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. 2012 May;83(3):366-71.
doi: 10.1016/j.mvr.2012.01.001. Epub 2012 Jan 9.

Brain-derived neurotrophic factor increases expression of MnSOD in human circulating angiogenic cells

Tongrong He  1 , Zvonimir S Katusic
Affiliations

Brain-derived neurotrophic factor increases expression of MnSOD in human circulating angiogenic cells

Tongrong He et al. Microvasc Res. 2012 May.

Abstract

Existing evidence suggests that brain-derived neurotrophic factor (BDNF) promotes survival and proliferation of endothelial cells, stimulates mobilization of hematopoietic progenitors, and induces angiogenesis in ischemic tissues. However, the mechanisms underlying vascular protective function of BDNF are poorly understood. We hypothesized that BDNF increases antioxidant capacity of circulating angiogenic cells. Human mononuclear cells were isolated from peripheral blood of 30 healthy male volunteers (48±2 years old), and cultured in endothelial growth medium-2 for 4-5 days. The attached cells (so called early endothelial progenitor cells [early EPCs], or circulating angiogenic cells) expressed BDNF receptors, tropomyosin-related kinase B and p75 neurotrophin receptor. Treatment of early EPCs with recombinant human BDNF for 24 h significantly increased manganese superoxide dismutase (MnSOD) expression, but had no effect on expression of other antioxidant enzymes including copper zinc SOD (CuZnSOD), catalase, and glutathione peroxidase-1. BDNF stimulated phosphorylation of IκB kinase (IKK)α/β and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK); however it did not activate p38, Erk, or AKT. Treatment with nuclear factor κB inhibitor, PDTC, or JNK inhibitor, SP600125, attenuated BDNF-augmented MnSOD protein expression. BDNF treatment inhibited apoptosis induced by a superoxide anion generator LY83583, and serum starvation-induced cell detachment. These findings suggest that BDNF protects EPCs by increasing expression of MnSOD thereby enhancing their antioxidant capacity.

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Figures

Fig 1
Fig 1
Phenotyping of EPCs. Mononuclear cells were cultured on fibronectin-coated plates in EGM2 for 4 days. A: Phase contrast image of day 4 EPCs (× 20 magnification). B–E: FACS analysis of cell surface markers on EPCs. Shown are representative data from at least 3 independent experiments for each marker. The open black-lined histograms represent tested antibodies, and filled histograms represent the control IgG. F: Human EPCs expressed receptors that bind to BDNF, TrkB and p75NTR. Positive controls (Post) for p75NTR and TrkB are SK-N-MC cell lysate and mouse brain, respectively. G: mRNA expressions of TrkB and p75NTR in day 4 EPCs and circulating mononuclear cells (MNC).
Figure 2
Figure 2
BDNF increased MnSOD expression in EPCs. A and B: Human EPCs were treated with BDNF (50 or 100 ng/ml) in EBM2 for 24 h. Protein samples were subjected to Western blotting. Blots are representative of 3 independent experiments. B: Optical density analysis of MnSOD protein levels; n=3, *P<0.05, compared with control. C: cells were cultured in EBM2 for 15 h, then incubated with BDNF for 7.5 h, mRNA levels were measured by RT-PCR; n=3, *P<0.05, compared to non-treatment.
Figure 3
Figure 3
BDNF induced phosphorylation of IKKα/β, and JNK. A–C: EPCs were cultured in EBM-2 for 20 h, then were treated with BDNF for indicated periods. All blots are representative of at least 3 independent experiments. A: n=6–9, P<0.05 compared to control. B: n=7–11, P<0.05, compared to control. D: EPCs were pretreated with PDTC or SP600125 for 1 h, and then incubated with BDNF for 24 h; n=8, *P<0.05, compared to other 3 groups.
Figure 3
Figure 3
BDNF induced phosphorylation of IKKα/β, and JNK. A–C: EPCs were cultured in EBM-2 for 20 h, then were treated with BDNF for indicated periods. All blots are representative of at least 3 independent experiments. A: n=6–9, P<0.05 compared to control. B: n=7–11, P<0.05, compared to control. D: EPCs were pretreated with PDTC or SP600125 for 1 h, and then incubated with BDNF for 24 h; n=8, *P<0.05, compared to other 3 groups.
Figure 4
Figure 4
BDNF protected EPCs from oxidative stress. A and B: EPCs were incubated with indicated treatments, then were subjected to TUNEL assay (A, n=5–8, *P<0.05, compared to other 2 groups), or Western blotting (B, n=5, *P<0.05, compared to other two groups, **P<0.05, compared to control). C: MNC were seed in 6-well plates (20×106 cells/well, in duplicates) and cultured in EGM2 for 4 days. The non-adherent cells were washed away, the attached cells (EPCs) were treated with EGM2 (control), EBM2, or EBM2 +BDNF for 48 h (treatment was refreshed every 24 h). The attached cells were collected by trypsinization and cell numbers were counted using a hemocytometer. Data are presented as % to control (EGM2 alone); n=7, *P<0.05.
Figure 4
Figure 4
BDNF protected EPCs from oxidative stress. A and B: EPCs were incubated with indicated treatments, then were subjected to TUNEL assay (A, n=5–8, *P<0.05, compared to other 2 groups), or Western blotting (B, n=5, *P<0.05, compared to other two groups, **P<0.05, compared to control). C: MNC were seed in 6-well plates (20×106 cells/well, in duplicates) and cultured in EGM2 for 4 days. The non-adherent cells were washed away, the attached cells (EPCs) were treated with EGM2 (control), EBM2, or EBM2 +BDNF for 48 h (treatment was refreshed every 24 h). The attached cells were collected by trypsinization and cell numbers were counted using a hemocytometer. Data are presented as % to control (EGM2 alone); n=7, *P<0.05.
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