PURPOSE: This investigation was undertaken to determine whether the matricellular protein SPARC is expressed in the human trabecular meshwork (TM) and cultured human trabecular meshwork cells. METHODS: Human donor trabecular meshwork and cultured cells obtained from trabecular meshwork were used in this study. Total RNA was obtained from TM and cultured TM endothelial cells, and RT-PCR was done with primers specific for SPARC. Western blotting was performed on donor TMs using an anti-SPARC monoclonal antibody prepared against rHuSPARC. Confocal microscopy was used to determine the distribution of SPARC in human anterior segments, and immunofluorescence on cultured TM cells was performed with the anti-SPARC antibody. RESULTS: SPARC mRNA was expressed both in TM and in cultured TM cells. Immunoblotting for SPARC showed a doublet with a molecular mass approximately 43 kDa. The ratio of the doublet bands varied with each of the samples; some of the cultured cells and the tissue samples exhibited more of the upper band, and other cultured cells contained almost equal amounts of the two bands. The upper band was shown to be a glycosylated form of SPARC. Immunofluorescence showed that SPARC was expressed in the cultured TM, and confocal microscopy with the anti-SPARC antibody demonstrated the presence of this protein in the TM and in other tissues in the anterior segment. CONCLUSIONS: Our data conclusively show that SPARC mRNA and protein are present in non-glaucomatous TM tissue and in cultured TM cells. Because of its effect on matrix metalloproteinases, SPARC may play a role in the regulation of intraocular pressure.
PURPOSE: This investigation was undertaken to determine whether the matricellular protein SPARC is expressed in the human trabecular meshwork (TM) and cultured human trabecular meshwork cells. METHODS:Humandonor trabecular meshwork and cultured cells obtained from trabecular meshwork were used in this study. Total RNA was obtained from TM and cultured TM endothelial cells, and RT-PCR was done with primers specific for SPARC. Western blotting was performed on donor TMs using an anti-SPARC monoclonal antibody prepared against rHuSPARC. Confocal microscopy was used to determine the distribution of SPARC in human anterior segments, and immunofluorescence on cultured TM cells was performed with the anti-SPARC antibody. RESULTS:SPARC mRNA was expressed both in TM and in cultured TM cells. Immunoblotting for SPARC showed a doublet with a molecular mass approximately 43 kDa. The ratio of the doublet bands varied with each of the samples; some of the cultured cells and the tissue samples exhibited more of the upper band, and other cultured cells contained almost equal amounts of the two bands. The upper band was shown to be a glycosylated form of SPARC. Immunofluorescence showed that SPARC was expressed in the cultured TM, and confocal microscopy with the anti-SPARC antibody demonstrated the presence of this protein in the TM and in other tissues in the anterior segment. CONCLUSIONS: Our data conclusively show that SPARC mRNA and protein are present in non-glaucomatous TM tissue and in cultured TM cells. Because of its effect on matrix metalloproteinases, SPARC may play a role in the regulation of intraocular pressure.
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