Original article
Ex vivo delivered stromal cell-derived factor-1α promotes stem cell homing and induces angiomyogenesis in the infarcted myocardium

https://doi.org/10.1016/j.yjmcc.2007.02.001Get rights and content

Abstract

We aimed to optimize non-viral transfection of human stromal cell derived factor (SDF-1α) gene into skeletal myoblasts (SkM) and, transplant these cells to establish transient SDF-1α gradient to favor extra-cardiac stem cell translocation into infarcted heart. Optimized conditions for transfection of SDF-1α gene into syngenic SkM were achieved using FuGene™6/phSDF-1α (3:2v/w, 4 h transfection) with 125 μM ZnCl2 (p < 0.001). After characterization for transgene overexpression by immunostaining, ELISA and PCR, the cells were transplanted in female rat model of myocardial infarction. Thirty-six rats were grouped (n = 12/group) to receive 70 μl DMEM without cells (group-1) or containing 1.5 × 106 non-transfected (group-2) or SDF-1α transfected SkM (group-3). On day 4 post-transplantation (in 4 animals/group), marked expression of SDF-1α/sry-gene (p = 0.003), total Akt, phospho-Akt and Bcl2 was observed in group-3. The number of CD31+, C-kit+ and CD34+ cells was highest in group-3 hearts (p < 0.01). Blood vessel density in group-3 was higher in both scar and peri-scar regions (p < 0.001) as compared with other groups. Echocardiography showed improved indices of left ventricle contractile function and remodeling in group-3 (p < 0.05) as compared with groups-1 and -2. We conclude that ex vivo SDF-1α transgene delivery promotes stem and progenitor cell migration to the heart, activates cell survival signaling and enhances angiomyogenesis in the infarcted heart.

Introduction

SDF-1α protein and gene delivery to the ischemic heart promote endothelial progenitor cells recruitment into the ischemic myocardium to promote angiogenesis [1], [2], [3]. SDF-1α specifically interacts with the CXCR4 receptor and orchestrates the mobilization and homing of hematopoietic stem cells from bone marrow (BM) to the ischemic heart [3], [4], [5], [6]. Physiologically, BM harbors a heterogenous population of cells positive for CXCR4 receptor. BM has the highest SDF-1α concentration gradient as compared with other tissues, and promotes homing of circulating hematopoietic stem cells into BM stromal adult heart; SDF-1α is expressed constitutively. In the early phase after MI, however, elevated SDF-1α levels have been reported in the infarct zone [7], [8]. This provides the required stimulus for mobilization of stem cells from BM niches to the damaged site as part of a natural repair process [9]. The other key players involved in tissue ischemia-induced mobilization of BM progenitors to the circulation include vascular endothelial growth factor, placenta growth factor, stem cell factor and granulocyte colony stimulating factor [10], [11], [12], [13]. The effect of intrinsic SDF-1α up-regulated expression is, however, transient and insufficient for cardiac repair [4], [6]. In order to overcome this deficiency, SDF-1α concentration gradient in favor of myocardial scar has been achieved by the delivery of SDF-1α protein or gene encoding for hSDF-1α. The delivery strategies include intramyocardial injection of SDF-1α protein [2], naked plasmid encoding for SDF-1α [3], [5], [6], transplantation of ex vivo genetically manipulated cardiac fibroblasts overexpressing SDF-1α [4] and mesothelial cell transplantation which intrinsically expresses copious amounts of SDF-1α [1], [14]. Moreover, intramuscular injections of GCSF (Filgrastim), the sulfated polysaccharide (Fucoidan) [15], [16] or “3 hydroxy-3-methylglutaryl-CoA reductase inhibitor” (Atorvastatin) [17], [18] have been reported to increase the plasma concentration of hSDF-1α to a level sufficient enough to induce mobilization of stem cells from BM and their homing into the injured tissue.

The study is aimed to explore the use of synthetic vectors (three cationic lipid vectors; Lipofectamine™2000, FuGene™6 and Polyethyleneimine-JetPEI™) with the addition of ZnCl2 for optimal transfection of hSDF-1α into SkM. We anticipate that overexpression of hSDF-1α is cytoprotective and enhances cell survival during the initial phase after transplantation. The donor SkM overexpressing hSDF-1α established a transient and localized hSDF-1α gradient in the heart that favored stem cell translocation into ischemic myocardium, and enhanced angiogenesis.

Section snippets

SkM purification and culture in vitro

Skeletal muscle biopsies from syngenic male Fischer rats were dissected and roughly cut into small fragments in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL). The tissue fragments were enzymatically dissociated using collagenase-IA (2 mg/ml, Gibco BRL) and dispase (2 mg/ml, Gibco BRL) for 1 h followed by trypsin-EDTA (0.25%, Gibco BRL) for 20 min at 37 °C. The cells were filtered through a 100-μm sieve (Cell Strainer Nylon) and collected by sedimentation at 1500 rpm for 5 min. The enzyme

Transfection of SDF-1α into SkM

SkM during passage 2–4 were > 90% pure for desmin expression. The efficiency of phSDF-1α transfection into SkM was markedly influenced by vector/plasmid ratio and optimal efficiency was obtained with FuGene™6/phSDF-1α (v/w 3:2), Lipofectamine™2000/phSDF-1α (v/w 2.5:1) and JetPEI™/phSDF-1α (N/P = 5). Short-term ZnCl2 exposure displayed dose-dependent cytotoxicity on SkM. However, cell viability observed with concentrations of < 125 μM ZnCl2 was significantly higher as compared with concentrations of

Discussion

The main findings of our study were that (1) presence of ZnCl2 significantly enhanced the transfection of phSDF-1α into SkM, (2) overexpression of hSDF-1α rendered the cells resistant to anoxia in vitro and promoted their survival after transplantation, (3) higher gradient of hSDF-1α at the site of the cell graft triggered homing of stem and progenitor cells into the infarcted myocardium and (4) transplantation of hSDF-1α overexpressing SkM promoted angiogenesis in the infarcted region and

Acknowledgments

This work was supported by National Institutes of Health grants # R37-HL074272, HL-23597 and HL-080686 [to M.A].

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