Figure 4.

VEGF-B Regulates the Expression of Molecules Linked to Neuronal Growth and Differentiation

(A) The mechanism of VEGF-B-induced nerve growth. Left: VEGF-B upregulates the expression of several neurogenic molecules, including ATF6; Nr4a2; and a secreted factor, MANF. VEGF-B may, therefore, directly stimulate nerve growth via these downstream mediators. In addition, VEGF-B and soluble Nrp-1 may interfere with endogenous inhibition of nerve growth. Right: VEGF-B may compete for binding to Nrp-1 with endogenous ligands such as Sema3a, and sNrp-1 may further enhance this effect by binding Nrp-1 ligands. VEGF-B increases the expression and nuclear localization of transcription factor ATF6 6 days after Ad GT in pig myocardium (p < 0.05). (B and C) The nuclei were more intensely stained in VEGF-B transduced area (C) as compared to AdLacZ control GT (B). (D and E) Nuclear orphan receptor Nr4a2 expression was low in control pig myocardium (D) but was increased in AdVEGF-B₁₈₆-transduced pig myocardium (E) (p < 0.05). In addition, nuclear localization of Nr4a2 was also observed in αMHC-VEGF-B mice (insert in E), while expression level was very low in WT controls (insert in D) (p < 0.05). (F) The downstream target of both ATF6 and Nr4a2, MANF expression was very low in control hearts, and only a few positive cells were observed in AdLacZ-transduced pig myocardium. (G) VEGF-B increased MANF expression in capillaries and in some cardiomyocytes and other cell types (p < 0.005). Together, these molecules have been shown to regulate neuron growth, differentiation, and survival, thus possibly mediating the rapid nerve growth and branching observed after VEGF-B overexpression. (H and I) Immunostainings of ATF6, Nr4a2, and MANF in pig (H) and Nr4a2 in mouse (I) were analyzed blindly with semiquantitative methods, and all were significantly increased (p < 0.05) in VEGF-B-overexpressing tissues. The intensity of the staining and the number of stained areas were blindly scaled from 0 to 3, with 0 representing no staining intensity, 1 representing low intensity and number of stained areas, 2 representing medium intensity and number of stained areas, and 3 representing high intensity and number of stained areas. Overexpression of AdsNrp-1 induces nerve growth in the myocardium. (J and K) AdsNrp-1 increased the number of GAP43-positive nerves 6 days after GT in pig myocardium (K) as compared to AdLacZ control (J). (L) The number of nerves per FOV was increased by 2.8-fold as compared to AdLacZ control (p < 0.01). This suggests that Nrp-1 has an inhibitory role in nerve patterning and that sNrp-1 disrupts this inhibitory signaling.