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The FnA-D region of Tenascin-C and neuronal growthA major focus of my laboratory has been to understand how axonal growth and regeneration are modulated by interactions with extracellular matrix molecules. in particular. tenascin-C. We have found that the alternately spliced region of tenascin-C. called fnA-D. when used by itself. increases neurite outgrowth in culture. FnA-D also provides directional cues to growing neurites. a function defined as neurite guidance. Neurites demonstrate a strong preference for growth surfaces coated with fnA-D when given a choice at an interface. FnA-D even influences extension onto surfaces coated with normally repulsive chondroitin sulfate proteoglycans. a major type of inhibitory molecule found in the glial scar. This suggests that fnA-D or motifs within fnA-D could be incorporated into a therapeutic cocktail to facilitate axonal regeneration in the injured CNS. We have localized the outgrowth promoting activity to the amino acid sequence VFDNFVLK and the guidance activity to the amino acid sequence DINPYGFTVSWMASE. both found within the fnA-D region of tenascin-C. We identified the α7β1 integrin as the receptor for the outgrowth promoting activity and are continuing to work to identify the receptor that mediates the guidance activity. Our observations have lead us to hypothesize that neurite outgrowth and guidance mediated by fnA-D are distinct processes. each of which can be manipulated independently to encourage directed neuronal regrowth. Engineering Nanofibrillar Surfaces for Spinal Cord RepairThe goal of this work is to test the effects of VFDNFVLK and DINPYGFTVSWMASE. in conjunction with a scaffold made of electospun nanofibers. in an animal model of neuronal regeneration. The nanofibers are composed of a slowly biodegradable form of polyamide and mimic the porosity and three dimensional geometry of the extracellular matrix on which neurons grow in vivo.We have developed techniques to covalently modify the nanofibers with VFDNFVLK and DINPYGFTVSWMASE. The peptide-modified nanofibers will be surgically implanted at the site of a spinal cord injury in a rat to provide a biomimetic surface. structurally reminiscent of the extracellular matrix. for neuronal attachment and growth. as well as a bridge for axonal elongation across the glial scar. In early experiments. the unmodified nanofibers permitted some axonal regrowth. even in the absence of peptides. and reduced glial scarring. They also promoted activation of Rac. a signaling molecule involved in axon extension. Our hypothesis is that the peptide/nanofiber combination will be an even more favorable substrate than nanofibers alone for axonal regrowth in vivo. resulting in longer or more regenerating axons. earlier regeneration. or improved functional recovery. Engineering Nanofibrillar Surfaces for Cell CultureResearch focused on deciphering the biochemical mechanisms that regulate cell function has largely depended on the use of tissue culture methods in which cells are grown on two dimensional plastic or glass surfaces. However. the flat surface of the tissue culture plate is a poor topological approximation of the more complex three dimensional architecture of the extracellular matrix and the basement membrane. a structurally compact form of the extracellular matrix. The three dimensional nanostructures formed by the nanofibrils of the extracellular matrix/basement membrane are essential for the reproduction of physiological patterns of cell adherence. cytoskeletal organization. migration. signal transduction. morphogenesis. proliferation. and differentiation in cell culture. The goal of this work is to develop and test a series of completely synthetic three dimensional nanofibrillar growth surfaces that structurally resemble the extracellular matrix/basement membrane. Such chemical and physical parameters as matrix compliancy and surface chemistry (covalent attachment of specific peptide recognition motifs derived from extracellular matrix proteins) will be modified to evaluate their role in the promoting specific neuronal. astrocytic. fibroblastic. and stem cell functions. We will also evaluate the causal relationship between changes in cellular function and morphology with the activation of the Rho family of small GTPases. Rho. Rac. and Cdc42. the mediators and propagators of ECM-cytoskeleton signaling. Our overall aim will be to develop a number of structurally defined. completely synthetic three dimensional nanofibrillar surfaces that can be utilized for more physiologically relevant studies of cell/tissue function in culture and also to provide matrices that are optimized for specific applications in regenerative medicine. such as wound healing. Selected PublicationsNur-E-Kamal A, Meiners S, Ahmed I, Azarova A, Lin CP, Lyu YL, Liu LF. (2007) Role of DNA topoisomerase IIbeta in neurite outgrowth. Brain Res. Apr 19; [Epub ahead of print] LaGier AJ, Yoo SH, Alfonso EC, Meiners S, Fini ME. (2007) Inhibition of human corneal epithelial production of fibrotic mediator TGF-beta2 by basement membrane-like extracellular matrix. Invest Ophthalmol Vis Sci. 48(3):1061-71. Ahmed I, Ponery AS, Nur-E-Kamal A, Kamal J, Meshel AS, Sheetz MP, Schindler M, Meiners S. (2007) Morphology, cytoskeletal organization, and myosin dynamics of mouse embryonic fibroblasts cultured on nanofibrillar surfaces. Mol Cell Biochem. Feb 9; [Epub ahead of print] Park JK, Fischer R, Dechend R, Shagdarsuren E, Gapeljuk A, Wellner M, Meiners S, Gratze P, Al-Saadi N, Feldt S, Fiebeler A, Madwed JB, Schirdewan A, Haller H, Luft FC, Muller DN.(2007) p38 mitogen-activated protein kinase inhibition ameliorates angiotensin II-induced target organ damage. Hypertension. 49(3):481-9. Meiners S, Ludwig A, Lorenz M, Dreger H, Baumann G, Stangl V, Stangl K. (2006) Nontoxic proteasome inhibition activates a protective antioxidant defense response in endothelial cells. Free Radic Biol Med. 40(12):2232-41. Schindler M. Nur-E-Kamal A. Ahmed I. Kamal J. Liu HY. Amor N. Ponery AS. Crockett DP. Grafe TH. Chung HY. Weik T. Jones E. Meiners S. (2006) Living in three dimensions: 3D nanostructured environments for cell culture and regenerative medicine. Cell Biochem Biophys. 45(2):215-27. Ahmed I. Liu HY. Mamiya PC. Ponery AS. Babu AN. Weik T. Schindler M. Meiners S. (2006) Three-dimensional nanofibrillar surfaces covalently modified with tenascin-C-derived peptides enhance neuronal growth in vitro. J Biomed Mater Res A. 76(4):851-60. Liu HY. Nur-E-Kamal A. Schachner M. Meiners S. (2005) Neurite guidance by the FnC repeat of human tenascin-C: neurite attraction vs. neurite retention. Eur J Neurosci. 22(8):1863-72. Nur-E-Kamal A. Ahmed I. Kamal J. Schindler M. Meiners S. (2006) Three-dimensional nanofibrillar surfaces promote self-renewal in mouse embryonic stem cells. Stem Cells. 24(2):426-33. Nur-E-Kamal A. Zhang A. Keenan SM. Wang XI. Seraj J. Satoh T. Meiners S. Welsh WJ. (2005) Requirement of activated Cdc42-associated kinase for survival of v-Ras-transformed mammalian cells. Mol Cancer Res. 3(5):297-305. Schindler M. Ahmed I. Kamal J. Nur-E-Kamal A. Grafe TH. Young Chung H. Meiners S. (2005) A synthetic nanofibrillar matrix promotes in vivo-like organization and morphogenesis for cells in culture. Biomaterials. 26(28):5624-31. Schindler. M.. Ahmed. I.. Kamal. J.. Nur-e-Kamal. A.. Grafe. T.H.. Chung. H.Y.. and Meiners. S. (2005). Three dimensional nanofibrillar surfaces promote in vivo-like organization and morphogenesis for cells in culture. Biomaterials 26:5624-5631. Nur-e-Kamal. A.. Ahmed. I.. Kamal. J.. Schindler. M.. and Meiners. S. (2005). Three dimensional nanfibrillar surfaces induce activation of Rac. Biochem. Biophys. Res. Commun. 331:428-434. Mercado. M.L.T.*. Nur-e-Kamal. A.*. Liu. H.-Y.*. Gross. S.*. Movahed. R.. and Meiners. S. (2004). Neurite outgrowth by the alternatively spliced region of tenascin-C is mediated by neuronal alpha7beta1 integrin. J. Neurosci. 24:238-247. Meiners. S. and Mercado. M.L.T. (2003). Functional peptide sequences derived from extracellular matrix glycoproteins and their receptors: strategies to improve neuronal regeneration. Molec. Neurobiol. 27:177-196. Meiners. S.. Nur-e-Kamal. M.S.A.. and Mercado. M.L.T. (2001). Identification of a neurite outgrowth promoting motif in the alternatively spliced region of tenascin-C. J. Neurosci. 21:7215-7225. Meiners. S.. Mercado. M.L.T.. and Geller. H.M. (2000). The multi-domain structure of extracellular matrix molecules: implications for nervous system regeneration. In: Progress in Brain Research. Vol. 128 (Neural Plasticity and Regeneration). F.J. Seil. ed.. Elsevier Science. pp. 23-32. Meiners. S.. Mercado. M.L.T.. Nur-e-Kamal. M.S.A.. and Geller. H.M. (1999). Tenascin-C contains domains that independently regulate neurite promotion and neurite guidance. J. Neurosci. 19:8443-8453. Meiners. S.. Powell. E.M.. and Geller. H.M. (1999). Neurite outgrowth promotion by the alternatively spliced region of tenascin-C is influenced by cell type-specific binding. Matrix Biology 18:75-87. |
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