Vascular endothelial growth factor (VEGF) as a key therapeutic trophic factor in bone marrow mesenchymal stem cell-mediated cardiac repair

Abstract

We recently demonstrated a novel effective therapeutic regimen for treating hamster heart failure based on injection of bone marrow mesenchymal stem cells (MSCs) or MSC-conditioned medium into the skeletal muscle. The work highlights an important cardiac repair mechanism mediated by the myriad of trophic factors derived from the injected MSCs and local musculature that can be explored for non-invasive stem cell therapy. While this therapeutic regimen provides the ultimate proof that MSC-based cardiac repair is mediated by the trophic actions independent of MSC differentiation or stemness, the trophic factors responsible for cardiac regeneration after MSC therapy remain largely undefined. Toward this aim, we took advantage of the finding that human and porcine MSCs exhibit species-related differences in expression of trophic factors. We demonstrate that human MSCs when compared to porcine MSCs express and secrete 5-fold less vascular endothelial growth factor (VEGF) in conditioned medium (40+/-5 and 225+/-17 pg/ml VEGF, respectively). This deficit in VEGF output was associated with compromised cardiac therapeutic efficacy of human MSC-conditioned medium. Over-expression of VEGF in human MSCs however completely restored the therapeutic potency of the conditioned medium. This finding indicates VEGF as a key therapeutic trophic factor in MSC-mediated myocardial regeneration, and demonstrates the feasibility of human MSC therapy using trophic factor-based cell-free strategies, which can eliminate the concern of potential stem cell transformation.

Figure 1

Left ventricular functional improvement mediated by human and porcine MSCs and MSC-conditioned medium. Echocardiography was performed in a blind-folded manner. Left ventricular ejection fraction (LVEF) was measured pre-injection, 2 weeks, and 4 weeks after initiation of injections. As reference, normal age-matched F1B hamsters maintain a stable LVEF of ~70%. Panel A: TO2 hamsters were injected with hMSCs and pMSCs. A saline injection control group was also included. Panel B: TO2 hamsters were injected with MSC-conditioned medium. Conditioned medium was also prepared from hMSCs overexpressing VEGF (designated as VEGF hMSC CM). n=6 per animal group. Anova analysis was performed for group comparisons. *P<0.005 vs. pre-injection; %P<0.05 vs. pre-injection; #P<0.005 vs. saline control.

Figure 2

Expression profiles of hMSC and pMSC trophic factors analyzed by qRT-PCR. Primer sequences were listed in Table 1. Total RNA isolated from hMSCs and pMSCs was analyzed by qRT-PCR. Threshold cycles were normalized based on the reference gene β2-microglobulin (B2M). n=3. *P<0.05 between hMSCs and pMSCs.

Figure 3

Measurement of VEGF concentrations by ELISA. Conditioned medium was prepared from hMSCs, pMSCs, and hMSCs overexpressing VEGF. The medium was assayed by a VEGF ELISA kit. VEGF concentrations were expressed as pg/ml shown above each bar. n=3. *P<0.001 compared to hMSC CM.