The Effect Of BDNF-TrkB Pathway For Cardiac Microvascular Endothelial Cells

Objectives:Cardiac microvascular endothelial cells (CMECs) play an important role in cardiac angiogenesis. Recent studies have shown that, in addition to its neuropoietic actions, brain derived neurotrophic factor (BDNF) promotes endothelial cell survival and induces angiogenesis in ischemic tissues. The receptor of BDNF is TrkB. However, the molecular mechanism regarding to BDNF-TrkB pathway in endothelial cells is still unknown. The present study is therefore designed to investigate (1) the effects of BDNF in proliferation of CMECs and the effects of replicative senescence on the expressions of BDNF receptors and the proliferation of CMECs; (2) the influence of replicative senescence on the expressions of TrkB isoforms in CMECs; (3) the karyotype analysis of CMECs after passage.Methods and Results:(1) Passaging of CEMCs results in chromosome loss, the proportion of cells with 42 chromosomes decreased from 63.3% (P20) to 18.6% (P100). (2) The expressions of TrkB isoforms in CMECs derived from young rat were analyzed by real-time PCR and all of them decreased after passage; only TrkB-Tl was detected in CMECs derived from old rat. (3) CMECs were treated by BDNF in order investigate the effect of BDNF on proliferation of CMECs by Alamar Blue reduction assay and flow cytometer. The cell growth curve suggested that BDNF promoted the proliferation of CMECs. The proportion of CMECs (P6) derived from young rat in S phase was 31.31±0.88% in 50ng/mL BDNF group vs 30.38±1.02% in control group (P<0.01). The proportion of CMECs (P14) derived from young rat in S phase was 28.92±1.46% in 50ng/mL BDNF group vs 25.95±0.39% in control group (P<0.01). The proportion of CMECs (P5) derived from old rat in S phase was 31.31±0.88% in 50ng/mL BDNF group vs 30.38±1.02% in control group. (4) The representive genes from the microarray analysis, which are related to cell migration, regulation of proliferation and cell cycle regulation, were analyzed. The expressions of these genes and TrkB isoforms in CMECs treated with BDNF were determined by real-time PCR. The results showed that Synedecan, BGN, Cav-1, RTN-4, MGP, Hoxd13, ACTG-2 were up-regulated and TrkB-FL, Nisch, FLNa, Spna2 were down-regulated. (5) TrkB-shiRNA vector was used to transfected into CMECs. The expressions of TrkB isoforms (TrkB-FL, TrkB-T1 and TrkB-T2) in CMECs was analyzed by real-time PCR. It was found that the expressions of TrkB isoforms were decreased in 2-day and 4-day after CMECs transfection with TrkB-shiRNA vector. In addition, the proliferation of transfected CMECs was decreased.Conclusions:(1) The hereditary character of passages CMECs was unstable. Passaging of CEMCs results in chromosome loss and the morphology of CMECs changed. (2) The expressions of TrkB isoforms in CMECs derived from young rat decreased after passage. Only the expression of TrkB-Tl is detected in CMECs derived from old rat. (3) BDNF at different concentrations could promote the proliferation of CMECs. Up-regulation of Synedecan, BGN, Cav-1, RTN-4, MGP, Hoxd13, ACTG-2 and down-regulation of Nisch, FLNa, Spna2 might relate to BDNF-mediated proliferation of CMECs. (4) Transfection of CMECs with TrkB-shiRNA vector was able to decrease expression of TrkB-FL, TrkB-T1 and TrkB-T2 and decreasec the proliferation of CMECs.