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Analysis of angiogenesis induced by local IGF-1 expression after myocardial infarction using microSPECT-CT imaging

https://doi.org/10.1016/j.yjmcc.2009.10.008Get rights and content

Abstract

Insulin-like growth factor-1 (IGF-1) has been found to exert favorable effects on angiogenesis in prior animal studies. This study explored the long-term effect of IGF-1 on angiogenesis using microSPECT-CT in infarcted rat hearts after delivering human IGF-1 gene by adeno-associated virus (AAV). Myocardial infarction (MI) was induced in Sprague-Dawley rats by ligation of the proximal anterior coronary artery and a total of 1011 AAV-CMV-lacZ (control) or IGF-1 vectors were injected around the peri-infarct area. IGF-1 expression by AAV stably transduced heart muscle for up to 16 weeks post-MI and immunohistochemistry revealed a remarkable increase in capillary density. A 99mTc-labeled RGD peptide (NC100692, GE Healthcare) was used to assess temporal and regional αv integrin activation. Rats were injected with NC100692 followed by 201Tl chloride and in vivo microSPECT-CT imaging was performed. After imaging, hearts were excised and cut for quantitative gamma-well counting (GWC). NC100692 retention was significantly increased in hypoperfused regions of both lacZ and IGF-1 rats at 4 and 16 weeks post-MI. Significantly higher activation of αv integrin was observed in IGF-1 rats at 4 weeks after treatment compared with control group, although the activation was lower in the IGF-1 group at 16 weeks. Local IGF-1 gene delivery by AAV can render a sustained transduction and improve cardiac function post-MI. IGF-1 expression contributes to enhanced αv integrin activation which is linked to angiogenesis. MicroSPECT-CT imaging with 99mTc-NC100692 and quantitative GWC successfully assessed differences in αv integrin activation between IGF-1-treated and control animals post-MI.

Introduction

Insulin-like growth factor-1 (IGF-1) is a 7.6 kDa polypeptide growth factor, which is expressed by many cells and tissues during embryonic and postnatal development and in adult animals [[1], [2], [3]]. 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 [2]. In addition, in vivo, IGF-1 action is modulated by a family of IGF-binding proteins present in the circulation [3]. IGF-1, secreted from the liver in response to growth hormone (GH), promotes postnatal growth in bone, muscle, fat, and other tissues [[1], [2], [3]]. Human and murine IGF-1 deficiency causes severe intrauterine and postnatal growth retardation, perinatal lethality, delayed development in a variety of organs [1], indicating that IGF-1 is a key regulator of cell development. It has recently been reported that overexpression of IGF-1 attenuates myocyte necrosis and apoptosis after infarction in transgenic mouse model [4] and rescues cardiac myocytes from apoptosis in dilated cardiomyopathy [5]. It has also been reported that overexpression of IGF-1 improves cardiomyocyte senescence in transgenic mouse model [6] and reduces cardiomyocyte atrophy induced by ischemia [7]. Accordingly, IGF-1 has been regarded as a pleiotropic growth factor affecting myocyte proliferation and regeneration in heart development and injury.

Recent reports have further indicated that IGF-1 has a strong effect on angiogenesis [[8], [9], [10]], which represents the formation of neovasculature from the endothelium of preexisting vessels. It has been reported that IGF-1 can induce angiogenesis in skeletal muscle and brain tissue [9,10]. Su et al. [8] demonstrated that IGF-1 potently induced endothelial cell migration in a Matrigel assay and capillary formation in organ ring culture assay, which are known as important components of the angiogenic response. As systemic IGF-1 peptide administration induces unfavorable side effects such as edema and tachycardia [11], Rabinovsky et al. performed local injection of expression plasmids containing the IGF-1 gene into the skeletal muscle following femoral artery ligation in mice and demonstrated that IGF-1 treatment evoked angiogenesis and increased blood flow in the skeletal muscle. Therefore, it is plausible that local IGF-1 expression would protect cardiomyocytes from ischemia through induction of angiogenesis under conditions of ischemic injury.

Recombinant adeno-associated virus (AAV) is currently recognized as an effective gene transfer vector for heart diseases [[12], [13], [14]]. The physical stability of AAV makes these vectors advantageous for in vivo use, and transgene expression can persist long term in a wide range of tissues including heart and skeletal muscle [[15], [16], [17]]. Recently, we reported that AAV markedly transduced cardiomyocytes in an infarcted rat heart and the transgene expression was observed up to 22 weeks [18].

Traditionally, the angiogenic response has been examined by evaluation of the physiological changes associated with the process. More recently, a number of investigators have demonstrated the feasibility of noninvasive evaluation of angiogenesis using image-based approaches targeted at cell surface receptors. We previously demonstrated the potential of single photon emission computed tomographic (SPECT) imaging with radiolabeled tracers targeted at αv integrins and X-ray computed tomography (CT) for evaluation of spatial and temporal changes in peripheral [19] and in myocardial angiogenesis in mice [20], rats, and dogs post-MI [21,22].

To date, there have been no reports showing the effect or mechanism of long-term IGF-1 expression on angiogenesis post-MI. In this study, we examined the IGF-1 effect on angiogenesis with both biochemical assay and microSPECT-CT imaging using 99mTc-labeled peptide (NC100692, GE Healthcare) targeted at αv integrins [23], known to be activated during angiogenic process. Here we show that AAV mediated long-term stable expression of human IGF-1 in rat heart post-MI, and overexpression of IGF-1 significantly improved cardiac function and enhanced angiogenesis in the early stage after infarction.

Section snippets

AAV vector production

To produce an AAV-CMV-IGF-1 construct, human IGF-1 cDNA, as defined in NCBI-X00173, was amplified by a standard polymerase chain reaction (PCR) method using IGF-1 specific PCR primers as shown below, using a human gene clone (clone ID: 984882, catalog no. 97002RG, Invitrogen™ Life Technologies):

  • 5′primer: 5′-CCGAATTCTTCAGAAGCAATGGGA-3′

  • 3′primer: 5′-CGGGATCCGTCTTCCTACATCCTG-3′

(underlines show original IGF-1 cDNA sequence).

After the fragment of sequence was confirmed, the cDNA of IGF-1 was inserted

AAV-mediated IGF-1 expression in cardiac cells and tissues

We initially overexpressed the AAV-IGF-1 vector containing human IGF-1 gene in cultured rat H9C2 cells. H9C2 cells were infected with 1000 particles per cell of AAV-IGF-1, and mRNA expression of human IGF-1 was examined by RT-PCR. IGF-1 was strongly expressed in a time dependent manner through the AAV vector system (Fig. 1A). To evaluate whether the exogenous IGF-1 expression affects the infected cells, we analyzed Akt, an important effector on angiogenesis [32], after infection of control

Discussion

In this study, we demonstrated that local delivery of IGF-1 gene by recombinant AAV in the setting of acute MI resulted in sustained IGF-1 expression, increased angiogenesis, reduced LV remodeling, and improved cardiac function. The effect of AAV-IGF-1 treatment on angiogenesis was examined using an established rat model of MI with both in vitro biochemical assays for a number of angiogenic factors, and in vivo microSPECT-CT imaging using a 99mTc-labeled peptide targeted at αv integrins, known

Disclosures

Marivi Mendizabal is an employee of GE Healthcare.

Acknowledgments

We thank Dr. Douglas Losordo for support. This work was supported by Starr foundation grant (R.A.) and NIH grant R01 HL65662 (A.S.).

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