The aim of this study was to identify the role of the precursor of the brain-derived neurotrophic factor (pro-BDNF) in myocardial hypoxia/reoxygenation injury (H/R) and to address the underlying mechanisms. For this purpose, myocardial microvascular endothelial cells (MMECs) exposed to a high concentration of glucose (30 mM) for 48 h were subjected to 4 h of hypoxia followed by 2 h of reoxygenation. Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining and flow-cytometric analysis were performed to detect apoptosis. Cell scratch and capillary-like-structure formation assays were employed to evaluate cell function. The levels of apoptosis-related proteins were evaluated by Western blotting and immunofluorescence assays. Our results showed that H/R resulted in MMEC injury, as indicated by significant increases in TUNEL-positive cell numbers and a reduction in MMEC migration and in capillary-like-structure formation coupled with increased pro-BDNF protein expression. In addition, overexpression of pro-BDNF in MMECs via a viral vector led to increased pro-BDNF expression, and this upregulation induced apoptosis. Mechanistic experiments revealed that H/R did not influence BDNF, JNK, and caspase 3 expression, but upregulated pro-BDNF, p75NTR, sortilin, phospho-JNK, and cleaved caspase 3 protein levels. In contrast, neutralization of endogenous pro-BDNF with an antibody significantly attenuated H/R-induced upregulation of pro-BDNF, p75NTR, sortilin, p-JNK, and cleaved caspase 3 protein levels, indicating that p75NTR-sortilin signaling and activation of JNK and caspase 3 may be involved in these effects. In conclusion, H/R-induced injury may be mediated by pro-BDNF, at least in part through the regulation of p75NTR-sortilin signaling and activation of JNK and caspase 3.
Diabetes mellitus (DM), a potent and prevalent risk factor of ischemic heart disease, has received increasing attention globally. Cardiovascular complications constitute the leading cause of morbidity and mortality among patients with DM [
In recent years, studies on the nerve growth factor (NGF) family have been focused on the nervous system [
Both ECs and endothelial progenitor cells express high-affinity receptors called Trk [
JNK, one of the members of the MAP kinase superfamily, is primarily involved in the induction of death receptor-initiated exogenous and mitochondrial apoptosis in vivo after exposure to various chemical or biological agents [
Currently, there is no evidence that pro-BDNF participates in the process of endothelial I/R injury, and the corresponding molecular mechanism is unclear. In the present study, we investigated the role of pro-BDNF in the regulation of hypoxia/reoxygenation- (H/R-) induced endothelial apoptosis, migration, and tube formation and next examined the expression of proteins related to apoptosis. These findings will lead to a novel therapeutic approach for myocardial I/R injury.
The human myocardial microvascular endothelial cell (MMEC) line was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in the DMEM high-glucose complete medium (Gibco, Waltham, MA, USA), supplemented with 10% of fetal bovine serum (HyClone, Logan, UT, USA), 100 U/mL penicillin (Sigma, St. Louis, MO, USA), and 100
To induce H/R injury as described previously [
The recombinant adenoviruses expressing the human pro-BDNF gene (Ad-GFP-pro-BDNF) or GFP control (Ad-GFP) were purchased from GenePharma (Shanghai, China) and were used to infect the ECs according to the manufacturer’s instructions. Transduction efficiency was verified via GFP expression and Western blotting. The neutralizing antibody to the recombinant prodomain of BDNF (10
Apoptosis was detected by the TUNEL assay (Roche Applied Science) and by corresponding flow-cytometric analyses according to the instructions of the manufacturer. For quantification, the TUNEL-positive cells were counted in at least five randomly chosen visual fields in three independent samples (500 counted cells in total). The flow-cytometric assay was then performed on a BD FACSCalibur instrument (Becton, Dickinson and Company, Lake Franklin, NJ, USA).
This analysis was conducted to determine the protein expression and phosphorylation levels. Cellular proteins were extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology). Proteins (lysate corresponding to 20
Cells were fixed with 4% paraformaldehyde at room temperature (RT) for 30 min and permeabilized or not permeabilized with 0.5% Triton X-100 (Sigma-Aldrich), and nonspecific binding was blocked by incubation with 5% donkey serum (Jackson ImmunoResearch Laboratories) at RT for 30 min. Coverslips were incubated overnight at 4°C with the following primary antibodies: rabbit anti-BDNF (Abcam, 1 : 500), rabbit anti-pro-BDNF (Alomone Labs, 1 : 400), anti-JNK (Santa Cruz Biotechnology, 1 : 100), anti-phosphorylated-JNK (p-JNK) (Santa Cruz Biotechnology, 1 : 100), rabbit anti-p75NTR (Santa Cruz Biotechnology, 1 : 100), and goat anti-sortilin (Abcam, 1 : 500) antibodies. A secondary antibody conjugated with Cy3 or fluorescein isothiocyanate was incubated for 2 h at RT. Nuclei were stained for 5 min with 4
We performed a cell scratch assay and capillary-like-structure formation experiments to evaluate the functional effects of pro-BDNF on MMECs in groups control, H/R, H/R + anti-pro-BDNF, and H/R + vehicle.
The assay of capillary-like-structure formation in vitro was performed as previously described [
All values are presented as
We first examined the effects of H/R on MMECs after exposure to high concentration of glucose (HG). Representative photographs were taken, and quantitative analysis of TUNEL positivity was performed to evaluate the proapoptotic effects. After exposure to HG, H/R caused a significant increase in the proportion of TUNEL-positive cells as compared to MMECs not subjected to H/R (control group), indicating that H/R induced MMEC apoptosis (Figures
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
Next, we examined the effect of H/R on pro-BDNF protein levels. The expression of pro-BDNF measured by immunostaining was observed in the cytoplasm and plasma membrane of MMECs. Of note, exposure to H/R caused overlapping signals of pro-BDNF staining and TUNEL staining among MMECs (Figures
To test whether an increase in pro-BDNF levels exerted proapoptotic actions on MMECs under HG conditions, we transfected MMECs with either Ad-pro-BDNF or with Ad-GFP as a negative control group (NON). A TUNEL assay of adenovirus-infected MMECs under HG conditions was then performed (Figures
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
The proapoptotic action of H/R seemed to be mediated at least in part by upregulation of pro-BDNF. We next evaluated the relation between pro-BDNF and H/R-induced apoptosis (Figures
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.
To address the functional effects of pro-BDNF on MMECs, capillary-like-structure formation experiments (Figure
To elucidate the molecular mechanisms behind the action of pro-BDNF under HG and H/R conditions, experiments were performed on several markers of apoptosis by immunostaining (Figure
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
Hyperglycemia, a common feature of both type 1 and type 2 diabetes, is a key factor that contributes to the development of DM-related vascular disease and notably microvascular disease [
One research group [
Pro-BDNF shows high-affinity binding to sortilin and performs its biological functions by acting on its receptors: p75NTR and sortilin [
Diabetic nephropathy is a serious microvascular complication of DM; H/R promoted oxidative stress in NRK-52E cells exposed to HG accompanied by increased levels of Nrf2 and HO-1 protein expression [
In summary, the major finding of our study is that inhibition of pro-BDNF may exhibit a beneficial effect against H/R by promoting MMEC migration and capillary-like-structure formation. Moreover, these effects are at least in part related to the decrease in MMEC apoptosis through p75NTR-sortilin-mediated activation of JNK and caspase 3. Our results may facilitate future studies on the therapeutic implications of pro-BDNF in the treatment of MIRI.
Brain-derived neurotrophic factor
Myocardial microvascular endothelial cells
Hypoxia/reoxygenation
Diabetes mellitus
Cardiac microvascular endothelial cells
Nerve growth factor
Tumor necrosis factor
c-Jun N-terminal kinase
Myocardial ischemia/reperfusion injury.
The data used to support the findings of this study are available from the corresponding author upon request.
All the experiments and procedures were approved by the Ethics Committee of the Central South University, Changsha, Hunan 410011, China.
The authors declare that they have no competing interests.
Fei Yu and Junmei Xu designed the study. Fei Yu and Yuezhu Liu conducted the experiments and collected the data. Fei Yu and Yuezhu Liu analyzed and interpreted the experimental data. Fei Yu and Junmei Xu prepared the manuscript.
This work was sponsored by the National Natural Science Foundation of China (Grant no. 81370254). The authors are grateful to Man Li for technical assistance.