Assessment of the Impact of Enforced Expression of Platelet Derived Growth Factor Beta in Human Mesenchymal Stem Cells and Contribution to Blood Flow Restoration.

Abstract 3568

Poster Board III-505

Both mesenchymal stem cells (MSC) and platelet derived growth factor beta (PDGFB) have been shown to promote angiogenesis in vivo and are therefore interesting therapeutic agents for the restoration of blood flow to ischemic tissues encountered in peripheral artery disease and critical limb ischemia. Careful evaluation of the mechanisms responsible for neovessel formation in stem cell-based therapeutic angiogenesis is critical for developing efficient treatment strategies. Previous studies have shown that MSCs correspond to pericytes, which are lost in PDGFB-null mice, suggesting that MSCs strongly rely on PDGFB signaling in vivo. Our major aim was to establish a highly sensitive method to assess whether overexpression of PDGFB in MSCs could modulate expression of pro-angiogenic cytokines and improve the restoration of bloodflow in an established murine xenograft model of hind limb ischemia. Human bone marrow derived MSCs (passage 2-5) were transduced with either a lentiviral vector containing the coding sequence of PDGFB or the same vector without PDGFB (control). As expected, overexpression of PDGFB strongly enhanced the proliferation of MSCs, reducing the doubling time from 51 to 20 hours (n=4, p<0.05). This effect correlated with a shift of 15% of cells from the G0/G1 to the S/G2/M phase, as compared to controls, when measured by flow cytometry of propidium iodide stained cells. Next, we induced a unilateral hind limb ischemia in NODSCIDbeta2null mice by excision of the femoral artery and performed intravenous transplantation of 2.5×10e5 MSCs (control MSC vs. PDGFB-MSC) the day following surgery. Recovery of blood flow to the affected limb was assessed at days 0 ((before transplantation), n=11), 4 (n=11), 7 (n=9), 10 (n=6), and 14 (n=6) by laser doppler imaging and values were normalized to the non-ischemic contralateral leg. Treated animals of both groups (control and PDGFB overexpressing MSC) exhibited a steadily progressive recovery in blood flow in the region proximal to the primary ligation site, reaching near normal levels by day 10. Surprisingly, no significant difference was seen in the blood flow recovery in animals receiving the PDGF over expressing MSCs. Donor MSCs (control and PDGFB) homed to the site of injury, as assessed by immunofluorescent staining of the ischemic muscle in the regions proximal and distal to the primary ligation site at days 4 and 7. However, the rapid recovery in blood flow did not appear to be attributed to a direct differentiation or integration of MSC into the vasculature, but rather through the expression of paracrine angiogenic factors. Interestingly, overexpression of PDGF reduced FGF2 mRNA expression levels in MSC (n=4, p=0.056), suggesting that high levels of PDGFB may indeed lead to a reduction of other key angiogenic factors, explaining the mild effect of PDGFB in vivo. Our results show that, although overexpression of PDGFB in bone marrow-derived MSCs enhanced the proliferation of MSC in vitro, it did not impact the speed or magnitude of blood flow recovery to the ischemic hind limb as compared to native MSCs, likely due to down modulation of other key angiogenic signals including FGF2.