Pro‐BDNF Contributes to Hypoxia/Reoxygenation Injury in Myocardial Microvascular Endothelial Cells: Roles of Receptors p75NTR and Sortilin and Activation of JNK and Caspase 3

Figures

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  Figure 1. Effects of H/R on the apoptosis and pro-BDNF expression among MMECs exposed to HG. (a, b) Representative images of the TUNEL assay of MMECs exposed to HG without (control) or with (H/R group) H/R. (c) The percentage of TUNEL-positive cells. H/R significantly increased the percentage of TUNEL-positive cells among MMECs, indicating the induction of apoptosis. (d, e) Immunostaining results on the pro-BDNF protein expression and a TUNEL assay. (f, g) Representative Western blots and quantitative analysis of pro-BDNF protein. H/R markedly increased the expression of pro-BDNF. The data were analyzed by the t-test. The error bars represent SEM. ∗P < 0.05 as compared with the control group.

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  Figure 2. Overexpression of pro-BDNF in MMECs and its effect on MMEC apoptosis. (a–e) MMECs were transfected with either pro-BDNF or Ad-GFP. Immunostaining, Western blotting, and quantitative analysis showed that the protein expression of pro-BDNF increased in MMECs after transduction with pro-BDNF. (f–h) Transfected cells were exposed to HG and then subjected to a TUNEL assay (f, g) and enumeration of TUNEL-positive cells (h) to evaluate apoptosis. Pro-BDNF overexpression markedly elevated the numbers of TUNEL-positive cells. The data were analyzed by the t-test. The error bars represent SEM. ∗P < 0.05 as compared with the control group or Ad-pro-BDNF group.

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  Figure 3. Effects of the anti-pro-BDNF antibody on apoptosis, migration, and capillary-like-structure formation among MMECs after exposure to HG and H/R. (a) Effects of pro-BDNF on apoptosis were analyzed by flow cytometry of MMECs after different treatments: control, H/R, H/R + anti-pro-BDNF, and H/R + vehicle. (b, c) The functional effects of pro-BDNF on MMECs were assessed by capillary-like-structure formation and cell scratch assays. (d) Relative apoptosis levels and fold changes are expressed in relation to the control group. The H/R group showed markedly increased relative apoptosis levels, decreased capillary-like-structure formation, and reduced cell migration when compared with the control group. These effects were reversed by treatment with the anti-pro-BDNF antibody to the levels similar to those in the control group. The data were subjected to one-way ANOVA. The error bars represent SEM. ∗P < 0.05 as compared with the control group; #P < 0.05 as compared with the H/R group or the H/R + vehicle group.

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  Figure 4. Effects of the anti-pro-BDNF antibody on the expression of p75NTR and sortilin and apoptosis-related proteins. (a) Representative immunofluorescent images of pro-BDNF (red, first column), p-JNK (green, second column), BDNF (red, fourth column), JNK (green, fifth column), sortilin (red, seventh column), and p75NTR (green, eighth column) in groups control, H/R, H/R + anti-pro-BDNF, and H/R + vehicle. (b–e) Representative Western blots and quantitative analysis of pro-BDNF, BDNF (b), p75NTR, sortilin (c), JNK, p-JNK (d), caspase 3 and cleaved-caspase 3 expression (e) in response to different treatments. All the data were normalized to β-actin, and fold changes are expressed in relation to the control group. Exposure of MMECs to H/R resulted in significantly higher expression levels of pro-BDNF, p75NTR, and sortilin and in activation of JNK and caspase 3 as compared with MMECs maintained under normal conditions (control). Nonetheless, there were no significant differences in BDNF, JNK, and caspase 3 expression levels after H/R. Treatment with the anti-pro-BDNF antibody significantly reversed the increase in the protein expression of pro-BDNF, p75NTR, sortilin, p-JNK, and cleaved…