Interactions between vascular endothelial growth factor and neuroglobin

Abstract

Vascular endothelial growth factor (VEGF) and neuroglobin (Ngb) participate in neuronal responses to hypoxia and ischemia, but the relationship between their effects, if any, is unknown. To address this issue, we measured Ngb levels in VEGF-treated mouse cerebrocortical cultures and VEGF levels in cerebrocortical cultures from Ngb-overexpressing transgenic mice. VEGF stimulated Ngb expression in a VEGFR2/Flk1 receptor-dependent manner, whereas Ngb overexpression suppressed expression of VEGF. These findings provide further insight into hypoxia-stimulated neuronal signaling pathways.

Figure 1. VEGF-induced Ngb protein expression

Mouse cerebrocortical neuron cultures were treated for 24 h with the indicated concentrations of VEGF, Ngb (M_r~17 kDa) levels were measured by immunoblotting (top), and the results were analyzed by densitometry, normalized to β-actin, and plotted as mean ± SEM (bottom). *p<0.05, **p<0.002 compared to 0 ng/ml VEGF (N=4).

Figure 2. VEGF receptor expression

Triplicate mouse cerebrocortical neuron cultures were probed for expression of VEGFR1/Flt1 (M_r~180 kDa, top) and VEGFR2/Flk1 (M_r~150 kDa, bottom), with mouse hippocampal neuron × N18TG2 neuroblastoma (HN33) cells (right lane) used as a positive control.

Figure 3. Effect of VEGFR2/Flk1 receptor kinase inhibitors on VEGF-induced Ngb protein expression

Mouse cerebrocortical neuron cultures were treated for 24 h with 20 ng/ml of VEGF, with or without 10 μM SU1498 (SU) or 20 nM VEGFR kinase inhibitor IV (Inh IV). Ngb levels were measured by immunoblotting (top), and the results were analyzed by densitometry, normalized to β-actin, and plotted as mean ± SEM (bottom). *p<0.05 (N=3), **p<0.01 (N=4) compared to control.

Figure 4. Effect of MAPK inhibition on VEGF-induced Ngb expression

Mouse cerebrocortical neuron cultures were treated for 24 h with 20 ng/ml of VEGF, with or without 1–100 μM PD90859 (PD), or with 100 μM PD90859 alone. Ngb levels were measured by immunoblotting (top), and the results were analyzed by densitometry, normalized to β-actin, and plotted as mean ± SEM (bottom). *p<0.05 (N=3) compared to control.

Figure 5. VEGF expression in wild type and Ngb-overexpressing transgenic cultures

VEGF levels in cerebrocortical neuron cultures from wild type and Ngb-overexpressing transgenic mice were measured by immunoblotting (top), and the results were analyzed by densitometry, normalized to β-actin, and plotted as mean ± SEM (bottom). *p<0.02 compared to wild type (N=4).

Figure 6. Overview of interactions between neuronal hypoxia-response mediators

Previously described, positive interactions are indicated by thin arrows. Positive and negative interactions described in this report are indicated by the thick pointed arrow and flat-tipped arrow, respectively.