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Published on: June 14, 2016
Increased matrix-metalloproteinase-2 and matrix-metalloproteinase-9 expression in the brain of dystrophic mdx mouse
1Department of Human Anatomy and Histology, University of Bari Medical School, Piazza Giulio Cesare, 11, Policlinico, I-70124 Bari, Italy. nico@histology.uniba.it
Abstract:
Brain edema and severe alterations of the glial and endothelial cells have recently been demonstrated in the dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy, and an increase in microvessel density in patients affected by Duchenne muscular dystrophy has also been shown. In order to further elucidate the mechanisms underlying the angiogenetic processes occurring in Duchenne muscular dystrophy, in this study we analyzed matrix-metalloproteinase-2 and -9 expression in the brain of 20-month-old mdx and control mice by means of immunohistochemistry, in situ hybridization, immunoblotting and gelatin zymography. Moreover, we studied vascular endothelial growth factor expression by means of Western blot and immunohistochemistry, and by dual immunofluorescence using anti-vascular endothelial growth factor and anti matrix-metalloproteinase-2 and-9 antibodies. Ultrastructural features of the brain choroidal plexuses were evaluated by electron microscopy. Spatial relationships between endothelium and astrocyte processes were studied by confocal laser microscopy, using an anti-CD31 antibody as a marker of endothelial cells, and anti-glial fibrillary acidic protein (GFAP) as a marker of glial cells. The results demonstrate that high expression of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 protein content occurs in mdx brain and in choroidal plexuses where, by in situ hybridization, matrix-metalloproteinase-2 and matrix-metalloproteinase-9 mRNA was localized in the epithelial cells. Moreover, matrix-metalloproteinase-2 mRNA was found in both mdx perivascular astrocytes and blood vessels, while matrix-metalloproteinase-9 mRNA was localized in mdx vessels. Through zymography, increased expression of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 was found in mdx brain compared with the controls. These enhanced matrix-metalloproteinase levels in mdx mice were found to be associated with increased vascular endothelial growth factor expression, as determined by immunoblotting and immunocytochemistry and with ultrastructural alterations of the mdx choroidal epithelial cells and brain vessels, as previously reported [Nico B, Frigeri A, Nicchia GP, Corsi P, Ribatti D, Quondamatteo F, Herken R, Girolamo F, Marzullo A, Svelto M, Roncali L (2003) Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice. Glia 42:235-251]. Indeed, in the mdx epithelial cells of the plexuses, the apical microvilli were located on the lateral membranes, whereas in the controls they were uniformly distributed over the free ventricular surface. Moreover, by dual immunofluorescence, a colocalization of vascular endothelial growth factor and matrix-metalloproteinase-2 and matrix-metalloproteinase-9 was found in the ependymal and epithelial cells of plexuses in mdx mice and, under confocal laser microscopy, mdx CD-31 positive vessels were enveloped by less GFAP-positive astrocyte processes than the controls. Overall, these data point to a specific pathogenetic role of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 in neurological dysfunctions associated with Duchenne muscular dystrophy.
Insights
Matrix metalloproteinase-2 and -9 (MMP-2/9) are upregulated in the brains of mdx mice, a model for Duchenne muscular dystrophy. These MMPs correlate with increased vascular endothelial growth factor and brain abnormalities, suggesting a role in neurological dysfunction.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Duchenne muscular dystrophy (DMD) is associated with brain edema and glial/endothelial cell alterations in mdx mice.
- Increased microvessel density is observed in DMD patients, suggesting angiogenesis is involved.
- The precise mechanisms of angiogenetic processes in DMD brains remain unclear.
Purpose of the Study:
- To investigate the expression and role of matrix metalloproteinases-2 and -9 (MMP-2/9) in the brain of mdx mice.
- To analyze the relationship between MMPs, vascular endothelial growth factor (VEGF), and brain structural changes in mdx mice.
- To elucidate the pathogenetic contribution of MMPs to neurological dysfunction in DMD.
Main Methods:
- Immunohistochemistry, in situ hybridization, immunoblotting, and gelatin zymography were used to analyze MMP-2/9 expression in mdx and control mouse brains.
- Western blot and immunohistochemistry assessed VEGF expression, with dual immunofluorescence for MMPs and VEGF.
- Electron microscopy evaluated ultrastructural features of choroid plexuses; confocal microscopy examined astrocyte-endothelium spatial relationships (GFAP/CD31).
Main Results:
- Significantly elevated MMP-2/9 protein and mRNA levels were detected in the brains and choroid plexuses of mdx mice compared to controls.
- Increased MMP expression correlated with enhanced VEGF levels and ultrastructural alterations in choroidal epithelial cells and brain vessels.
- Mdx mice showed altered spatial relationships between endothelial cells (CD31) and astrocyte processes (GFAP), with co-localization of VEGF and MMPs in plexus cells.
Conclusions:
- Matrix metalloproteinase-2 and -9 are upregulated in the mdx mouse brain, indicating their involvement in DMD-related neurological changes.
- Elevated MMPs are associated with increased VEGF and structural brain abnormalities, suggesting a key pathogenetic role.
- These findings highlight MMP-2/9 as potential targets for addressing neurological dysfunctions in Duchenne muscular dystrophy.
