Molecular and Cellular Biochemistry

, Volume 339, Issue 1, pp 107–118

Combination of chemokine and angiogenic factor genes and mesenchymal stem cells could enhance angiogenesis and improve cardiac function after acute myocardial infarction in rats

Authors

    • Department of Cardiology, Institute of Clinical MedicineRenmin Hospital, Yunyang Medical College
    • Department of PhysiologyYunyang Medical College
    • Hubei Key Laboratory of Embryonic Stem Cell ResearchYunyang Medical College
    • Department of Cardiology, Institute of Clinical MedicineRenmin Hospital, Yunyang Medical College
    • Hubei Key Laboratory of Embryonic Stem Cell ResearchYunyang Medical College
  • Fei Zheng
    • Department of Cardiology, Institute of Clinical MedicineRenmin Hospital, Yunyang Medical College
    • Hubei Key Laboratory of Embryonic Stem Cell ResearchYunyang Medical College
  • Xia Kong
    • Department of Cardiology, Institute of Clinical MedicineRenmin Hospital, Yunyang Medical College
    • Hubei Key Laboratory of Embryonic Stem Cell ResearchYunyang Medical College
  • Linyun Guo
    • Department of Cardiology, Institute of Clinical MedicineRenmin Hospital, Yunyang Medical College
    • Hubei Key Laboratory of Embryonic Stem Cell ResearchYunyang Medical College
  • Jianye Yang
    • Department of Cardiology, Institute of Clinical MedicineRenmin Hospital, Yunyang Medical College
    • Hubei Key Laboratory of Embryonic Stem Cell ResearchYunyang Medical College
  • Lei Zhang
    • Department of Cardiology, Institute of Clinical MedicineRenmin Hospital, Yunyang Medical College
    • Hubei Key Laboratory of Embryonic Stem Cell ResearchYunyang Medical College
  • Yongzhang Huang
    • Department of Cardiology, Institute of Clinical MedicineRenmin Hospital, Yunyang Medical College
    • Hubei Key Laboratory of Embryonic Stem Cell ResearchYunyang Medical College
Article

DOI: 10.1007/s11010-009-0374-0

Cite this article as:
Tang, J., Wang, J., Zheng, F. et al. Mol Cell Biochem (2010) 339: 107. doi:10.1007/s11010-009-0374-0

Abstract

Gene and stem-cell therapies hold promise for the treatment of ischemic cardiovascular disease. Combined stem cell, chemokine, and angiogenic growth factor gene therapy could augment angiogenesis, and better improve heart function in the infarcted myocardium. In order to prove this action, we established the animal model of myocardial infarction (MI) was by occlusion of the left anterior descending artery in rats. Seven days after surgery, 5.0 × 106 Ad-EGFP–MSC, 5.0 × 106 Ad-SDF-1–MSC, 5.0 × 106 Ad-VEGF–MSC, or 5.0 × 106 Ad-SDF–VEGF–MSC (Ad-SDF-1–VEGF–MSC) suspension in 0.2 ml of serum-free medium was injected into four sites in the infarcted hearts. Results showed that MSCs transfected with Ad-VEGF and Ad-SDF-1 produced more SDF-1 and VEGF protein than MSCs alone, the increased protein levels of VEGF and SDF-1 activated Akt in MSCs transfected with Ad-VEGF and Ad-SDF-1, and improved the survival capability of the MSCs in vitro and in vivo. These transplanted cells showed that the characteristic phenotype of cardiomyocyte (e.g., cTnt) and endothelial cells (e.g., CD31). Four weeks after transplantation, reduced infarct size and fibrosis, greater vascular density, and a thicker left ventricle wall were observed in Ad-SDF–VEGF–MSC group. Measurement of hemodynamic parameters showed an improvement in left ventricular performance in Ad-SDF–VEGF–MSC group compared with other groups. These results demonstrated that combination of chemokine and angiogenic factor gene and stem cells could enhance angiogenesis and improves cardiac function after acute myocardial infarction in rats.

Keywords

Myocardial infarctionSDF-1VEGFCell therapyAngiogenesis

Abbreviations

MSCs

Mesenchymal stem cells

VEGF

Vascular endothelial growth factor

SDF-1α

Stromal cell-derived factor-1 alpha

EGFP

Enhanced green fluorescent protein

MI

Myocardial infarction

Ad

Adenoviral

LVSP

Left ventricle systolic pressure

LVEDP

Left ventricle end-diastolic pressure

+dP/dtmax and −dP/dtmax

Rate of rise and fall of ventricular pressure

Introduction

Mesenchymal stem cells (MSCs) are pluripotent adult stem cells that reside primarily within the bone marrow. MSCs can be obtained in relatively large numbers through standard clinical procedures, and MSCs are easily expanded in culture. Recent studies have shown the potential of MSCs to differentiate into cardiomyocytes, endothelial cells, and vascular smooth muscle cells in vitro and in vivo. Animal studies and preliminary clinical investigations showed that therapeutically delivered MSCs safely improve heart function after acute myocardial infarction (MI). The multi-lineage potential of MSCs, in combination with their immunoprivileged status, makes MSCs a promising source of cell therapy for cardiovascular diseases [1].

Therapy using MSCs has limitations: age and other risk factors for cardiovascular disease reduce the availability of MSCs and impair their potential for differentiation and proliferation [2]. In order to overcome these limitations, MSCs could be genetically modified to improve their vasculogenic properties. Researchers have genetically modified MSCs with vascular endothelial growth factor (VEGF), stromal cell-derived factor-1alpha (SDF-1α), or Akt genes to enhance their vasculogenic function [3, 4]. These studies suggested that combination of angiogenic factor genes with MSCs for therapeutic neovascularization may be a potent strategy for treatment of severe ischemic diseases.

Vascular endothelial growth factor (VEGF) has been identified as a key component in the development of blood vessels, but VEGF alone may be insufficient to achieve functional and mature development of the vasculature. VEGF-induced vessels are often leaky and do not connect appropriately to the existing vasculature [5, 6]. SDF-1α is responsible for maturation of blood vessels. SDF-1α is not only a mobilization signal capable of recruiting CXCR4-positive progenitor cells into hypoxic tissues but also a retention signal for angiocompentent bone marrow-derived stem cells. It also recruits pericytes and smooth muscle cells to stabilize and mature newly formed blood vessels [7]. We hypothesized that a combination of VEGF165/SDF-1α genes and MSCs would augment angiogenesis and improve myocardial function in the infarcted myocardium. We investigated the effect of MSCs transfected with VEGF165 and the SDF-1α gene on neovascularization and remodeling of the myocardium in a model of myocardial infarction in rats.

Methods

Isolation and culture of MSCs and multi-differentiation of MSCs

Isolation and primary culture of MSCs has been described elsewhere [8]. Briefly, rat MSCs were isolated from bone marrow by density-gradient centrifugation. They were cultured in low-glucose Dulbecco’s modified Eagle’s medium DMEM (Gibco, USA) supplemented with 10% fetal calf serum (FCS; Hyclone, Co), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. The culture medium was removed and replaced with fresh medium twice a week. At confluence, cells were harvested for passage with 0.25% trypsin (Solon, OH, USA) containing 0.02% ethylene diamine tetra-acetic acid (EDTA; Sigma, St. Louis, MO, USA). Experiments were carried out using cells from the fourth passage.

For osteogenic differentiation, a 70% subconfluent culture of MSCs from passage two was used. The medium was replaced by osteogenic medium with 10−7 M dexamethasone, 0.2 mM ascorbic acid, and 10 mM β-glycerophosphate (all Sigma). After 15 days in osteogenic medium, cell colonies displayed bone-like nodular aggregates of matrix mineralization. von Kossa staining for calcium enabled visualization of mineral deposition. For adipogenic differentiation, the medium was replaced by adipogenic medium with 1 μM dexamethasone, 0.2 mM metacen, and 0.5 mM isobutyl methylxanthine (IBMX) and 10 μg/ml insulin (all Sigma). The medium was replaced every 3 days for 21 days. Fat cells could be visualized by oil-red staining.

MSCs transfected with adenoviral vectors and evaluation

Ad-VEGF (adenovirus expressing human VEGF165 and EGFP), and Ad-SDF-1 (adenovirus expressing human SDF-1α and EGFP), adenoviral vector containing hunan VEGF165 or human SDF-1α gene under the control of cytomegalovirus (CMV) promoter, and Ad-EGFP, an adenoviral vector containing the EGFP gene under the control of CMV promoter, have been used in studies from our research team [9, 10].

The transfection of MSC with adenovirus was carried out as previously described [9]. Briefly, MSCs were plated (10,000 cells/cm2) in six-well plates or T75 flasks and cultured overnight. Cells were exposed to fresh culture medium for MSCs containing Ad-SDF-1, Ad-VEGF, or Ad-EGFP at 100 multiplicities of infection (MOI; defined as pfu/cell) for 48 h. Cells were analyzed for transgene expression or used for ex-vivo gene therapy. Cell viability was determined by the Trypan blue exclusion method. Expression of the EGFP transgene in MSCs was evaluated under fluorescence microscopy. FACS was used to quantify the transfection efficiency of the adopted MOI according to the expression of EGFP [9].

Mesenchymal stem cells (MSCs) were incubated with Ad-SDF-1 or Ad-VEGF for 48 h, and the cell lysate assayed for SDF-1α, VEGF (1:250; Santa Cruz) and phosphorylated Akt (pAkt) (1:250; Cell Signaling) by Western blot and ELISA analysis, respectively.

Labeling of MSCs

Mesenchymal stem cells (MSCs) were stably transfected with Ad-SDF-1, Ad-VEGF, or Ad-EGFP as previously described. Two days after infusion, sterile 4,6-diamidino-2-phenylindole (DAPI) solution was added to the culture medium on the day of implantation at a final concentration of 50 μg/ml for 60 min. MSCs were rinsed six times in Hanks balanced salt solution to remove excess unbound DAPI. MSCs were harvested (approximately 5.0 × 106 cells for each implantation) and resuspended in the minimum volume of serum-free DMEM.

Cell implantation and the MI model

A rat myocardial infarction (MI) model leading to left ventricle dysfunction was used in present study. Male SD rats (250–300 g) were randomized to Ad-EGFP–MSC, Ad-VEGF–MSC, Ad-SDF–MSC, Ad-SDF–VEGF–MSC, and control groups. All procedures were performed in accordance with the Guidelines of the Hubei Council of Animal Care and approved by the Animal Use Subcommittee at the Yunyang Medicial Colllege, China.

Myocardial infarction (MI) was achieved by ligation of the left anterior descending (LAD) coronary artery as previously described [8]. Briefly, male SD inbred rats were anesthetized with ketamine (50 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). Tracheal ventilation was carried out with room air using a Colombus ventilator (HX-300, Taimeng Instruments, Chengdu, China). After left lateral thoracotomy in the fourth intercostal space, the LAD coronary artery was ligated. Before chest closure, infarction was confirmed by observation of injury demarcation with blanching of the myocardium and electrocardiography. Rats were randomized for each group. Seven days after MI induction, 5.0 × 106 Ad-EGFP–MSC, 5.0 × 106 Ad-VEGF–MSC, or 5.0 × 106 Ad-SDF–VEGF–MSC suspensions in 0.2 ml of serum-free medium were injected separately into four sites (0.05 ml per site) for each MI heart in the transplantation group with a 30-gauge tuberculin syringe. Two injection sites were in the myocardium bordering the infracted area, and two were within the infracted area. Control group received identical MI surgery, but only an equivalent volume of cell-free medium was injected. Penicillin (150, 000 U/ml, i.v.) was given before each operation and buprenorphine hydrochloride (0.05 mg/kg, s.c.) was administered twice a day for the first 48 h after surgery.

Cell apoptosis and survival assays in vitro

Mesenchymal stem cells (MSCs) were transfected with adenoviral vectors (Ad-SDF-1, Ad-VEGF, and Ad-EGFP) at the 100 MOI. The cells were incubated overnight under normoxic conditions (21% O2, 25% CO2, and balance N2), and the media were replaced. Then, the cells were exposed to hypoxia (1.0% O2) for 24 h. Cell apoptosis was assayed using terminal deoxynucleotidyl transferase biotin-dUPT nick end labeling (TUNEL). In brief, a TUNEL staining kit (Dead End Fluorometric TUNEL system, Promega, Madison, WI, USA) was used to visualize cell death in sections. After 10 min of fixing by 4% paraformaldehyde and pretreatment at −20°C with ethanol/acetic acid (2:1) and 0.2% Triton X-100, the sections were incubated in an equilibration buffer. The TdT enzyme and nucleotide mix were then added at proportions specified by the kit for 75 min at room temperature. Slides were washed twice with standard saline citrate for 15 min and followed by three washings with PBS.

The transplanted MSCs labeled with DAPI were identified in tissue sections by blue fluorescence. Survival cells number of DAPI-labeled MSCs in transplanted sites was given by the number of blue fluorescent cellular nuclei per HPW (×200). Cell survival change rate = treated MSC number/control MSCs number. Simultaneously, the quantitative analysis of survival MSCs was evaluated by real-time PCR of EGFP gene. Quantitative RT-PCR was performed using SYBR Green Reaction Mix (Eurogentec) on an ABI PRISM 7900HT Detection System (Applied Biosystems). Each sample was run in duplicate. The expression of EGFP gene within the different tissue samples was quantified relative to GAPDH expression. The primer sequences were as follows: EGFP (729 bp) Forward: 5′-ATGGTGAGCAAGGGCGAGGAGCTG-3′; Reverse: 5′-TTACTTGTACAGCTCGTCCATGCCGAG-3′; GAPDH (623 bp) Forward: 5′-CCAAAAGGGTCATCATCTCC-3′, Reverse: 5′-GTAGGCCATGAGGTCCACCAC-3′.

Western blotting

Western blot was performed with rabbit polyclonal antibody raised against VEGF, SDF-1 (1:250; Santa Cruz), and pAkt (1:250, cell signaling). The LV tissues were obtained from individual rats in different groups at different time points after cell transplantation (for each time point, n = 5). The tissues were homogenized on ice in 0.1% Tween-20 with a protease inhibitor A total of 50 μg of protein was loaded onto a 12% SDS gel and transferred to a polyvinylidene fluoride membrane (Millipore) following electrophoresis. After being blocked for 120 min, the membrane was incubated with the primary antibody followed by corresponding horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG 1:5,000 dilution, Santa Cruz). The immunoblots were detected by enhanced chemiluminescence reaction (Amersham pharmacia biotech, American) and measured with densitometry [8].

Measurement of hemodynamic parameters

Four weeks after treatment, hemodynamic measurements in vivo were done. Rats were anesthetized with pentobarbital sodium (60 mg/kg, i.p.). The carotid artery and femoral artery were isolated. Two catheters were filled with heparinized (10 U/mL) saline solution and connected to a Statham pressure transducer (Gould, Saddle Brook, NJ, USA). The carotid arterial catheter was advanced into the left ventricle to record ventricular pressure. The femoral artery catheter was inserted into an isolated femoral artery to monitor mean arterial pressure (BP) and heart rate (HR). These hemodynamic parameters were monitored simultaneously and recorded on a thermal pen-writing recorder (RJG-4122, Nihon Kohden, Japan) and on an FM magnetic tape recorder (RM-7000, Sony, Tokyo, Japan). The heart was rapidly removed from killed rats (each group, n = 11–13) after the measurements were taken [8].

Histology and morphometric measurement

At day-28 after treatment, the wet weight of rats was measured. Left ventricular (LV) function was assessed. Hearts were quickly removed and cut into six transverse slices (apex to base) and processed for histology. Briefly, left ventricles were sliced transversely into 5–6 sections (thickness, 2 mm) and incubated for 30 min in a 1% solution of buffered triphenyltetrazolium chloride (TTC) at 37°C. Slices were photographed with a digital camera with magnification to identify the infarcted myocardium (unstained by TTC) and the non-ischemic zones (stained brick-red). Two experienced technicians, who were blinded to the study protocol, did all the measurements for the treatment group. The thickness of the wall of the left ventricle (LV-wall thickness) was measured at three widely spaced locations within the scar segment, as well as the non-infarcted region, for each slide and the mean value calculated. In order to estimate the overall degree of ventricular remodeling, the expansion index was determined on day-28 using 10% paraformaldehyde-fixed tissue as previously described. Expansion index, as defined by Hochman and Choo, which is expressed as [(LV cavity area/total LV area) × (septum thickness/scar thickness)]. Analyses of LV-wall thickness and expansion index were done on two middle slides and the mean values calculated for each heart [8, 11].

Immunohistochemical analysis

Rats were killed 7 and 28 days after treatment (each time point, n = 5). After quick removal, hearts were immersion-fixed in 4% paraformaldehyde and embedded in optimum cutting temperature (OCT) compound (Fisher Scientific, Fair Lawn, NJ, USA). Serial transverse sections of 5 μm length were cut across the entire long axis of the heart and mounted on slides. Specimens were incubated with a monoclonal anti-mouse CD31 (1:200, ABCAM) antibody, alpha-smooth muscle actin (alpha-SMA; 1:250; Santa Cruz) antibody or anti-cTnt (1:250, NeoMarkers) antibody at 4°C overnight. Positive stains were shown as red fluorescence with tetramethyl rhodamine isothiocyanate (TRITC) IgG.

Specimens were also stained with anti-alpha-SMA antibody to confirm that mature vessels were similarly stained with anti-vwVIII factor antibody. The number of alpha-SMA-positive vessels was counted in a similar way to that for capillary density. Five fields from three sections were randomly selected from each sample, and the number of capillaries counted manually by two pathologists unaware of rat grouping. Capillary density was calculated as mean number of capillaries high-power field (×200). The transplanted MSCs labeled with DAPI were identified in tissue sections by blue fluorescence. The number of DAPI-labeled MSCs, and cTnt-positive myocardium density was counted in a similar way to that for capillary density.

Collagen volume fraction

In order to analyze collagen accumulation, Masson’s trichrome staining was done to delineate collagen content as percentage of infarction and peri-infarction area. Sixteen areas of high-power fields (×200) in each section were visualized under light microscopy and photographed at the same exposure time. The Collagen volume fraction (CVF) in the infarction and peri-infarction area was calculated as the percentage of stained tissue in the sum of muscle area and connective tissue by a densitometric method using Image Pro5.02 software.

Data analysis

Data are mean ± SD. Statistical significance between two groups was determined by unpaired Student’s t-test. Results for more than two experimental groups were evaluated by one-way ANOVA to specify differences between groups. P < 0.05 was considered significantly different.

Results

Characterization of MSCs

In order to characterize the phenotype of MSCs, the expression surface molecules were analyzed by FACS. Almost all cultured cells expressed CD29 and CD90, though they did not express CD34 and 45 (Fig. 1—Supplementary material). And, cultured MSCs (passage 1–5) showed that the characterization of CD90+, CD105+, CD166+, c-Kit+, CXCR4+, Oct4+, Nanglow, CD34low, CD133, CD31, and alpha or beta-MHC by reverse transcription polymerase chain reaction (RT–PCR; Fig. 1—Supplementary material). Expression of early cardiac markers (Nkx2.5/Csx, GATA-4, and MEF2C) gradually decreased; MSCs did not express these early cardiac markers after passage 3. The ability of MSCs to differentiate into osteocytes and adipocytes was tested in all cultures from various donors. When cultured in osteogenic medium for 15 days, the morphology changed: on day-1, a nearly confluent spindle-shaped layer was seen; days 5–7, cells formed nodular aggregates; and cells began to mineralize their matrix and were positive for Kossa staining on days 12–15 (Fig. 1—Supplementary material). They could also differentiate into adipocytes, and cells accumulated different numbers of lipid vacuoles (Fig. 1—Supplementary material) after cultivation in adipogenic medium.

Mortality

Among, the 150 rats used in this study, 21 died during the first 7 days after the surgery due to the severe MI, and 15 died by accident in 28 days after cell transplantation.

Transfection efficiency and expression of Ad-VEGF and Ad-SDF-1 in MSCs in vitro

This study constructed two Ad vectors expressing the coding regions of human VEGF165 (Ad-VEGF) and of human SDF-1α (Ad-SDF-1) under the control of a constitutive CMV promoter. The transfection efficiency for these viral vectors transfected into MSCs was high. The transfection efficiency for Ad-EGFP, Ad-SDF-1, and Ad-VEGF was 95%, 92%, and 93%, respectively (Fig. 2—Supplementary material).

In order to determine if cultured MSCs transfected with Ad-VEGF and Ad-SDF-1 could release soluble VEGF and SDF-1 protein, we collected conditioned medium from MSCs and assayed for VEGF and SDF-1 by ELISA. The SDF-1 levels secreted in the culture medium for the Ad-SDF-1–VEGF–MSC were 5-fold higher than those in the MSCs [(576.6 ± 70.2) vs. (102.4 ± 9.8) pg/ml, P < 0.05]. And, the VEGF levels secreted in the culture medium for the Ad-SDF-1–VEGF–MSC group were 6-fold higher than those in the control group. [(781.5 ± 103.7) vs. (137.5 ± 10.4) pg/ml, P < 0.05]. These data indicated that the Ad-delivered human SDF-1 and VEGF genes were working in MSCs.

Engraftment of MSCs without immune rejection

In order to confirm the engraftment and survival of MSCs in the implanted area, cultured MSCs were labeled with DAPI prior to transplantation, the labeling efficiency being 100% (Fig. 2—Supplementary material). Under fluorescence microscopy, the DAPI-labeled MSCs were observed in the myocardium 1 and 4 weeks after injection (Fig. 4—Supplementary material).There were some polymorphonuclear cells at the injection sites due to inflammation after surgery. However, there was no evidence of lymphocytic infiltration or immune rejection determined by a pathologist.

VEGF and SDF-1α enhanced the survival capability of MSCs in vitro and in vivo

In vitro, cell apoptosis under hypoxia was measured by TUNEL. The apoptosis of Ad-SDF-1–MSC, Ad-VEGF–MSC, and Ad-SDF-1–VEGF–MSC were 38%, 41%, and 30% that of Ad-EGFP-MSCs (Fig. 3—Supplementary material), respectively. We investigated if the Akt survival-promoting pathway is stimulated to uncover the survival mechanisms of MSCs transfected with Ad-VEGF or Ad-SDF-1. Three days after MSCs were transfected with Ad-VEGF or Ad-SDF-1, pAkt was measured by Western blotting. MSCs transfected with Ad-VEGF or Ad-SDF showed that the increased pAkt (Fig. 1a, b). Furthermore, PI3 K/Akt inhibitor wortmannin (100 nM) could abolish the anti-apoptosis effect of VEGF or SDF-1 on MSCs (Fig. 3—Supplementary material).
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Fig. 1

Expression of SDF-1 and VEGF protein in the infarcted hearts. a Western blotting analysis of phosphorylated Akt (pAkt) in MSCs transfected with Ad-VEGF and/or Ad-SDF-1. b Semi-quantitative analysis of pAkt protein levels by optical density value. Results are mean ± SD, n = 5, alpha-tubulin served as an internal control.* P < 0.05 versus other groups; # P < 0.05 versus Ad-EGFP–MSC and Ad-SDF-1–VEGF–MSC; # P > 0.05 versus Ad-VEGF–MSC. c Western blotting analysis of pAkt, SDF-1, and VEGF protein in the infarcted hearts. d Semi-quantitative assay of pAkt, SDF-1, and VEGF protein levels by optical density value. Results are mean ± SD, n = 5, alpha-tubulin served as an internal control. # P < 0.05 versus other groups; & P < 0.05 versus other groups; * P < 0.05 versus control, Ad-EGFP–MSC and Ad-SDF-1–MSC groups; $ P < 0.05 versus control, Ad-EGFP–MSC and Ad-VEGF–MSC groups;@ P < 0.05 versus control, Ad-EGFP–MSC and Ad-SDF-1–VEGF–MSC groups

Similarly, Western blotting analysis showed that the increased pAkt levels in the infarcted heart of Ad-SDF-1–MSC, Ad-VEGF–MSC, and Ad-SDF-1–VEGF–MSC transplantation (Fig. 1c, d). And, the number of DAPI labeling cells of the Ad-SDF-1–MSC, Ad-VEGF–MSC, and Ad-SDF-1–VEGF–MSC groups were 4.6 (±0.4)-, 4.3 (±0.5)-, and 7.6 (±0.6)-fold that of the Ad-EGFP–MSC group, respectively (P < 0.05), one week after cell implantation (Fig. 4—Supplementary material). Furthermore, more MSCs were detected in the infarcted hearts injected with Ad-SDF-1–VEGF–MSC than in the hearts injected with Ad-SDF-1–MSC or Ad-VEGF–MSC (P < 0.05) (Fig. 4—Supplementary material). More importantly, the quantitative analysis of survival MSCs showed that the similar results by real-time PCR of EGFP gene (Fig. 4—Supplementary material).

Expression of SDF-1 and VEGF protein in the infarcted hearts

Vascular endothelial growth factor (VEGF) and SDF-1 and protein increased in the Ad-EGFP–MSC, Ad-SDF-1–MSC, Ad-VEGF–MSC, and Ad-SDF-1–VEGF–MSC groups compared with control group at 7 days of treatment, and expression of VEGF and SDF-1 was more obviously increased in the ischemic myocardium in the Ad-SDF-1–VEGF–MSC group than in other groups (Fig. 1c, d).

Angiogenesis induced by Ad-SDF-1–VEGF–MSC in vitro and in vivo

In order to test the potential effect of SDF-1 or VEGF on MSCs differentiation toward the endothelial cell and cardiocytes, MSCs transfected with Ad-VEGF or Ad-SDF-1 were cultured for 14 days. After day-3 of treatment, MSCs started to express endothelial cell markers such as CD31 (Fig. 5—Supplementary material). We did not find that MSCs could differentiate into cardiomyocytes by RT–PCR and immunohistochemistry (Fig. 6—Supplementary material).

Immunofluorescence demonstrated that transplanted MSCs were positive for the cardiac marker cardiac troponin T. This result suggests that a few transplanted MSCs can differentiate into cardiomyocytes (Fig. 2). The area and number of cardiac myocytes within the infarct zone increased 2- to 4-fold in the Ad-SDF-1–VEGF–MSC group than in other groups. Simultaneously, some transplanted MSCs formed vascular structures in the myocardium, and were positive for CD31. The number of CD31+ cells was 3- to 5-fold more in the infarction area of the heart receiving Ad-SDF-1–VEGF–MSC than in other groups (Fig. 2). Combined administration of Ad-SDF-1/VEGF with MSC in the injured heart was positive for alpha-SMA and participated in vessel formation as mural cells (Fig. 2).
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Fig. 2

Effects of transplantation of MSCs transfected with Ad-VEGF and/or Ad-SDF-1 on myogenesis and angiogenesis. ad Transplanted MSCs showed that the cTnt-positive phenotype of cardiomyocytes 2 weeks after transplantation (white arrow). eh Transplanted MSCs showed that the CD31 positive phenotype of endothelial cells 2 weeks after transplantation (white arrow). il Transplanted MSCs showed that the alpha-SMA positive phenotype of vascular smooth muscle 2 weeks after transplantation (white arrow). DAPI showed that blue fluorescence and cTnt, CD31, and alpha-SMA showed that red fluorescence (×400)

Given that vwVIII factor antibody specifically identifies capillaries, we used vwVIII factor antibody to detect capillaries 28 days after intramyocardial transplantation. Mean values of capillary density in combined administration of Ad-SDF-1/VEGF with MSC in the injured heart were significantly higher than that of other groups (Fig. 3). An increase in larger vessels with positive anti-alpha-SMA antibody in the Ad-SDF-1–VEGF–MSC-, Ad-VEGF–MSC- and Ad-SDF-1–MSC-treated groups were also found (Fig. 4). There were significantly higher (P < 0.01) numbers of positive anti-alpha-SMA stained cells in Ad-SDF-1–VEGF–MSC (22.7 ± 4.5), Ad-VEGF–MSC (16.3 ± 2.2), and Ad-SDF-1–MSC groups (17.9 ± 1.9) compared with Ad-EGFP–MSC group (12.2 ± 1.7).
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Fig. 3

Effect of transplantation of MSCs transfected with Ad-VEGF and/or Ad-SDF-1 on capillary density. ae Representative image of vwVIII factor-positive capillary density in infarcted myocardium 4 weeks after transplantation. f Semi-quantitative assay of capillary density; Results are mean ± SD, n = 6 per group and * P<0.05 versus other groups; # P > 0.05 versus Ad-VEGF–MSC group

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Fig. 4

Effect of transplantation of MSCs transfected with Ad-VEGF and/or Ad-SDF-1 on alpha-SMA positive vessel density. ae Representative image of alpha-SMA positive vessel density in infarcted myocardium 4 weeks after transplantation. f Semi-quantitative assay of alpha-SMA positive vessel density; Results are mean ± SD, n = 6 per group and * P < 0.05 versus other groups; # P > 0.05 versus Ad-VEGF–MSC group

Effect of Ad-SDF-1–VEGF–MSC on histology and morphology of the heart

Representative Masson’s trichrome-stained sections showed that much more collagen in the myocardium, and decreased accumulation of collagen in the LV-wall-infarcted area and peri-infarction area 28 days after MI in the combination of VEGF/SDF-1 gene and MSCs than in the control and Ad-EGFP–MSC group. This change was more typical in the Ad-SDF-1–VEGF–MSC group than in the Ad-EGFP–MSC, Ad-VEGF–MSC, and Ad-SDF-1–MSC groups (Fig. 5a–j). Semi-quantitative analysis indicated that CVF in the infarcted area was significantly reduced in the Ad-SDF-1–VEGF–MSC group compared with the Ad-EGFP–MSC, Ad-VEGF–MSC, Ad-SDF-1–MSC, and control groups (32.7 ± 4.1% vs. 53.9 ± 5.4, 46.7 ± 6.3, 47.2 ± 4.3, and 81.5 ± 6.7%). The marked increases in mean LV-wall thickness were observed in the infarcted hearts of Ad-SDF-1–VEGF–MSC group (Fig. 5k). Infarct size was significantly reduced in the Ad-SDF-1–VEGF–MSC group compared with other groups (Fig. 5l). Quantitative analysis of cavitary dilatation in all hearts of the four groups revealed that Ad-SDF-1–VEGF–MSC rats displayed significantly less ventricular dilatation than other groups. The expansion index was significantly lower in the Ad-SDF-1–VEGF–MSC group than in other groups (Fig. 5m).
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Fig. 5

Effects of transplantation of MSCs transfected with Ad-VEGF and/or Ad-SDF-1 on histology and morphology of the heart. aj representative Masson’s trichrome-stained sections in the area of infarcted left ventricular tissue 4 weeks after treatment (blue, collagen; ae, ×3; fj, ×400). k Effects of transplantation of MSCs transfected with Ad-VEGF and/or Ad-SDF-1 on left ventricle wall thickness. Results are mean ± SD, n = 10/group, * P < 0.05 versus other groups; # P > 0.05 versus Ad-SDF-1–MSC group;& P < 0.05. vs. other groups. l Effects of transplantation of MSCs transfected with Ad-VEGF and/or Ad-SDF-1 on in infarct size. n = 10/group, * P < 0.05 versus other groups; # P > 0.05 versus Ad-VEGF–MSC group; & P < 0.05 versus other groups. m expansion index analyses in the MSCs transfected with Ad-SDF-1/Ad-VEGF groups were lower than control or Ad-EGFP-MSC groups. Results are mean ± SD, n = 10, * P < 0.05 versus other groups. # P > 0.05 versus Ad-VEGF–MSC group; & P < 0.05 versus other groups

Measurement of hemodynamic parameters

In order to test if transplantation of a combination of the VEGF/SDF-1 gene and MSCs into injured myocardium would further improve cardiac function, we measured hemodynamic parameters 4 weeks after transplantation. Measurement of hemodynamics in vivo showed that LV function was significantly improved in Ad-SDF-1–MSC, Ad-VEGF–MSC, Ad-EGFP–MSC, and Ad-SDF-1–VEGF–MSC groups than control group. And, in vivo measurement showed that LV function was significantly greater in Ad-SDF-1–MSC, Ad-VEGF–MSC, and Ad-SDF-1–VEGF–MSC groups than in Ad-EGFP–MSC groups. Furthermore, improvement of LV function was more obviously in Ad-SDF-1–VEGF–MSC group than in Ad-SDF-1–MSC and Ad-VEGF–MSC groups. However, the differences in left ventricular systolic pressure (LVSP), rate of rise and fall of ventricular pressure (±dP/dtmax) and left ventricular end-diastolic pressure (LVEDP) between Ad-VEGF–MSC and Ad-SDF-1–MSC groups were not obvious (Fig. 6).
https://static-content.springer.com/image/art%3A10.1007%2Fs11010-009-0374-0/MediaObjects/11010_2009_374_Fig6_HTML.gif
Fig. 6

Effects of transplantation of MSCs transfected with Ad-VEGF and/or Ad-SDF-1 on hemodynamic parameters. Left ventricular (LV) function under baseline resting conditions 4 weeks after transplantation; LVSP left ventricle systolic pressure, LVEDP left ventricle end-diastolic pressure, and +dP/dtmax and −dP/dtmax is rate of rise and fall of ventricular pressure, respectively. There were statistically significant differences among control, and Ad-EGFP–MSC and MSCs transfected with Ad-VEGF and/or Ad-SDF-1 groups (mean ± SD). Results are mean ± SD, n = 10, * P < 0.05 versus other groups; & P > 0.05 versus Ad-VEGF–MSC group; # P < 0.05 versus other groups

Discussion

Therapies using genes and stem cells hold promise for the treatment of ischemic cardiovascular disease. Locally delivered MSCs have promoted successful treatment of MI. This therapeutic intervention reduced the infarcted area and improved cardiac hemodynamics. Animal studies and preliminary clinical investigations showed that therapeutically delivered MSCs safely improve heart function after an acute MI. Recently, transfer of stem cells combined with VEGF and/or angiopoietin-1 genes has been shown to be more potent than stem cells alone in promoting vascular regeneration in ischemic heart and limbs [2, 12]. Mobilization of stem cells with cytokines has been demonstrated to potentiate VEGF or SDF-1 gene therapy for MI [13, 14]. Combination of VEGF and SDF-1 proteins synergistically induce neoangiogenesis in human ovarian cancers [15]. Combination therapy using stem cells and VEGF and SDF-1 genes for acute infarcted myocardium has not been reported. This study demonstrated, for the first time, that MSCs transfected with SDF-1 and VEGF genes are more potent than MSCs alone or MSCs transfected with SDF-1 or VEGF genes alone in reducing infarct size and fibrosis, increasing capillary density, and thickness of the left ventricle wall, and improving cardiac function in acute MI in rats.

Some of the engrafted MSCs were stained by cardiac protein such as cTnT, and some of the transplanted MSCs were also stained with CD31 and alpha-SMA. Combination of MSCs with the SDF-1 and/or VEGF gene in the injured heart could more efficiently differentiate into cardiomyocytes and vascular lineages in vivo. Although, MSCs alone expressed basal SDF-1 and VEGF protein, MSCs are superior to angiogenic growth factor genes for improving myocardial performance in a mouse model of acute MI [16]. MSCs transfected with SDF-1 and VEGF increased more SDF-1 and VEGF protein synthesis than MSCs alone. VEGF and SDF-1 not only improves survival of MSCs in infarcted hearts but also promotes differentiation of MSCs into cardiomyocytes and vascular lineages [8, 1620]. More surviving MSCs could provide a higher microenvironmental concentration of IGF-1 (insulin-like growth factor-1), HGF (hepatocyte growth factor), and bFGF (Basic fibroblast growth factor) besides VEGF and SDF-1 in transplantation sites [8, 9, 2123]. And, these growth factors could not only promote the survival of cardiomyocytes but also contribute to the regeneration of the infarcted myocardium through activation of cardiac stem cells mediated by growth factor-receptor systems [10, 24, 25]. Interestingly, MSCs participated in maintaining the bone-marrow stem cell niche [26]. In our study, we also found that the number of c-kit+ cardiac stem cells in the infarcted hearts was obviously increased in Ad-SDF-1–VEGF–MSC group than other groups (data not shown). Therefore, it is attractive to speculate that MSCs also participate in maintaining stem cell niches in the heart besides differentiation and paracrine signaling of MSCs. These mechanisms may explain the superiority of combined stem cell and gene therapy over stem cell or gene therapy alone.

Although, stem cells showed that a strong chemotactic response to SDF-1 in vitro, overexpression of SDF-1 alone, without concomitant expression of VEGF, was insufficient to retain stem cells. Overexpression of SDF-1 prevented the egress of stem cells from VEGF-overexpressing tissues when VEGF expression was switched-off. Pericytes and smooth muscle cells (i.e., mural cells) are entrapped around endothelial cells and have an accessory role in vessel growth (e.g., by releasing SDF-1) as well as in maturation and stability of vessels [5, 7]. MSCs express basal SDF-1 and VEGF protein, and tissue VEGF and SDF-1 increase in MI [8, 22, 23]. SDF-1 was induced in ischemic, inflamed, and malignant tissues in a VEGF-dependent manner. The CXCR4 (SDF-1 receptor) inhibitor AMD3100 abrogated entrapment of stem cells in tissues overexpressing VEGF [7]. A combination of SDF-1 and VEGF could enhance this effect of migration of exogenous stem cells (e.g., bone marrow-derived stem cells, circulating stem cells) and/or endogenous stem cells (e.g., cardiac stem cells) into infarcted hearts. Simultaneously, vascular cells, endogenous, and exogenous stem cells may sense SDF-1 and VEGF, and migrate to these areas, where they promote tissue formation by a mechanism that is probably different from angiogenic sprouting and which may resemble embryonic vasculogenesis [12, 27]. These mechanisms may explain why there are only marginal differences between the Ad-SDF-1–MSC group and Ad-VEGF–MSC group, and the superiority of the Ad-SDF-1–VEGF–MSC group over Ad-VEGF–MSC or Ad-SDF-1–MSC groups.

In addition to its potent angiogenic functions, VEGF and SDF-1 can stimulate proliferation, delay senescence, suppress apoptosis, and promote survival of various cells [17, 20, 28]. Activation of the pro-survival factor Akt is involved in VEGF- and SDF-1-mediated angiogenesis and cytoprotective responses [17, 2729]. VEGF and SDF-1 inhibited the apoptosis of endothelial cells and cardiac cells through Akt signaling pathways [17, 2831]. The increased protein levels of VEGF and SDF-1 activated Akt in the MSCs transfected with Ad-VEGF and/or Ad-SDF-1 and myocardium. Cardiomyocyte density, capillary density, and MSCs survival rate in the Ad-SDF-1–VEGF–MSC group was higher than that in Ad-VEGF–MSC or Ad-SDF-1–MSC groups. The possible mechanism may be involved in the higher therapeutic effect of implanted Ad-SDF-1–VEGF–MSC than Ad-VEGF–MSC or Ad-SDF-1–MSC transplantation. Ad-SDF-1–VEGF–MSC may provide a higher microenvironmental concentration of VEGF and SDF-1 in the transplantation sites. These changes could not only benefit the survival, proliferation, and differentiation of MSCs but also contribute to cardiomyocyte survival and angiogenesis mediated by SDF-1 and VEGF [8, 17, 2730]. These mechanisms may explain the superiority of Ad-SDF-1–VEGF–MSC over Ad-VEGF–MSC or Ad-SDF-1–MSC.

Several recent studies showed that overexpressing-SDF-1-MSCs did not differentiate into mature cardiac myocytes immediately or 24 h after MI, but these cells could improve heart function through increased cardiac myocyte survival, vascular density, and recruited cardiac stem cells [29, 32]. In our study, combination of MSCs with SDF-1 and/or VEGF gene in the injured heart could enable differentiation into cardiomyocytes and vascular lineages in vivo 7 days after MI. We presumed that this difference would be related to the time of cell transplantation after MI. Several studies showed that cell transplantation 1–2 weeks post-MI exerted better effects on increased survival of engrafted cells, angiogenesis, and functional cardiomyocytes in injured hearts than immediately, at 24 h or 4 weeks. Scar formation had not occurred and inflammation was reduced at these times, which should facilitate integration of transplanted cells and functional recovery [33].

We demonstrated that combination therapy with MSCs and gene therapy is superior to MSCs or gene therapy alone. It is known that MSCs express and secrete SDF-1, VEGF, and other cytokines important for angiogenesis, and SDF-1 activity is essential for endothelial cell survival, vascular branching and pericyte recruitment [24, 26]. Stem cells, combined with SDF-1 and VEGF genes transfer, could be synergistically more powerful than either alone for therapeutic angiogenesis and vasculogenesis. Although, there was no “gene therapy only” control group in the present study, the combination of SDF-1 and VEGF may have been very potent even without the cells. Interestingly, results from clinical trials, in which VEGF or SDF-1 was delivered in protein and gene forms were not impressive, which could be related with the characteristic of patient in clinical trials. These patients could be mostly people with coronary artery disease, diabetes mellitus, hyperlipidemia, aging, and many other factors. In this study, we used healthy rat and did not estimate the quality of MSCs, including their migratory and proliferation potential. However, aging and risk factors for coronary artery disease, diabetes, and hyperlipidemia have been found to contribute to a functional impairment of endogenous stem cells including MSCs, cardiac stem cells, endothelial progenitor cells, and hemopoietic stem cells [3436]. In addition, age and disease affect the tissue environment, in which the transplanted cells are injured [35]. Combination of genes and stem cells therapy could not only help the functional improvement (such as paracrine, survival, migratory, and proliferation potential) of transplanted MSCs but also contribute to the sustainable expression of the gene. Therefore, the combination therapy may provide useful approaches for enhancement of cell therapy for cardiovascular diseases.

In summary, combined therapy with MSCs and chemokine and angiogenic growth factor genes reduces infarct size, prevents remodeling, increases capillary density, and improves heart function in acute infarcted myocardium. Combined stem cells and chemokine and angiogenic growth factor genes therapy may represent a new approach to treat patients with ischemic disease refractory to conventional treatment.

Acknowledgments

We thank Professor Yongsheng Ren for his helpful instruction in heart function evaluation. We are grateful to Long Chen for help with immunofluorescence staining. We thank international science editing corporation for his helpful instruction in the article edition. This study was supported by grants from the National Natural Science Foundation of China (30700306), Hubei Natural Science Foundation (2005ABA079), Hubei Health Department Science Foundation (JX3B29), Hubei Education Department Science Foundation (Q200524003, B200624006), Yun Yang Medical College Science Foundation (2005QDJ01), and Shiyan Renmin Hospital Science Foundation, China.

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