Changes in Human Trabecular Meshwork Cells Induced by Overexpression of SPARC

Abstract

Abstract: : Purpose:To analyze the effect of overexpression of SPARC (osteonectin/secreted protein acidic and rich in cysteine) on the cytoskeleton and focal adhesions in primary cultures of human trabecular meshwork (HTM). Methods:A replication-deficient recombinant adenovirus was generated by homologous recombination in E. Coli with the SPARC protein coding region. HTM primary cultures were infected at different multiplicity of infection (m.o.i.). The expression of the protein and its effect on cell morphology and adhesion properties were monitored at 48 hours post-infection. Results:The recombinant adenovirus codes for a protein with identical sequence to the human SPARC protein. This protein can be detected by WB with SPARC specific antibodies in primary cultures infected with the recombinant adenovirus. Additionally it is secreted normally into the extracellular media. The amount of protein expressed is proportional to the m.o.i of the adenovirus. Overexpression of SPARC induces cell rounding and partial detachment of confluent monolayers of HTM cells. Conclusion:The recombinant virus is able to transfer a protein with identical properties to the endogenous protein. Since SPARC is constitutively expressed in the cells of the outflow pathway its ability to induce cell shape changes in HTM cells is consistent with a possible role in outflow facility modulation.

Keywords: 601 trabecular meshwork • 307 adenovirus • 339 cell adhesions/cell junctions