PlGF repairs myocardial ischemia through mechanisms of angiogenesis, cardioprotection and recruitment of myo-angiogenic competent marrow progenitors

Abstract

Rationale: Despite preclinical success in regenerating and revascularizing the infarcted heart using angiogenic growth factors or bone marrow (BM) cells, recent clinical trials have revealed less benefit from these therapies than expected.

Objective: We explored the therapeutic potential of myocardial gene therapy of placental growth factor (PlGF), a VEGF-related angiogenic growth factor, with progenitor-mobilizing activity.

Methods and results: Myocardial PlGF gene therapy improves cardiac performance after myocardial infarction, by inducing cardiac repair and reparative myoangiogenesis, via upregulation of paracrine anti-apoptotic and angiogenic factors. In addition, PlGF therapy stimulated Sca-1(+)/Lin(-) (SL) BM progenitor proliferation, enhanced their mobilization into peripheral blood, and promoted their recruitment into the peri-infarct borders. Moreover, PlGF enhanced endothelial progenitor colony formation of BM-derived SL cells, and induced a phenotypic switch of BM-SL cells, recruited in the infarct, to the endothelial, smooth muscle and cardiomyocyte lineage.

Conclusions: Such pleiotropic effects of PlGF on cardiac repair and regeneration offer novel opportunities in the treatment of ischemic heart disease.

Figure 1. pPlGF1 upregulates angiogenic factors in ischemic myocardium.

A : Real-time PCR revealed that gene expression of hPlGF was detectable in fibrosis and peri-infarct areas 4 days post pPlGF1 gene transfer. B , C , Real-time PCR revealed that gene expression of rPlGF and rVEGF in fibrosis and peri-infarct areas was significantly enhanced in the pPlGF1 group compared with controls. D , E , Real-time PCR revealed that expression of rAng-1 in peri-infarct and remote areas and that of rAng-2 in all areas significantly augmented in the pPlGF1 group compared with controls. *, P<0.05 vs PBS; †, P<0.05 vs Mock. (n = 8 in each group).

Figure 2. Angiographical or histological evaluation of LV remodeling and myocardial neovascularization after MI.

A , Representative microangiographic images 28 days after PBS, Mock or pPlGF1 injection (7.0×7.0 mm; scale bar; 100 µm) (arrow: ligation point). B , Representative histochemical staining for isolectin B4 at day 28 (×20). C , Representative Masson-trichrome staining at day 28. D , Capillary density in rats receiving pPlGF1, Mock or PBS at day 28. **, P<0.01 vs PBS; ††, P<0.01 vs Mock. E , Ratio of fibrosis area/entire LV area (% fibrosis area) at day 28. **, P<0.01 vs PBS; ††, P<0.01 vs Mock. (n = 8 in each group). LV functional evaluation by echocardiography. F , Representative recording of M-mode echocardiography 5 and 28 days after pPlGF1, Mock or PBS injection (arrow, endocardium in lateral wall; arrowhead, endocardium in septal wall). G , Changes in echocardiographic parameters between day 5 and day 28 after gene transfer (n = 8 in all groups). FS, fractional shortening; RWMS, regional wall motion score. **, P<0.01 vs PBS; ††, P<0.01 vs Mock. (n = 8 in each group).

Figure 3. pPlGF1 reduces cardiomyocyte (CMC) apoptosis.

A : Cardiac apoptosis was detected by the TdT-mediated dUTP nick end-labeling (TUNEL) assay (green) and cardiac troponin-I (cTn-I) staining. White arrows showed TUNEL-positive CMCs in infarcted myocardium (apoptotic CMCs). B : Bar graph indicates % apoptotic CMCs, which was calculated as the ratio of the number of TUNEL-positive CMCs to the number of total CMCs in the infarcted myocardium. The % apoptotic CMCs on day 4 significantly decreased following pPlGF1 injection compared to Mock and PBS administration. *, P<0.05 vs PBS; †, P<0.05 vs Mock. C : Real-time PCR demonstrated significant upregulation of endogenous IGF-1 in infarcted and peri-infarct areas and IGFR at peri-infarct area after pPlGF1 gene transfer compared with the controls. D : Immunoblotting for phospho-Akt and phospho-p44 or p42 of MAPK revealed enhanced expression of these proteins in the pPlGF1 group than controls. E : Immunoblotting indicated greater expression of Bcl-x_L protein in the pPlGF1 group than in the controls. (n = 6 in each group).

Figure 4. pPlGF1 auguments mobilization of BM progenitors into PB and their recruitment into ischemic myocardium.

A , Representative recording of serial FACS analyses of PB GFP⁺/lin⁻ cells isolated from xeno-rats in each group. The number of PB GSL cells 7 days after MI was significantly increased in the pPlGF1 group than controls. *, P<0.05 vs PBS; †, P<0.05 vs Mock. B , Double immunofluorescent staining for Sca-1 and GFP (green) in infarcted myocardium of each group at day 7. Arrows show the double positive cells, which indicate BM-derived immature cells were incorporated into the ischemic area. C , The bar graph showing the number of Sca-1+/GFP+ cells in the ischemic area indicates enhanced recruitment of the BM-derived immature cells by pPlGF1 gene transfer at day 7. **, P<0.01 vs PBS; ††, P<0.01 vs Mock. D , Migratory response of PB-SL cells toward different dosages of PlGF by modified Boyden chamber migration assay. *, P<0.05 vs 0 ng/ml (control). E , Real-time PCR demonstrated that pPlGF1 significantly augmented expression of rSDF-1 mRNA at the peri-infarct area 4 days after MI compared with controls. *, P<0.05 vs PBS; †, P<0.05 vs Mock. F–H , Representative immunofluorescent staining for SDF-1 in PBS (F), Mock (G) or pPLGF1 (H) group 7 days after gene transfer. Red fluorescence indicates SDF-1 protein in cytoplasms. I , Quantification of SDF-1+ cells at peri-infarcted area in rats receiving pPlGF1, Mock or PBS at day7. SDF-1+ cells were significantly increased following pPlGF1 gene transfer. **, P<0.01 vs PBS; *, P<0.05 vs Mock; ††, P<0.01 vs Mock. (n = 8 in each group).

Figure 5. Representative double immunofluorescent staining for immature cardiomyogenic or vasculogenic markers and GFP or Sca-1 at day 7.

A , B , Double immunofluorescent staining for GFP and GATA4 (A) or MEF2C (B) at day 7. BM-derived immature CMCs were identified as cells positive for cytoplasmic GFP (green) and nuclear GATA4 (A) or MEF2C (B) following pPlGF1 transfer. White arrows show nuclei of immature BM-derived CMCs (A and B). C , D, Double immunofluorescent staining for Sca-1 and GATA4 (C) or MEF2C (D) at day 7. BM-derived immature CMCs were identified as cells positive for Sca-1 and nuclear GATA4 (green) (C) or MEF2C (green) (D) following pPlGF1 transfer. White arrows show nuclei of immature BM-derived CMCs (C and D). (n = 6 in each group). Histological evaluation of development of BM-derived stem/progenitor cells into CMCs in rat ischemic myocardium at day 28. E , F : Representative double immunofluorescent staining for cTn-I and GFP at day 28. BM-derived CMCs were identified as double positive cells for cTn-I (green) and GFP. E, merge in pPlGF1 group, ×10; F, merge in pPlGF1 group, ×40. White arrows show nuclei of BM-derived CMCs. G , H: Representative double immunofluorescent staining for cardiac troponin-I (cTn-I) and GFP in Mock and PBS groups at day 28. BM-derived CMCs were identified as double positive cells for cTn-I (green) and GFP (red). G, merge in Mock group, ×40; H, merge in PBS group, ×40. (n = 6 in each group).