VEGF/SDF-1 promotes cardiac stem cell mobilization and myocardial repair in the infarcted heart

Abstract

Aims: The objective of this study was to investigate whether vascular endothelial growth factor (VEGF) secreted by mesenchymal stem cells (MSC) improves myocardial survival and the engraftment of implanted MSC in infarcted hearts and promotes recruitment of stem cells through paracrine release of myocardial stromal cell-derived factor-1α (SDF-1α).

Methods and results: VEGF-expressing MSC ((VEGF)MSC)-conditioned medium enhanced SDF-1α expression in heart slices and H9C2 cardiomyoblast cells via VEGF and the vascular endothelial growth factor receptor (VEGFR). The (VEGF)MSC-conditioned medium markedly promoted cardiac stem cell (CSC) migration at least in part via the SDF-1α/CXCR4 pathway and involved binding to VEGFR-1 and VEGFR-3. In vivo, (VEGF)MSC-stimulated SDF-1α expression in infarcted hearts resulted in massive mobilization and homing of bone marrow stem cells and CSC. Moreover, VEGF-induced SDF-1α guided the exogenously introduced CSC in the atrioventricular groove to migrate to the infarcted area, leading to a reduction in infarct size. Functional studies showed that (VEGF)MSC transplantation stimulated extensive angiomyogenesis in infarcted hearts as indicated by the expression of cardiac troponin T, CD31, and von Willebrand factor and improved the left ventricular performance, whereas blockade of SDF-1α or its receptor by RNAi or antagonist significantly diminished the beneficial effects of (VEGF)MSC.

Conclusion: Exogenously expressed VEGF promotes myocardial repair at least in part through SDF-1α/CXCR4-mediated recruitment of CSC.

Figure 1

Transplantation of ^VEGFMSC enhanced SDF-1α expression in infarcted hearts. (A) Immunostaining of SDF-1α. The expression of SDF-1α and cardiomyocyte marker cTnt was examined in both infarction and peri-infarction areas from ^VEGFMSC-transplanted hearts. DAPI stained nuclei. Bar represents 50 µm. (B) VEGF induced SDF-1α expression in infarcted heart tissues. Sham-operated and infarcted heart tissues without (Ctrl) or with ^LacZMSC or ^VEGFMSC transplantation were isolated 7 days after treatment. Total proteins were extracted followed by western blotting to detect the expression of SDF-1α and VEGF as indicated. α-Tubulin served as an internal control (n= 5). (C) Quantitative analysis of SDF-1α and VEGF expression (n = 9). *P < 0.01 vs. Ctrl; ^#P < 0.01 vs. LacZ. (D) Comparison of VEGF-induced expression of SDF-1α from the (peri) infarcted tissue and transplanted ^VEGFMSC. SDF-1α and VEGF expression from rat (peri) infarcted heart and transplanted ^VEGFMSC was examined by ELISA (n = 5). For rat SDF-1α (rSDF-1α) expression, ^▾P < 0.01 vs. Ctrl; *P < 0.05 vs. LacZ. For ^VEGFMSC-expressed human SDF-1α (hSDF-1α) expression, ^#P <0.01 vs. LacZ. For Rat VEGF (rVEGF) expression, ^▴P < 0.01 vs. Ctrl; ^&P < 0.01 vs. LacZ. For ^VEGFMSC secreted human VEGF (hVEGF) expression, ^$P < 0.01vs. LacZ.

Figure 2

^VEGFCM stimulated CXCR4 expression, leading to increased migration of CSC. (A and B) Flow cytometric analysis showed that untreated CSC expressed VEGF receptors Flt-1 and Flk-1. (C–E) ^VEGFCM stimulated CXCR4 expression in CSC 24 h after ^VEGFCM treatment. CSC were untreated (Control, C), or treated with ^VEGFMSC-conditioned medium (^VEGFCM, D) or ^VEGFCM and VEGFR1 inhibitor AMG (E) followed by flow cytometry. AMG inhibited the expression of ^VEGFCM-induced CXCR4. (F) Quantitative analysis of CXCR4 expression by flow cytometry. CSC were treated with ^CtrlCM or ^VEGFCM with or without VEGFR specific inhibitors for VEGFR1 (AMG), VEGFR2 (VEGFR2-I), and VEGFR3 (MAZ). *P < 0.05 vs. control, AMG, VEGFR2-I. and MAZ; ^#P < 0.05 vs. AMG and MAZ (n = 5). (G) ^VEGFCM promoted CSC migration. *P < 0.05 vs. ^CtrlCM; ^▴P <0.01 vs. ^CtrlCM; ^&P < 0.05 vs. ^CtrlCM+AMG and ^CtrlCM+MAZ (n = 15); ^@P < 0.01 vs. ^CtrlCM; ^▾P <0.01 vs. ^CtrlCM; ^$P < 0.05 vs. ^VEGFCM+AMG and ^VEGFCM+MAZ. (H) ^VEGFCM-mediated CSC migration was blocked by CXCR4 inhibitor AMD3100. ^CTLCM was used as a migration control (n = 15). *P <0.01vs. ^CtrlCM; ^#P <0.01 vs. ^LacZCM; ^$P <0.01 vs. ^CtrlCM and ^LacZCM; ^&P <0.01 vs. ^VEGFCM.

Figure 3

^VEGFMSC induced endogenous CSC mobilization through SDF-1α in infarcted hearts. PKH26-labelled ^VEGFMSC were implanted in the ischaemic myocardium. Seven days later, the mobilization of endogenous CSC to the heart region with ^VEGFMSC engraftment was examined by immunostaining of stem cell markers c-kit and MDR1 as shown in green. Mobilized cells were shown as green (white arrow). DAPI stained nuclei (blue) (400×). (A) c-Kit⁺ stem cells in the infarcted hearts after MI. (B and C) c-Kit⁺ stem cells (green) induced by ^LacZMSC was significantly reduced by AMD3100 treatment (C) when compared with the vehicle-treated group (B). (D–F) c-Kit⁺(D) and MDR1⁺(E) stem cells (green) induced by ^VEGFMSC was significantly reduced by AMD3100 treatment (F) when compared with the vehicle-treated groups. (G) Control shRNA (shCtrl) had no inhibition effect on the mobilization of CSC induced by ^VEGFMSC. (H) SDF-1α shRNA (shSDF) significantly inhibited the mobilization of CSC induced by ^VEGFMSC. (I and J) The numbers of the c-kit⁺ and MDR1⁺ cells in infarcted hearts were counted and statistical analysis was performed (n = 5). Bar represents 25 µm. ^#P =0.0001 vs. Ctrl (no implantation); ^▾P <0.01 vs. Ctrl; *P <0.05 and ^▴P <0.01 vs. LacZ; ^&P <0.01, and ^$P <0.01 vs. VEGF; ^@P >0.01 vs. VEGF.

Figure 4

^VEGFMSC stimulated exogenous CSC migration in vivo. (A–D): ^VEGFMSC stimulated CSC migration via CXCR4. PKH26-labelled CSC were implanted into the atrioventricular groove of the infarcted hearts (A and B). ^VEGFMSC were transplanted into the infarcted and peri-infarction areas of the hearts that was simultaneously implanted with CSC untreated (C and D) and treated (E and F) by AMD or ^VEGFMSC with SDF-1α shRNA (shSDF-1) (G and H) were transplanted into the infarcted and peri-infarction areas of the hearts that was simultaneously implanted with CSC. (I and J) Quantitative analysis of the migration of PKH26-labelled CSC to the infarcted and peri-infarction areas (n = 5). Bar represents 25 µm. *P <0.01 vs. CSC-implanted group; ^&<0.01 and ^#P <0.01 vs. VEGF/CSC-implanted groups.

Figure 5

^VEGFMSC promoted angiomyogenesis in infarcted myocaridum. (A–C) ^VEGFCM-H9C2 or ^VEGFCM-HS promoted the differentiation of CSC into cardiomyocytes and endothelial cells in vitro. CSC were cultured in conditioned medium of H9C2 cells (A) or HS (B and C) that were cultured in ^VEGFMSC-conditioned medium. The differentiation of cardiomyocytes and endothelial cells was detected by the expression of cTnt (A and B) and endothelial marker CD31 (C), respectively. Bar represents 25 µm. (D–H) ^VEGFMSC induced angiogenesis through SDF-1α/CXCR4 in vivo. ^LacZMSC (LacZ), ^VEGFMSC (VEGF), ^VEGFMSC with AMD3100 (VEGF/AMD) or ^VEGFMSC transduced with adenovirus expressing SDF-1α shRNA (VEGF/shSDF) were implanted in the infarcted myocardium. 28 days later, the myocardium was harvested and the angiogenesis in infarcted areas was detected by the expression of vwFVIII with immunostaining (green). Bar represents 50 µm. (I) Quantitative analysis of blood vessel density in infarcted areas (n = 10). ^#P <0.01 vs. Ctrl; ^▾P <0.01 vs. Ctrl; *P <0.01 vs. LacZ; ^▴P <0.01 vs. LacZ; ^&P <0.01 vs. VEGF; ^$P <0.01 vs. VEGF; ^@P >0.05 vs. VEGF.

Figure 6

^VEGFMSC transplantation improved haemodynamics. (A) ^VEGFMSC partially restored structural damage of myocardium caused by ischaemia. 28 days after infarction, hearts were removed and Masson's trichrome staining was performed. (B–E) ^VEGFMSC reduced infarction size and improved the left ventricular (LV) function of infarcted hearts. ^LacZMSC (LacZ), ^VEGFMSC (VEGF), or ^VEGFMSC transduced with adenovirus expressing SDF-1α shRNA (VEGF/shSDF) were implanted in the infarcted myocardium. AMD3100 treatment was described in Methods. LV function of infarcted hearts was measured under baseline resting conditions 4 weeks after treatment. (B) Infarction size (n = 10). (C) Left ventricular systolic pressure (LVSP) (n = 10). (D) Left ventricular end-diastolic pressure (LVEDP). (E and F) Rate of rise and fall of ventricular pressure [+dP/dt_max (E) and –dP/dt_max (F)] (n = 10), ^#P <0.05 vs. Ctrl; ^▾P <0.05 vs. Ctrl; *P <0.05 vs. LacZ; ^▴P< 0.05 vs. LacZ; ^&P < 0.05 vs. VEGF; ^$P < 0.05 vs. VEGF; ^@P>0.05 vs. VEGF.