Hepatoma-Derived Growth Factor Secreted from Mesenchymal Stem Cells Reduces Myocardial Ischemia-Reperfusion Injury

Figures

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  Figure 1. MSC-CdM reduce cardiac reperfusion injury. Wild-type mice were given 5 mg/kg CF-CdM, 5 mg/kg MSC-CdM, or vehicle i.v. 15 min before 45 min of ischemia. MSC-CdM: conditioned medium derived from MSC; CF-CdM: conditioned medium derived from cardiac fibroblasts. (a) Ratio of area at risk (AAR) to left ventricular (LV) area, ratio of infarct size (I) to AAR, and ratio of infarct size to LV after 24 h of reperfusion. Data represent mean ± standard error of mean (SEM) of values from five mice. (b) The maximum rates of rise and decline of left-ventricular pressure (+dp/dtmax and −dp/dtmax) assessed at 24 h reperfusion. Data are mean ± SEM of values from six mice. (c) TUNEL staining at 24 h reperfusion; apoptotic nuclei were stained (red), and cardiomyocytes were detected by cardiac troponin I staining (green). Bar = 50 μM. Data are mean ± SEM of values from three hearts per group, with at least 3000 nuclei examined per heart. ∗P < 0.05.

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  Figure 2. Secretome patterns and different HDGF expression between MSCs and fibroblasts. (a) iTRAQ analysis was applied and 1596 proteins were identified. Hierarchical clustering displayed as a heatmap. Red indicates an increase in expression, and green indicates decreases in expression compared with fibroblasts. (b) HDGF mRNA levels of MSCs and fibroblasts were assessed by RT-qPCR. Data represent mean ± SEM of values from three determinations. (c) Western blot assays on proteins precipitated from the MSC-CdM and CF-CdM. Representative of three independent experiments. ∗∗P < 0.01.

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  Figure 3. HDGF contributed to the protective effects of MSC-CdM. Conditioned medium that collected from MSCs transfected with vector (MSCnull-CdM) or HDGF shRNA lentivirus (MSCHDGF KD-CdM) were treated to H9C2 cells subjected to 9 h of hypoxia followed by 4 h of reoxygenation. (a) Cell death was evaluated with flow cytometry analysis. Annexin V−/PI−: viable cells; Annexin V+/PI−: early apoptotic cells; Annexin V+/PI+: late apoptotic or necrotic cells; Annexin V−/PI+: necrotic cells. (b) Cleaved caspases-3 as detected by Western blotting. Data represent mean ± SEM of values from three determinations. ∗P < 0.05.

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  Figure 4. HDGF reduced apoptosis and activated PKCε pathway. H9C2 cells treated by recombinant mouse HDGF (100 nmol/L) or vehicle control were subjected to 9 h of hypoxia followed by 4 h of reoxygenation. (a) Cell death was evaluated with flow cytometry analysis. (b) Cleaved caspases-3 as detected by Western blotting. Data represent mean ± SEM of values from three determinations. (c) Phosphorylation of PKCε as detected by Western blotting. Data represent mean ± SEM of values from three determinations. (d) Mitochondria were isolated from H9C2 cells and activity of ALDH2 in mitochondria was measured. Data represent mean ± SEM of values from three mice. (e) 4-HNE protein adducts in H9C2 cells were assessed. Data represent mean ± SEM of values from four determinations. ∗P < 0.05.

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  Figure 5. PKCε contributed to HDGF-induced reduction of reperfusion injury. PKCε dominant negative mice (PKCε-DN) and wild-type (WT) littermates with or without 50 μg/kg recombinant mouse HDGF treatment intramyocardially were subjected to 45 min of cardiac ischemia followed by 24 h reperfusion. (a) Ratio of area at risk to left ventricle area (AAR/LV), ratio infarct size to AAR ratio (I/AAR), and ratio of infarct size to LV area (I/LV) of hearts were assessed. Data represent mean ± SEM of values from five mice. (b) The maximum rates of rise and decline of left-ventricular pressure (+dp/dtmax and −dp/dtmax) assessed at 24 h reperfusion. Data are mean ± SEM of values from six mice. (c) Quantitative analysis of TUNEL positive cells at 24 h reperfusion. Data are mean ± SEM of values from three hearts per group, with at least 3000 nuclei examined per heart. (d) Mitochondria were isolated from heart tissue after reperfusion injury and the activity of ALDH2 in mitochondria was measured. Data represent mean ± SEM of values from three mice. (e) 4-HNE protein adducts in heart tissues was assessed. Data represent mean ± SEM of values from three mice. ∗P < 0.05.