HIF activation and VEGF overexpression are coupled with ZO-1 up-phosphorylation in the brain of dystrophic mdx mouse

Abstract

In Duchenne muscular dystrophy (DMD) metabolic and structural alterations of the central nervous system are described. Here, we investigated in the brain of 10 mdx mice and in five control ones, the expression of hypoxia inducible factor-1alpha (HIF-1alpha) and we correlated it with the expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2) and of the endothelial tight junction proteins zonula occludens-1 (ZO-1) and claudin-1. Results showed an activation of mRNA HIF-1alpha by reverse transcription polymerase chain reaction (RT-PCR) and a strong HIF1-alpha labeling of perivascular glial cells and cortical neurons by immunohistochemistry, in mdx mouse. Moreover, overexpression of VEGF and VEGFR-2, respectively, in neurons and in endothelial cells coupled with changes to endothelial ZO-1 and claudin-1 expression in the latter were detected by immunoblotting and immunohistochemistry, in the mdx brain. Furthermore, by immunoprecipitation, an up-phosphorylation of ZO-1 was demonstrated in mdx endothelial cells in parallel with the reduction in ZO-1 protein content. These data suggest that the activation of HIF-1alpha in the brain of dystrophic mice coupled with VEGF and VEGFR-2 up-regulation and ZO-1 and claudin-1 rearrangement might contribute to both blood-brain barrier opening and increased angiogenesis.

Figure 1

Immunocytochemistry of hypoxia inducible factor‐1α (HIF‐1α) in the brain of mdx (A,C,D,E) and control (B,F) mice. Astrocytes scattered in the neuropil (A, arrow), perivascularly arranged (C,D, arrows), glia limitans membrane (A, arrowhead) and neurons (D,E arrowhead) are HIF‐1α strongly labeled in mdx brain. No reaction is detectable in the controls (B,F) where the glia limitans membrane (B, arrowhead), and neurons (F, arrowhead) are unlabeled. Scale bar: 50 µm (A,B); 25 µm (C,D,E,F).

Figure 2

Dual immunofluorescence of hypoxia inducible factor‐1α (HIF‐1α, red) and glial fibrillary acidic protein (GFAP, green) in mdx (A–C) and control (D) mice. Mdx astrocytes show a strong GFAP (A green, arrowhead), HIF‐1α (B red, arrowhead) and both GFAP and HIF‐1α (C orange, arrowhead) expression. No HIF‐1α expression is found in the control (D), where the astrocytes are only GFAP labeled. Scale bar: 25 µm.

Figure 3

Dual immunofluorescence of hypoxia inducible factor‐1α (HIF‐1α, green) and vimentin (red) in mdx (A,B) and control (C) mice. Mdx astrocytes show HIF‐1α labeling (A green, arrowhead), while no vimentin expression is found in the mdx (B) and in the control (C), where the astrocytes are also HIF‐1α negative. Scale bar: 25 µm.

Figure 4

Immunocytochemistry of vascular endothelial growth factor receptor‐2 (VEGFR‐2, A,B) and vascular endothelial growth factor (VEGF, C,D) in the brain of mdx (A,C) and control (B,D) mice. Numerous VEGFR‐2 labeled vessels (A) and VEGF cortical neurons (C, arrowheads) are recognizable in mdx brain, compared with controls (B,D) where a few vessels are faintly VEGFR‐2 labeled (B) and no VEGF stained neurons are present (D). Note in A, (inset, arrowhead) a dotted VEGFR‐2 staining of mdx vessels. Scale bar: 50 µm (A,B); 10 µm (A, inset), 25 µm (C,D).

Figure 5

Zonula occludens‐1 (ZO‐1) and Claudin‐1 immunofluorescence in control (A,C) and mdx (B,D) mice. Banded expression of ZO‐1 (green) and claudin‐1 (red) in control vessel (A,C arrows), and diffuse vascular expression of both proteins in mdx vessels (B,D). Scale bar: 12.5 µm.

Figure 6

Analysis of hypoxia inducible factor‐1α (HIF‐1α), vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor‐2 (VEGFR‐2) mRNA level in brain of mdx and control (ctrl) mice. A. Semiquantitative RT‐PCR analysis shows enhanced HIF‐1α, VEGF and VEGFR‐2 mRNA levels (bp) in mdx brain as compared with the control. The position of the beta‐actin mRNA marker (700 bp) is shown. B. Densitometric analysis of the bands revealed a significant increase in HIF‐1α, VEGF and VEGFR‐2 compared with the controls (166 ± 19.3 vs. 112 ± 15, P < 0.001; 149.5 ± 29 vs. 125 ± 12, P < 0.05; 114.7 ± 14.7 vs. 93 ± 12, P < 0.01). The error band represents the standard deviation of ten experiments. OD = optical density.

Figure 7

Immunoblotting analysis vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor‐2 (VEGFR‐2) protein expression in brain homogenates of mdx and control (ctrl) mice. A. 60 µg of total protein extract was run on a reducing SDS‐PAGE gel and immunoblotted with a polyclonal antibody raised against VEGF and a polyclonal antibody raised against VEGFR‐2 respectively. B. Quantification of VEGF and VEGFR‐2 expression after Western‐blot analysis. The histograms show a significant increase in VEGF and VEGFR‐2 in mdx brain compared with the control (203 ± 29 vs. 133.8 ± 20, P < 0.0001; 132.2 ± 12.4 vs. 70.5 ± 15.8, P < 0.0001). The error band represents the standard deviation of ten experiments. OD = optical density.

Figure 8

Immunoblotting analysis of zonula occludens‐1 (ZO‐1) and Claudin‐1 protein expression (A,C) and immunoblotting precipitation of ZO‐1 (B) in brain homogenates of mdx and control mice. A,C. 60 µg of total protein extract was run on a reducing SDS‐PAGE gel and blotted to a polyvinyl difluoride (PVDF) membrane. B. 250 µg of brain proteins were subjected to immunoprecipitation, using anti‐ZO‐1 antibody and the precipitate was analyzed by Western blot with a polyclonal anti‐phosphotyrosine antibody. A reduction in ZO‐1 content (A) and a ZO‐1 up‐phosphorylation (B) in mdx brain, as compared with the control, are recognizable; no difference in claudin‐1 content (C) is detectable. Abbreviations: ctrl = control; IP = immunoprecipitate; IB : ptyr = immunoblotting : phosphotyrosine.