Vascular endothelial growth factors A and C are induced in the SVZ following neonatal hypoxia-ischemia and exert different effects on neonatal glial progenitors

Abstract

Episodes of neonatal hypoxia-ischemia (H-I) are strongly associated with cerebral palsy and a wide spectrum of other neurological deficits in children. Two key processes required to repair damaged organs are to amplify the number of precursors capable of regenerating damaged cells and to direct their differentiation towards the cell types that need to be replaced. Since hypoxia induces vascular endothelial growth factor (VEGF) production, it is logical to predict that VEGFs are key mediators of tissue repair after H-I injury. The goal of this study was to test the hypothesis that certain VEGF isoforms increase during recovery from neonatal H-I and that they would differentially affect the proliferation and differentiation of subventricular zone (SVZ) progenitors. During the acute recovery period from H-I both VEGF-A and VEGF-C were transiently induced in the SVZ, which correlated with an increase in SVZ blood vessel diameter. These growth factors were produced by glial progenitors, astrocytes and to a lesser extent, microglia. VEGF-A promoted the production of astrocytes from SVZ glial progenitors while VEGF-C stimulated the proliferation of both early and late oligodendrocyte progenitors, which was abolished by blocking the VEGFR-3. Altogether, these results provide new insights into the signals that coordinate the reactive responses of the progenitors in the SVZ to neonatal H-I. Our studies further suggest that therapeutics that extend VEGF-C production and/or agonists that stimulate the VEGFR-3 will promote oligodendrocyte progenitor cell development to enhance myelination after perinatal brain injury.

Figure 1. VEGF-A and C protein expression

A, B. ELISAs were performed for VEGF-A (A) and VEGF-C (B) on protein samples isolated from microdissected SVZs (subventricular zones) at time points varying from 12 h – 7 d after neonatal H-I (hypoxia-ischemia) (n=4 per sample). Error bars = SEM. * denotes p < 0.05 using student’s t-test between IL (ipsilateral) and both CL (contralateral) and NHC (non-hypoxic control) hemisphere protein samples. C. ELISAs were performed on supernatants from glial progenitor spheroids (GP), astrocytes and microglia. Spheroids were continuously grown in 2% oxygen and astrocytes and microglia were exposed to 2% oxygen and glucopenic conditions for 2 h (hypoxic-glucopenic, HG). Media was collected 24 h after returning the cells to atmospheric, normoglycemic (control) conditions, n=4 for each condition. Error bars = SEM. * denotes significant t-test (p < 0.05) between control and HG conditions for each cell type.

Figure 2. VEGFs A and C differentially affect glial generation from spheroids in vitro

Spheroids were gently dissociated into single cells and plated for 6 h prior to the addition of growth factors for 96 h. A–B, Representative image of cells maintained in N2B2, VEGF-A and VEGF-C and stained for O4 (red), GFAP (green) and DAPI (blue) in B. Graphs depict averaged data from counting at least 100 cells from 4 wells for each treatment. Error bars = SEM. * denotes p < 0.05 compared to control conditions using student’s t test. E. BrdU (10 µM) was added to the media during the last 4 h of incubation with growth factors. Cells were stained for O4, GFAP, BrdU and DAPI (C). Graphs depict averaged data from counting at least 100 cells from 4 wells for each treatment. Data are representative of 2–3 independent experiments. Error bars = SEM. * denotes p < 0.05 ANOVA with Fisher’s PLSD post-hoc test.

Figure 3. VEGF-A and C cooperate to stimulate astrocyte proliferation but only VEGF-C alone stimulates oligodendrocyte progenitor proliferation

A) Astrocytes from neonatal mixed glial cultures were plated onto chamber slides overnight in N2B2 alone or supplemented with 8 ng/mL VEGF-A and/or 100 ng/mL VEGF-C. After 18 h BrdU was added and 2 hours later the cells were fixed and stained for BrdU, GFAP and DAPI. B) Oligodendrocyte progenitor cells (OPCs) were generated from mixed glial cultures and plated onto chamber slides overnight in N2B2 with 0.5% serum, followed by a 6 h starvation period. Then media was supplemented with 8 ng/mL VEGF-A and/or 100 ng/mL VEGF-C and 10uM BrdU was added during the final 2 h of an 18 h treatment. Chamber slides were fixed and stained for BrdU, O4 and DAPI. Graphs represent averaged data from counting at least 100 cells from 4 wells for each treatment. Data are representative of 2–3 independent experiments. Error bars = SEM. * denotes p < 0.05 compared to control conditions using ANOVA analysis with Fisher’s post-hoc test.

Figure 4. VEGFR-3 is expressed by neural precursors and blocking VEGFR-3, but not VEGFR-1 or R2, abolishes VEGF-C stimulated oligodendrocyte proliferation

A. Neurospheres and spheroids were generated from neonatal rat SVZs and cultured for 7 DIV in 2%O₂, 5% CO₂, 93% N₂. Astrocytes and oligodendrocyte progenitor cells (OPCs) were generated from mixed glial cell cultures from neonatal rat brains. Fibroblasts (3T3) were grown in 10% serum containing medium. Expression of VEGFR-3 (Flt4) (175kDa) was evaluated by Western Blot. Data are representative of 3 independent experiments. B–U. Early OPCs were incubated with either VEGF-C (100ng/ml) or neutralizing antibodies to VEGFR-1 (5µg/ml), VEGFR-2 (0.2µg/ml) or VEGFR-3 (7.5µg/ml) in N2B2 medium. Cells were double immunostained for Ki67 with either A2B5 or O4 and counterstained with DAPI, (B–U). VEGF-C promoted proliferation of both early OPCs (C,H) and also late OPCs (M,R), compared with N2B2 control medium (B,G,L,Q). The neutralizing antibody to VEGFR-3 completely blocked VEGF-C-stimulated OPC proliferation (F, K, P, U). In contrast, neutralizing antibodies to VEGFR-1 or R2 did not inhibit VEGF-C function (D, I, N, S; and E, J, M, T). Scale bar represents 50 µM. V+W. Quantification of both A2B5+ (V), and O4+ OPCs (W), *P<0.05 by ANOVA followed by X post-hoc test.

Figure 5. Microvasculature changes in SVZ after neonatal H-I

Paraffin-embedded sections spanning 4 to 48 h of recovery from H-I were stained with hematoxylin and eosin and blood vessels were identified by morphology (red arrows) in IL (B) and CL (D) hemispheres (D). Morphometric analysis was performed on 40X images of corresponding vessels in the SVZ using IP Lab (Scanalytics, Inc. Fairfax, Virginia USA). Sections from 4h brains were stained for BrdU (red), tomato lectin (green) and DAPI (blue) to label proliferating blood vessels (white arrowheads) in IL (C) and CL hemispheres (E). IL = ipsilateral hemisphere, CL = contralateral hemisphere, NHC = non-hypoxic control. N = 8 vessels measured from 3 random, nonadjacent SVZ sections and 4 animals at each time point. Error bars = SEM. * indicates p < 0.05 compared to both CL and NHC. (V = ventricles)