PubMed Health⌕ Search

Biomedical subjects

Allan R Shepard

Publications and source records attributed to Allan R Shepard.

4 recordsLinked to original sources
PubMed2006

TGFbeta2-induced changes in human trabecular meshwork: implications for intraocular pressure.

PURPOSE: Transforming growth factor (TGF)-beta2 levels are elevated in glaucomatous human aqueous humor. TGFbeta is a cytokine that alters extracellular matrix (ECM) metabolism, and excess ECM has been proposed to increase aqueous outflow resistance in the trabecular meshwork (TM) of glaucomatous eyes. This study was undertaken to investigate effects of TGFbeta2 on secretion of fibronectin and the protease inhibitor plasminogen activator inhibitor (PAI)-1 from human TM cell cultures and perfused human ocular anterior segments. METHODS: Total RNA was isolated from pooled human TM cell monolayers and used for a gene microarray expression analysis. Supernatants from treated human TM cells were analyzed by ELISA for fibronectin or PAI-1 content. TGFbeta2 effects on intraocular pressure (IOP) were evaluated in a perfused organ culture model using human anterior segments, and eluates were analyzed for fibronectin and PAI-1 content. RESULTS: Overnight treatment of TM cells with TGFbeta2 upregulated multiple ECM-related genes, such as PAI-1. TGFbeta2 also increased secretion of both fibronectin and PAI-1 from TM cells. TGFbeta2 effects on TM cells were blocked by inhibitors of the TGFbeta type I receptor. In perfused human anterior segments, TGFbeta2 treatment elevated IOP and increased eluate fibronectin and PAI-1 content. CONCLUSIONS: TGFbeta2 effects on IOP may be transduced by TGFbeta type-I receptor-mediated changes in TM secretion of ECM-related factors such as fibronectin and PAI-1. Modulation of TGFbeta2-induced changes in the ECM may provide a novel and viable approach to the management of glaucoma.

Aged↗
PubMed2005

Evaluation of inducible nitric oxide synthase in glaucomatous optic neuropathy and pressure-induced optic nerve damage.

PURPOSE: To determine whether inducible nitric oxide synthase (NOS-2) is involved in glaucomatous optic neuropathy. METHODS: Chronic elevation of rat intraocular pressure (IOP) leading to optic nerve damage was induced by episcleral injection of hypertonic saline, which caused sclerosis and blockade of aqueous humor outflow pathways. Expression of NOS-2 in the retina and optic nerve head (ONH) was evaluated by immunohistochemistry, gene array analysis, and quantitative PCR (Q-PCR). Immunohistochemistry was also used to assess the NOS-2 level in the ONH from primary open-angle glaucoma (POAG) and nonglaucomatous human eyes. Finally, an NOS-2 inhibitor, aminoguanidine, administered orally in the drinking water, was tested for its effect on optic nerve injury in rats with ocular hypertension. RESULTS: Chronically elevated IOP in the rat produced optic nerve damage that correlated with pressure change (r(2) = 0.77), but did not increase NOS-2 immunoreactivity in the optic nerve, ONH, or ganglion cell layer. Retinal and ONH NOS-2 mRNA levels did not correlate with either IOP level or severity of optic nerve injury. Similarly, there was no difference in NOS-2 immunoreactivity in the optic nerve or ONH between POAG and nonglaucomatous eyes. Furthermore, aminoguanidine treatment did not affect the development of pressure-induced optic neuropathy in the rat. CONCLUSIONS: As demonstrated by several independent methods, glaucomatous optic neuropathy was not associated with a significant change in the expression of NOS-2 in the retina, ONH, or optic nerve.

Aged↗
PubMed2003

Characterization of rabbit myocilin: Implications for human myocilin glycosylation and signal peptide usage.

BACKGROUND: Mutations in the gene encoding human myocilin (MYOC) have been shown to cause juvenile- and adult-onset glaucoma. In addition, myocilin has been associated with glucocorticoid-induced ocular hypertension and steroid-induced glaucoma. To better understand the role myocilin plays in steroid-induced glaucoma and open-angle glaucoma, we examined rabbit myocilin for use in the rabbit animal model of steroid-induced glaucoma. RESULTS: We have cloned the rabbit ortholog of human MYOC. Rabbit MYOC consists of three exons and an open reading frame encoding a 490 amino acid, 54,882-Da protein, which is 14 amino acids shorter at the N-terminus than human myocilin but 84% identical overall. Rabbit myocilin migrates as a single electrophoretic band, vs. double-banded human myocilin, by SDS-PAGE/immunoblot analysis. We determined that the differential migration exhibited is due to an N-glycosylation site that is present in human (Asn57), monkey and mouse myocilin but absent in rabbit (Ser43), rat and bovine myocilin. Rabbit myocilin is secreted in vitro in trabecular meshwork cell culture and in vivo in aqueous humor. Secretion of human myocilin is shown to be dependent on the signal peptide and independent of the extra 14 amino acids not found in rabbit myocilin. Many of the amino acids in myocilin that are mutated in glaucoma patients are conserved across species. CONCLUSION: We have cloned the rabbit MYOC cDNA and determined that rabbit myocilin is secreted but not N-linked glycosylated. Knowledge of the rabbit MYOC cDNA sequence will facilitate future studies in the rabbit animal model examining the role of myocilin in steroid-induced glaucoma and the gain-of-function hypothesis in open-angle glaucoma.

Amino Acid Sequence↗