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Characterization of extracellular matrix macromolecules in primary cultures of equine keratinocytesAbstract: Physical and proteolytic tissue separation in combination with media containing a calcium concentration of 0.6 mM in combination with additional media supplements proved optimal for proliferation and subculture of equine lamellar keratinocytes on collagen coated substratum. Immunofluorescence and immunoblotting studies confirmed that equine skin and lamellar keratinocytes produce Ln-332 in vitro and processing of this molecule follows that of other species. As well, matrix components including integrin alpha-6 (α6) and the hemidesmsome proteins, bullous pemphigoid antigen 1 (BP180) bullous pemphigoid antigen 2 (BP230) and plectin are also expressed.Isolation of equine keratinocytes and study of the matrix and adhesion related molecules produced by them provides a valuable tool for future work in the veterinary field.The basement membrane (BM) is a thin layer of extracellular matrix (ECM) of which one of the major components is laminin (Ln). Laminins are a large family of heterotrimeric glycoproteins composed of at least 16 isoforms that play many roles in cell function, including cell adhesion and migration. Ln-332 (α3β3γ2) is a major isoform found in epithelial BMs [1].Attachment of the epithelial cell to the underlying BM is mediated through hemidesmosomes (HD). The transmembrane integrin α6β4 links the epithelial cell to Ln-332 in the BM while the bullous pempigoid antigen 1 (BP180) also plays a role in cell attachment. The cytoplasmic proteins plectin and the bullous pemphigoid antigen 2 (BP230) connect the cytokeratin intermediate filament skeleton to the HD complex [2,3].Extracellular matrix proteins are affected in diseases of multiple species. Recently, a mutation in the Ln-332 γ2 subunit in some Belgian horse foals has found to result in blistering of the skin, mouth epithelia and loss of the hooves [4]. A variety of human genetic and autoimmune bullous diseases also exist. Epidermolysis bullosa (EB) is a group of diseases resulting in blistering in the BM
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