VEGF is critical for stem cell-mediated cardioprotection and a crucial paracrine factor for defining the age threshold in adult and neonatal stem cell function

Abstract

Bone marrow mesenchymal stem cells (MSCs) may be a novel treatment modality for organ ischemia, possibly through the release of beneficial paracrine factors. However, an age threshold likely exists as to when MSCs gain their beneficial protective properties. We hypothesized that 1) VEGF would be a crucial stem cell paracrine mediator in providing postischemic myocardial protection and 2) small-interfering (si)RNA ablation of VEGF in adult MSCs (aMSCs) would equalize the differences observed between aMSC- and neonatal stem cell (nMSC)-mediated cardioprotection. Female adult Sprague-Dawley rat hearts were subjected to ischemia-reperfusion injury via Langendorff-isolated heart preparation (15 min equilibration, 25 min ischemia, and 60 min reperfusion). MSCs were harvested from adult and 2.5-wk-old neonatal mice and cultured under normal conditions. VEGF was knocked down in both cell lines by VEGF siRNA. Immediately before ischemia, one million aMSCs or nMSCs with or without VEGF knockdown were infused into the coronary circulation. The cardiac functional parameters were recorded. VEGF in cell supernatants was measured via ELISA. aMSCs produced significantly more VEGF than nMSCs and were noted to increase postischemic myocardial recovery compared with nMSCs. The knockdown of VEGF significantly decreased VEGF production in both cell lines, and the pretreatment of these cells impaired stem cell-mediated myocardial function. The knockdown of VEGF in adult stem cells equalized the myocardial functional differences observed between adult and neonatal stem cells. Therefore, VEGF is a critical paracrine mediator in facilitating postischemic myocardial recovery and likely plays a role in mediating the observed age threshold during stem cell therapy.

Figures

Fig. 1.

Adult stem cells produce more VEGF than neonatal stem cells after 72 h of culture. Transfection of cells with VEGF small-interfering (si)RNA significantly decreased VEGF production as seen by ELISA. Transfecting mesenchymal stem cells (MSCs) with Scramble siRNA did not significantly alter VEGF production compared with controls (A). Transfection of VEGF siRNA also significantly decreased adult and neonatal stem cell VEGF RNA expression as seen by PCR (B).

Fig. 2.

Intracoronary infusion of adult MSCs (aMSCs) provided significantly better cardioprotection compared with neonatal MSCs (nMSCs). This was seen in end-diastolic pressure (EDP; A), cardiac contractility (+dP/dt; B), and rate of myocardial relaxation (−dP/dt; C) at end reperfusion. These data allowed for the important appreciation that an age threshold may exist as to when stem cells can provide beneficial tissue protection (n = 5 animals/group).

Fig. 3.

Infusion of aMSCs that were transfected with VEGF siRNA significantly impaired postischemic myocardial recovery. End-diastolic pressure (A) was higher, and +dP/dt (B) and −dP/dt (C) were lower during reperfusion after the application of MSCs treated with VEGF siRNA compared with Scramble siRNA groups. No differences in myocardial function were observed between Scramble siRNA-transfected and nontransfected adult stem cell groups at end reperfusion (n = 5 animals/group).

Fig. 4.

Transfection of VEGF siRNA into nMSCs impaired stem cell-mediated myocardial function as noted by increased EDP (A) and a decreased percentage of baseline +dP/dt (B) and −dP/dt (C) during recovery. No significant differences in myocardial function were noted between Scramble siRNA-transfected and nontransfected nMSC groups (n = 5 animals/group).

Fig. 5.

Transfection of VEGF siRNA into aMSCs equalized the myocardial functional differences seen between adult and neonatal stem cell therapy. This was seen in EDP (A), +dP/dt (B), and −dP/dt (C). In fact, the percent recovery of +dP/dt and −dP/dt was lower in the VEGF siRNA aMSCs compared with Scramble siRNA-transfected nMSCs (n = 5 animals/group). #P < 0.05 vs. adult VEGF siRNA MSC groups.