in a transgenic mouse model (Li et al., 1997) and rescues cardiac myocytes from apoptosis in dilated cardiomyopathy (Welch et al., 2002). It has also been reported that overexpression of IGF-1 reduces cardiomyocyte senescence in a transgenic mouse model (Torella et al., 2004) and mice overexpressing IGF-1 exhibit less cardiomyocyte atrophy after ischemia (Schulze et al., 2005). Taken together, IGF-1 is a pleiotropic growth factor that induces myocyte proliferation and regeneration in heart development and injury. We have reported that local IGF-1 overexpression markedly improved cardiac function after myocardial infarction (MI) through direct AAV injection to heart muscle (Dobrucki et al., 2010).

The aims of this study were to further develop cardiac therapies, using genetically modified peripheral blood-derived EPCs. We sought to investigate whether transplantation of EPCs expressing IGF-1 after ex vivo AAV gene transfer improved cardiac function in rats post-MI.

Materials and Methods

Materials

Genistein, hydroxyurea (HU), N-acetyl-l-leucyl-l-leucyl-l-norleucinal (LLnL), and soluble yellow tetrazolium MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were obtained from Sigma-Aldrich (St. Louis, MO).

AAV vector production

Standard recombinant DNA approaches were used to incorporate the human IGF-1 cDNA (NCBI database, accession number X00173) open reading frame under the control of the cytomegalovirus (CMV) promoter as previously described (Kusano et al., 2007). Briefly, human IGF-1 cDNA was amplified by polymerase chain reaction (PCR) with the following specific primers: 5′-CCGAATTCTTCAGAAGCAATGGGA-3′ and 5′-CGGGATCCGTCTTCCTACATCCTG-3′, from a human gene clone (clone ID 984882, cat. no. 97002RG; Invitrogen Life Technologies, Carlsbad, CA) (Dobrucki et al., 2010). AAV vectors of serotype 2 were prepared at a titer of 1 × 10¹² vector genomes/ml as shown previously (Kusano et al., 2007).

AAV transduction and detection

Mononuclear cells were isolated from rat peripheral blood by density-gradient centrifugation, and then cultured in endothelial cell (EC) basal medium-2 (Clonetics EBM-2; Lonza Walkersville, Walkersville, MD) for 7 days. The majority of these ex vivo-expanded cells were of endothelial lineage and, as such, they constituted the ex vivo-expanded EPC-enriched fraction (Kawamoto et al., 2001). EPCs were transduced with AAV-CMV-lacZ (titer, 3 × 10³ genomes per cell) or AAV-CMV-IGF-1, fixed in by 4% paraformaldehyde (PFA) 1 week later, and stained for β-galactosidase (β-Gal) activity (Aikawa et al., 2002). Quantification of β-Gal activity in these cells was performed with a mammalian β-galactosidase assay kit (Pierce/Thermo Fisher Scientific, Waltham, MA).

To detect the AAV genome, total DNA was extracted from tissues, using a Gentra Puregene DNA isolation kit (Qiagen, Valencia, CA). A fragment of the AAV genome (286 bp) was amplified, using a forward primer within the inverted terminal repeat (ITR) region and a reverse primer within the CMV promoter as described previously (Iwakura et al., 2005).

RT-PCR for mRNA expression

Total RNA was extracted from heart tissue with RNA STAT (Tel-Test, Friendswood, TX) according to the manufacturer's instructions. First-strand cDNA was generated with a TaqMan MultiScribe reverse transcription kit (Applied Biosystems, Foster City, CA) primed with a mix of oligo(dT) and random hexamers. Gene expression was determined by TaqMan real-time (RT) quantitative PCR on a 7300 sequence detection system (Applied Biosystems), using TaqMan PCR master mix (Applied Biosystems). TaqMan primer–probe sets to detect IGF-1 expression (Biosearch Technologies, Novato, CA) were designed with Primer Express software (Applied Biosystems) with the following sequences: forward, 5′-CCATGTCCTCCTCGCATCTC; reverse, 5′-CGTGGCAGAGCTGGTGAAG; probe, 5′-ACCTGGCGCTGTGCCTGCTCA. PCR conditions were as follows: hold for 2 min at 50°C, 10 min at 95°C, followed by a two-step PCR for 40 cycles of 95°C for 15 sec and 60°C for 60 sec with fluorescence monitoring at the end of each elongation step. Relative mRNA expression of target genes was calculated by the comparative C _T method (Kusano et al., 2007). All target sequences were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in multiplexed reactions performed in duplicate. Differences in C _T values were calculated for each target mRNA by subtracting the mean value of GAPDH (Kusano et al., 2007).

Quantification of cell number, proliferation, and apoptosis

The MTT-formazan assay (Vistica et al., 1991) was used to assess the metabolic activity of cultured cells according to the instructions of the manufacturer (Sigma-Aldrich). The optical density of the solution was measured at 545 nm with a microwell plate reader. Rat EPCs transduced with AAV were cultured with neonatal rat cardiomyocytes (1:4 ratio) for 7 days (Yoon et al., 2005). For detection of apoptosis, the cells were first probed with an anti-α-actinin antibody (cat. no. A7811; Sigma-Aldrich). Nuclear staining for DNA fragmentation was performed by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method (cat. no. 11684795910; Roche Molecular Biochemicals, Indianapolis, IN). Bromodeoxyuridine (BrdU) was detected with sheep anti-BrdU antibody (diluted 1:50) (cat. no. M20105S; BIODESIGN International, Saco, ME) followed by streptavidin–fluorescein isothiocyanate (FITC) (diluted 1:100) (cat. no. SA-5001; Vector Laboratories, Burlingame, CA). BrdU-positive cells were counted in the periinfarct area, where the scar tissue constituted less than 20% of the visual field (magnification, × 200) (Yoon et al., 2005).

Animal studies

All procedures were performed in accordance with Caritas St. Elizabeth's Medical Center (Boston, MA) Institutional Animal Care and Use Committee (Approval number 248). Sprague-Dawley rats (7- to 8-week-old males; Jackson Laboratory, Bar Harbor, ME) were anesthetized with an intraperitoneal injection of ketamine (40–90 mg/kg) and xylazine (5–10 mg/kg) and respiration was controlled with an animal ventilator during thoracotomy. Peripheral blood (1 ml) was obtained from the heart of each rat and EPCs were cultured ex vivo. One week after transduction, the EPCs were used for autologous transplantation. Myocardial infarction was induced by ligating the proximal left anterior descending coronary artery with a 6-0 Prolene suture. Under direct visualization, transduced EPCs (1 × 10⁴ cells in 20 ml) were injected immediately into five sites of the myocardium surrounding the infarcted area, using a 30-gauge needle.

Echocardiography

Transthoracic echocardiography (SONOS 5500; Philips Healthcare, Amsterdam, The Netherlands) was performed on day 0 (before thoracotomy) and 12 weeks after myocardial infarction. Left ventricular diastolic dimension (LVDd), left ventricular systolic dimension (LVDs), and fractional shortening (FS) were measured at the mid-papillary muscle level (Kusano et al., 2007).

IGF-1 ELISA

Blood samples were taken periodically from the tail vein of anesthetized rats for the measurement of plasma human IGF-1 levels, using a human IGF-1 ELISA kit (Quantikine; R&D Systems, Minneapolis, MN) (Dobrucki et al., 2010).

Angiogenesis, apoptosis and proliferation in heart tissue

For the in vivo assay, hearts were harvested 12 weeks after myocardial infarction and transplantation of EPCs. To detect β-galactosidase, freshly excised tissues were flash frozen in O.C.T. compound (Sakura Finetek USA, Torrance, CA) and sectioned. Slides were incubated with an anti-β-Gal antibody (cat. no. G8021; Sigma-Aldrich) and anti-CD31 antibody (cat. no. 555445; BD Biosciences, San Jose, CA) for the detection of AAV-lacZ-transduced EPCs. Additional staining for capillaries was performed with rat-specific endothelial cell marker isolectin B4 (Vector Laboratories) as described previously (Dobrucki et al., 2010). Capillary density was scored morphometrically by histological examination of five randomly selected fields of tissue sections of periinfarct left ventricular myocardium. Capillaries were defined as tubular structures positive for isolectin B4. For apoptosis and proliferation assays, fixed heart tissue was first probed with α-actinin antibody (cat. no. A7811; Sigma-Aldrich). Nuclear staining for DNA fragmentation was performed by the TUNEL method to detect apoptosis, and Ki-67 staining was performed with rabbit polyclonal antibody against Ki-67 (cat. no. NCL-Ki67p; Novocastra Laboratories, Newcastle, UK) for cell proliferation, followed by 4′,6-diamidino-2-phenylindole (DAPI) staining (cat. no. 10236276001; Roche) to identify the number of nuclei in the periinfarct area as previously described (Kusano et al., 2007).

Statistical analysis

The mean and standard error (SE) were determined for replicate samples. An unpaired Student t test was applied to calculate the statistical significance between the means of two groups. Analysis of variance was performed to calculate the statistical significance for multiple samples. A p value less than 0.05 was considered significant.

Results

AAV-mediated transduction of EPCs augmented with genistein

We first examined the efficiency of transduction of peripheral blood-derived EPCs, using an AAV vector system (Kawamoto et al., 2001, 2003; Iwakura et al., 2005). Without pretreatment, the transduction efficiency was 23 ± 1.2% (see Supplementary Fig. 1A at www.liebertonline.com/hum). AAV transduction may be enhanced by reagents such as the anticancer drug hydroxyurea (HU) (Qing et al., 1998), a tyrosine kinase inhibitor, genistein (Qing et al., 1998), and a calpain inhibitor, LLnL (Ding et al., 2003). To optimize AAV transduction of EPCs in the context of our ex vivo expansion and transplantation approach, we evaluated those reagents by the MTT–formazan assay to assess cell viability (Vistica et al., 1991). After exposure to various concentrations of the three agents, each treatment decreased cell proliferation and viability in a concentration-dependent manner and we determined that pretreatment with 10 mM HU, 200 mM LLnL, and 100 μM genistein resulted in similar acceptable effects on proliferation and viability (see Supplementary Fig. 1B). Using the optimal concentrations for pretreatment reagents, we next examined the effects on AAV transduction. Preincubation with genistein (100 μM) achieved improved augmentation of AAV-lacZ transduction in EPCs compared with HU or LLnL (see Supplementary Fig. 1C). Moreover, to confirm the genistein effect on AAV transduction, AAV-IGF-1-mediated transduction of EPCs was shown by RT-PCR to be augmented by 7.2-fold after preincubation with genistein (see Supplementary Fig. 1D).

IGF-1-transduced EPCs inhibit apoptosis in and augment proliferation of neonatal cardiac myocytes

Reports demonstrate that IGF-1 has potent antiapoptotic and cell proliferation effects (Laviola et al., 2007). To determine whether IGF-1-expressing EPCs could directly exert this antiapoptotic effect, we cocultured neonatal rat cardiomyocytes with AAV-transduced EPCs (Yoon et al., 2005), and induced apoptosis by exposure to 100 mM hydrogen peroxide (H₂O₂) (Aikawa et al., 2000). In the presence of IGF-1-expressing EPCs, the number of H₂O₂ induced TUNEL-positive cardiomyocytes was significantly reduced compared with the control group (2.1 ± 0.2 vs. 4.6 ± 0.4; Fig. 1A). Next, to determine whether IGF-1-expressing EPCs could directly exert a cell proliferation effect, we measured the incorporation of BrdU, using the same coculture system (Yoon et al., 2005). When we cocultured neonatal cardiomyocytes with IGF-1-EPCs, the BrdU uptake of cardiomyocytes was significantly increased compared with the control group (4.1 ± 0.3 vs. 1.1 ± 0.1; Fig. 1B). Taken together, overexpression of IGF-1 in EPCs inhibited cardiomyocyte apoptosis induced by oxidant stress and augmented cardiomyocyte proliferation.

FIG. 1.

(A) Apoptosis assay of cardiac myocytes. Using a coculture system between AAV-infected endothelial progenitor cells (EPCs) and neonatal rat cardiomyocytes, the number of both α-actinin- and TUNEL-positive cell was counted after stimulation with H₂O₂. (B) Proliferation assay of cardiac myocytes. Using a coculture system between AAV-infected EPCs and neonatal rat cardiomyocytes, the number of both α-actinin- and BrdU-positive cells was counted (n = 5). *p < 0.05 control lacZ EPC group.

IGF-1-overexpressing EPCs improve cardiac function post-MI

For autologous EPC transplantation, we separately cultured autologous rat EPCs according to our published protocol (Kawamoto et al., 2001, 2003). After being cultured for 1 week, EPCs were transduced with AAV-lacZ control vector or AAV-IGF-1. We ligated the left anterior descending coronary artery of rats to induce myocardial infarction (MI), and transduced EPCs (1 × 10⁴ total) were delivered by direct injection to five different portions of the periinfarct area (Kusano et al., 2007). The physiological effects of IGF-1-expressing EPCs after MI in rats were assessed by transthoracic echocardiography. At baseline before MI, left ventricular diastolic dimension (LVDd), left ventricular systolic dimension (LVDs), and fractional shortening (FS) were similar between the two groups (Fig. 2). As expected, LVDd and LVDs were substantially increased and FS was decreased 12 weeks after MI (Fig. 2). However, LVDd and LVDs were significantly lower in the IGF-1-EPC treatment group (0.89 ± 0.04 and 0.71 ± 0.03 cm, respectively) compared with the lacZ-EPC control group (0.99 ± 0.03 and 0.78 ± 0.06 cm, respectively) (Fig. 2). FS of the IGF-1-EPC group by 12 weeks (25.7 ± 0.2%) was significantly higher compared with the control group (18.2 ± 0.7%) (Fig. 2). These results indicate that IGF-1-expressing EPCs improved cardiac function and remodeling post-MI in rats.

FIG. 2.

Serial changes in echocardiographic parameters such as left ventricular diastolic dimension (LVDd), left ventricular systolic dimension (LVDs), and fractional shortening (FS) from baseline (day 0) to 12 weeks. Lines with blue triangles indicate rat heart transplanted with AAV-IGF-1-EPC, and lines with red squares indicate rat heart transplanted with control AAV-lacZ-EPCs (n = 7). *p < 0.05 versus control lacZ-EPC group.

Angiogenesis is induced by transplantation of IGF-1-overexpressing EPCs

Twelve weeks after cell transplantation, to confirm the in vivo induction of IGF-1 expression by AAV in an infarcted heart, we measured the concentration of human IGF-1 (hIGF-1) in the serum of rats by hIGF-1 ELISA (Dobrucki et al., 2010). Although there were no elevations of hIGF-1 in the lacZ-EPC control group, circulating hIGF-1 levels were significantly increased at both 1 and 3 months after transplantation of IGF-1-EPCs (Fig. 3A). These results suggest that the AAV-IGF-1 vector significantly transduced EPCs and subsequently improved expression of IGF-1 in the transplanted EPCs of the infarcted heart, and that transduced EPCs were an effective tool in delivery of gene to the targeted site.

FIG. 3.

(A) IGF-1 expression. The serum concentration of human IGF-1 in rats transplanted with either lacZ-EPCs or IGF-1-EPCs was measured by ELISA. (B) Capillary density. After isolectin B4 staining, the capillary vessels were counted in rats receiving lacZ-EPCs or IGF-1-EPCs (n = 7). *p < 0.05 versus control lacZ-EPC group.

Reports have further indicated that IGF-1 has a strong effect on angiogenesis (Su et al., 2003; Lopez-Lopez et al., 2004; Rabinovsky and Draghia-Akli, 2004). Su and colleagues demonstrated that exposure to IGF-1 peptides potently induced endothelial cell migration in a Matrigel assay and capillary formation in an in vitro culture assay (Su et al., 2003), which are known to be important components of the proangiogenic response. Therefore, we examined angiogenesis between the lacZ-EPC group and the IGF-EPC group 12 weeks after MI. Immunohistochemical analysis revealed that capillary density of the heart in the IGF-1-EPC group was significantly higher than in the lacZ control group (151 ± 9 vs. 75 ± 5, respectively; Fig. 3B).

IGF-1-EPCs suppress cardiac apoptosis and induce cardiac proliferation post-MI

It has been reported that overexpression of IGF-1 attenuates the activation of myocyte necrosis and apoptosis after infarction in the transgenic mouse model (Li et al., 1997) and rescues cardiac myocytes from apoptosis in dilated cardiomyopathy (Welch et al., 2002). Accordingly, we compared cardiomyocyte apoptosis, by TUNEL assay analysis, between lacZ-EPC- and IGF-1-EPC-transplanted heart muscle post-MI. Twelve weeks after infarction, we harvested the tissue and performed α-actinin, TUNEL, and DAPI staining for cardiomyocytes, DNA fragments, and nuclei, respectively. The number of TUNEL-positive cells was significantly decreased in the IGF-1-EPC-treated group compared with the control lacZ-EPC group (16 ± 0.7 vs. 33 ± 1.2, respectively; Fig. 4A), indicating that AAV-IGF-1-EPCs may contribute to protect cardiac myocytes post-MI. It has also been reported that overexpression of IGF-1 increases the activation of cardiomyocyte proliferation after infarction in the transgenic mouse model (Li et al., 1997) and the number of proliferating myocytes in dilated cardiomyopathy (Welch et al., 2002). We reported that AAV-mediated growth hormone (GH) expression enhances cardiomyocyte proliferation post-MI in rats, as indicated by staining for the Ki-67 cell cycle marker protein (Kusano et al., 2007). In comparison with the lacZ control group, the number of Ki-67-positive myocytes was increased in the IGF-1 EPC group about 2.0-fold compared with the control group (420 ± 25 vs. 210 ± 55, respectively; Fig. 4B). These results, in combination with those of the in vitro assay (see Supplementary Fig. 1B), indicate that IGF-1-EPCs may contribute to promote cardiomyocyte proliferation post-MI.

FIG. 4.

(A) Cardiomyocyte apoptosis. Twelve weeks after infarction, the number of the TUNEL-positive cells was counted and is presented per 10⁵ cells. (n = 7) *p < 0.05 versus control lacZ-EPC group. (B) Cardiomyocyte proliferation. Twelve weeks after infarction, the number of the Ki-67-positive cells was counted and is presented per 10⁵ cells (n = 7). *p < 0.05 versus control lacZ-EPC group.

Genetically modified EPCs are limited to the targeted tissue

To verify gene transfer and to determine the distribution of gene marking, total DNA was isolated from major organs in recipient rats. The presence of AAV sequences was analyzed by PCR as described previously (Iwakura et al., 2005). Agarose gel electrophoresis demonstrated a 286-bp band, derived from the inverted terminal repeat (ITR) sequence of the AAV genome and extending into the CMV promoter sequence (Iwakura et al., 2005), in the heart 12 weeks after transplantation (see Supplementary Fig. 2 at www.liebertonline.com/hum). Conversely, there were no AAV genomes in other organs (see Supplementary Fig. 2). Gene marking of EPCs was limited to the targeted heart only, without migration and subsequent expression in other tissues.

Discussion

Although AAV2 is capable of transducing a variety of cell types (Van Vliet et al., 2008), endothelial cells (ECs) have been reported to be relatively resistant (Pajusola et al., 2002). We confirmed a relatively low transduction efficiency of EPCs despite the use of high-titer AAV vectors (see Supplementary Fig. 1A), which suggests that EPCs behave similarly to ECs. Accordingly, we sought to augment AAV transduction by using chemical reagents. In infected cells, the incoming AAV single-stranded DNA (ssDNA) must be converted into double-stranded DNA (dsDNA) to serve as a transcriptional template (Vincent-Lacaze et al., 1999; Nakai et al., 2000). Studies indicate that cellular permissiveness correlates with the phosphorylation status of FK506-binding protein-2 (FKBP2), which specifically interacts with the D sequence and inhibits AAV second-strand DNA synthesis (Qing et al., 2001; Zhong et al., 2004). Both genistein and hydroxyurea have been reported to promote dsDNA formation through inhibition of FKBP2 (Qing et al., 2001; Zhong et al., 2004). Moreover, a proteasome inhibitor, LLnL, has been shown to increase AAV transduction efficiency by protecting infected AAV vectors against degradation (Ding et al., 2003). To retain adequate EPC viability, we first used the MTT assay to determine the optimal pretreatment concentrations, which were as follows: 10 mM for hydroxyurea, 200 mM for LLnL, and 100 μM for genistein (see Supplementary Fig. 1B). At the optimal concentration, genistein yielded the greatest improvement in AAV transduction (see Supplementary Fig. 1C and D). In addition, another AAV serotype, AAV1 (Van Vliet et al., 2008), showed definite improvement after EPCs were pretreated with genistein, as transduction was lower than that of AAV serotype 2 (data not shown). Other reagents such as ethanol (Wheeler et al., 2000) and histone deacetylase inhibitors (Okada et al., 2006) may also augment AAV transduction efficacy. We have previously reported that transplantation of 1 × 10⁵ EPCs improved cardiac function post-MI in rats (Kawamoto et al., 2001). In preliminary experiments, we observed that 1 × 10⁴ EPCs transplanted into infarcted rat heart resulted in minimal differences between the control group and the treated group (data not shown). Accordingly, we genetically modified EPCs with AAV2 and transplantation of IGF-1-expressing EPCs (total, 1 × 10⁴) led to significant improvement of cardiac function in the same rat infarction model (Fig. 2). In addition, we confirmed the persistence of AAV gene expression by detecting IGF-1 expression in the heart 12 weeks post-MI (see Supplementary Fig. 2).

Transplantation of EPCs induces angiogenesis in infarcted heart after autocrine and paracrine activation of angiogenic factors such as vascular endothelial growth factor (VEGF) and IGF-1 (Kawamoto et al., 2001, 2003). In this study AAV-IGF-1-transduced EPCs induced higher capillary density in the heart compared with control AAV-lacZ-EPCs (Fig. 3B), and thus secreted IGF-1 may provide an additional improvement beyond EPCs alone. In our in vitro study, using a coculture system, IGF-1-expressing EPCs decreased apoptosis and increased cell proliferation in neonatal rat cardiomyocytes (Fig. 1A and B). These results also support the salutary effect of IGF-1. In addition, transplantation of AAV-IGF-1-EPCs reduced cardiomyocyte apoptosis (Fig. 4A) and promoted cardiomyocyte proliferation (Fig. 4B) compared with control lacZ-EPCs post-MI, presumably via a paracrine mechanism. This approach of using a combination of cell and gene therapy can achieve better results than individual therapy because the two modes of therapy will support each other. In addition, assimilation of EPCs into the vasculature at the site of treatment could lead to long-term IGF-1 production, long after the time of delivery and beyond the effects conferred by the paracrine properties of EPCs. Moreover, because Thum and colleagues demonstrated that age-dependent impairment of EPCs is improved by growth hormone (Thum et al., 2007), it is possible that IGF-1 may act similarly on functionally impaired EPCs in elderly patients with MI.

The AAV vector system is safe and continues to offer hope for human gene therapy, but its promiscuous tropism may lead to undesirable tissue expression. Both cardiac-specific promoters and cardiac-selective serotypes are being explored to avoid this potential problem (Aikawa et al., 2002; Inagaki et al., 2006). In our experiments various rat organs were examined by PCR and no AAV genomes were detected other than in heart tissue injected with lacZ-EPCs (see Supplementary Fig. 2). Thus, it is possible to restrict AAV-mediated gene expression in vivo by this method of AAV-transduced cell transplantation, in contrast to direct in vivo injection of AAV vectors. Human clinical trials of progenitor cells continue to show promise to improve cardiac function (Losordo et al., 2007). Our study provides evidence that both stem cells and progenitor cells may act as effective tools for gene delivery by AAV, and that expression of IGF-1 may improve the efficacy of this transplantation approach.

Footnotes

Acknowledgments

This work was supported by the Uehara Memorial Foundation (R.A.) and by a Starr Foundation grant (R.A.). Additional support was provided by the Leukaemia Foundation of Australia, the National Health and Medical Research Council of Australia, an anonymous Foundation, and by Cure the Future (J.E.J.R.).

Author Disclosure Statement

None declared.

References

Aikawa

, Nawano

, Gu

, Katagiri

, Asano

, Zhu

, Nagai

, Komuro

2000. Insulin prevents cardiomyocytes from oxidative stress-induced apoptosis through activation of PI3 kinase/Akt. Circulation, 102:2873–2879.

Aikawa

, Huggins

G.S.

, Snyder

R.O.

2002. Cardiomyocyte-specific gene expression following recombinant adeno-associated viral vector transduction. J. Biol. Chem., 277:18979–18985.

Aiuti

, Bachoud-Lévi

A.C.

, Blesch

, Brenner

M.K.

, Cattaneo

, Chiocca

E.A.

, Gao

, High

K.A.

, Leen

A.M.

, Lemoine

N.R.

, McNeish

I.A.

, Meneguzzi

, Peschanski

, Roncarolo

M.G.

, Strayer

D.S.

, Tuszynski

M.H.

, Waxman

D.J.

, Wilson

J.M.

2007. Progress and prospects: gene therapy clinical trials (part 2) Gene Ther., 14:1555–1563.

Chng

, Larsen

S.R.

, Zhou

, Wright

J.F.

, Martiniello-Wilks

, Rasko

J.E.J.

2007. Specific adeno-associated virus serotypes facilitate efficient gene transfer into human and non-human primate mesenchymal stromal cells. J. Gene Med., 9:22–32.

Delafontaine

, Song

Y.H.

, Li

2004. Expression, regulation, and function of IGF-1, IGF-1R, and IGF-1 binding proteins in blood vessels. Arterioscler. Thromb. Vasc. Biol., 24:435–444.

Ding

, Yan

, Zak

, Saavedra

, Rodman

D.M.

, Engelhardt

J.F.

2003. Second-strand genome conversion of adeno-associated virus type 2 (AAV-2) and AAV-5 is not rate limiting following apical infection of polarized human airway epithelia. J. Virol., 77:7361–7366.

Dobrucki

L.W.

, Tsutsumi

, Kalinowski

, Dean

, Gavin

, Sen

, Mendizabal

, Sinusas

A.J.

, Aikawa

2010. Analysis of angiogenesis induced by local IGF-1 expression after myocardial infarction using microSPECT-CT imaging. J. Mol. Cell. Cardiol., 48:1071–1079.

Inagaki

, Fuess

, Storm

T.A.

, Gibson

G.A.

, McTiernan

C.F.

, Kay

M.A.

, Nakai

2006. Robust systemic transduction with AAV9 vectors in mice: Efficient global cardiac gene transfer superior to that of AAV8. Mol. Ther., 14:45–53.

Iwakura

, Dean

, Hamada

, Eaton

, Qin

, Losordo

D.W.

, Aikawa

2005. Use of recombinant adeno-associated viral vectors as a tool for labeling bone marrow cells. J. Mol. Cell. Cardiol., 38:799–802.

10.

Kawamoto

, Gwon

H.C.

, Iwaguro

, Yamaguchi

J.I.

, Uchida

, Masuda

, Silver

, Ma

, Kearney

, Isner

J.M.

, Asahara

2001. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation, 103:634–637.

11.

Kawamoto

, Tkebuchava

, Yamaguchi

, Nishimura

, Yoon

Y.S

, Milliken

, Uchida

, Masuo

, Iwaguro

, Ma

, Hanley

, Silver

, Kearney

, Losordo

D.W

, Isner

J.M

, Asahara

. 2003. Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia. Circulation, 107:461–468.

12.

Khan

A.S.

, Sane

D.C.

, Wannenburg

, Sonntag

W.E.

2002. Growth hormone, insulin-like growth factor-1 and the aging cardiovascular system. Cardiovasc. Res., 54:25–35.

13.

Kusano

, Tsutsumi

, Dean

, Gavin

, Ma

, Silver

, Thorne

, Zhu

, Losordo

D.W.

, Aikawa

2007. Long-term stable expression of human growth hormone by rAAV promotes myocardial protection post-myocardial infarction. J. Mol. Cell. Cardiol., 42:390–399.

14.

Laviola

, Natalicchio

, Giorgino

2007. The IGF-I signaling pathway. Curr. Pharm. Des., 13:663–669.

15.

, Li

, Wang

, Leri

, Jana

K.P.

, Liu

, Kajstura

, Baserga

, Anversa

1997. Overexpression of insulin-like growth factor-1 in mice protects from myocyte death after infarction, attenuating ventricular dilation, wall stress, and cardiac hypertrophy. J. Clin. Invest., 100:1991–1999.

16.

Liu

J.L.

, Leroith

1999. Insulin-like growth factor I is essential for postnatal growth in response to growth hormone. Endocrinology, 140:5178–5184.

17.

Liu

, Pachori

A.S.

, Ward

C.A.

, Davis

J.P.

, Gnecchi

, Kong

, Zhang

, Murduck

, Yet

S.F.

, Perrella

M.A.

, Pratt

R.E.

, Dzau

V.J.

, Melo

L.G.

2006. Heme oxygenase-1 (HO-1) inhibits postmyocardial infarct remodeling and restores ventricular function. FASEB J., 20:207–216.

18.

Lopez-Lopez

, Leroith

, Torres-Aleman

2004. Insulin-like growth factor I is required for vessel remodeling in the adult brain. Proc. Natl. Acad. Sci. U.S.A., 101:9833–9838.

19.

Losordo

D.W.

, Schatz

R.A.

, White

C.J.

, Udelson

J.E.

, Veereshwarayya

, Durgin

, Poh

K.K.

, Weinstein

, Kearney

, Chaudhry

, Burg

, Eaton

, Heyd

, Thorne

, Shturman

, Hoffmeister

, Story

, Zak

, Dowling

, Traverse

J.H.

, Olson

R.E.

, Flanagan

, Sodano

, Murayama

, Kawamoto

, Kusano

K.F.

, Wollins

, Welt

, Shah

, Soukas

, Asahara

, Henry

T.D.

2007. Intramyocardial transplantation of autologous CD34⁺ stem cells for intractable angina: A phase I/IIa double-blind, randomized controlled trial. Circulation, 115:3165–3172.

20.

Manno

C.S.

, Pierce

G.F.

, Arruda

V.R.

, Glader

, Ragni

, Rasko

J.J.

, Ozelo

M.C.

, Hoots

, Blatt

, Konkle

, Dake

, Kaye

, Razavi

, Zajko

, Zehnder

, Rustagi

P.K.

, Nakai

, Chew

, Leonard

, Wright

J.F.

, Lessard

R.R.

, Sommer

J.M.

, Tigges

, Sabatino

, Luk

, Jiang

, Mingozzi

, Couto

, Ertl

H.C.

, High

K.A.

, Kay

M.A.

2006. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat. Med., 12:342–347.

21.

Nakai

, Storm

T.A.

, Kay

M.A.

2000. Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo. J. Virol., 74:9451–9463.

22.

Okada

, Uchibori

, Iwata-Okada

, Takahashi

, Nomoto

, Nonaka-Sarukawa

, Ito

, Liu

, Mizukami

, Kume

, Kobayashi

, Ozawa

2006. A histone deacetylase inhibitor enhances recombinant adeno-associated virus-mediated gene expression in tumor cells. Mol. Ther., 13:738–746.

23.

Pajusola

, Gruchala

, Joch

, Luscher

T.F.

, Yla-Herttuala

, Bueler

2002. Cell-type-specific characteristics modulate the transduction efficiency of adeno-associated virus type 2 and restrain infection of endothelial cells. J. Virol., 76:11530–11540.

24.

Qing

, Khuntirat

, Mah

, Kube

D.M.

, Wang

X.S.

, Ponnazhagan

, Zhou

, Dwarki

V.J.

, Yoder

M.C.

, Srivastava

1998. Adeno-associated virus type 2-mediated gene transfer: correlation of tyrosine phosphorylation of the cellular single-stranded D sequence-binding protein with transgene expression in human cells in vitro and murine tissues in vivo. J. Virol., 72:1593–1599.

25.

Qing

, Hansen

, Weigel-Kelley

K.A.

, Tan

, Zhou

, Srivastava

2001. Adeno-associated virus type 2-mediated gene transfer: role of cellular FKBP52 protein in transgene expression. J. Virol., 75:8968–8976.

26.

Rabinovsky

E.D.

, Draghia-Akli

2004. Insulin-like growth factor I plasmid therapy promotes in vivo angiogenesis. Mol. Ther., 9:46–45.

27.

Schulze

P.C.

, Fang

, Kassik

K.A.

, Gannon

, Cupesi

, Macgillivray

, Lee

R.T.

, Rosenthal

2005. Transgenic overexpression of locally acting insulin-like growth factor-1 inhibits ubiquitin-mediated muscle atrophy in chronic left-ventricular dysfunction. Circ. Res., 97:418–426.

28.

E.J.

, Cioffi

C.L.

, Stefansson

, Mittereder

, Garay

, Hreniuk

, Liau

2003. Gene therapy vector-mediated expression of insulin-like growth factors protects cardiomyocytes from apoptosis and enhances neovascularization. Am. J. Physiol. Heart Circ. Physiol., 284:H1429–H1440.

29.

Thum

, Hoeber

, Froese

, Klink

, Stichtenoth

D.O.

, Galuppo

, Jakob

, Tsikas

, Anker

S.D.

, Poole-Wilson

P.A.

, Borlak

, Ertl

, Bauersachs

2007. Age-dependent impairment of endothelial progenitor cells is corrected by growth-hormone-mediated increase of insulin-like growth-factor-1. Circ. Res., 100:434–443.

30.

Torella

, Rota

, Nurzynska

, Musso

, Monsen

, Shiraishi

, Zias

, Walsh

, Rosenzweig

, Sussman

M.A.

, Urbanek

, Nadal-Ginard

, Kajstura

, Anversa

, Leri

2004. Cardiac stem cell and myocyte aging, heart failure, and insulin-like growth factor-1 overexpression. Circ. Res., 94:514–524.

31.

Van Vliet

K.M.

, Blouin

, Brument

, Agbandje-McKenna

, Snyder

R.O.

2008. The role of the adeno-associated virus capsid in gene transfer. Methods Mol. Biol., 437:51–91.

32.

Vincent-Lacaze

, Snyder

R.O.

, Gluzman

, Bohl

, Lagarde

, Danos

1999. Structure of adeno-associated virus vector DNA following transduction of the skeletal muscle. J. Virol., 73:1949–1955.

33.

Vistica

D.T.

, Skehan

, Scudiero

, Monks

, Pittman

, Boyd

M.R.

1991. Tetrazolium-based assays for cellular viability: A critical examination of selected parameters affecting formazan production. Cancer Res., 51:4501.

34.

Welch

, Plank

, Witt

, Glascock

, Schaefer

, Chimenti

, Andreoli

A.M.

, Limana

, Leri

, Kajstura

, Anversa

, Sussman

M.A.

2002. Cardiac-specific IGF-1 expression attenuates dilated cardiomyopathy in tropomodulin-overexpressing transgenic mice. Circ. Res., 90:641–648.

35.

Wheeler

M.D.

, Kono

, Rusyn

, Arteel

G.E.

, McCarty

, Samulski

R.J.

, Thurman

R.G.

2000. Chronic ethanol increases adeno-associated viral transgene expression in rat liver via oxidant and NFκB-dependent mechanisms. Hepatology, 32:1050–1059.

36.

Yoon

Y.S.

, Wecker

, Heyd

, Park

J.S.

, Tkebuchava

, Kusano

, Hanley

, Scadova

, Qin

, Cha

D.H.

, Johnson

K.L.

, Aikawa

, Asahara

, Losordo

D.W.

2005. Clonally expanded novel multipotent stem cells from human bone marrow regenerate myocardium after myocardial infarction. J. Clin. Invest., 115:326–338.

37.

Zhong

, Li

, Yang

, Chen

, Li

, Qing

, Weigel-Kelley

K.A.

, Yoder

M.C.

, Shou

, Srivastava

2004. Improved transduction of primary murine hepatocytes by recombinant adeno-associated virus 2 vectors in vivo. Gene Ther., 11:1165–1169.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.08 MB