Abstract
Hyaluronan (HA) is a major component of the extracellular matrix (ECM) and is synthesized by three HA synthases (HAS). Similarities between the HAS2 knockout mouse and the hdf mutant mouse, which has a mutation in the versican gene, suggest that HA and versican expression may be linked. In this study, the relationship between HA synthesis and levels of versican, fibronectin and several other ECM components in trabecular meshwork cells from the anterior segment of the eye was investigated. HA synthesis was inhibited using 4-methylumbelliferone (4MU), or reduced by RNAi silencing of each individual HAS gene. Quantitative RT-PCR and immunoblotting demonstrated a reduction in mRNA and protein levels of versican and fibronectin. Hyaluronidase treatment also reduced versican and fibronectin levels. These effects could not be reversed by addition of excess glucose or glucosamine or exogenous HA to the culture medium. CD44, tenascin C and fibrillin-1 mRNA levels were reduced by 4MU treatment, but SPARC and CSPG6 mRNA levels were unaffected. Immunostaining of trabecular meshwork tissue after exposure to 4MU showed an altered localization pattern of HA-binding protein, versican and fibronectin. Reduction of versican by RNAi silencing did not affect HA concentration as assessed by ELISA. Together, these data imply that HA concentration affects synthesis of certain ECM components. Since precise regulation of the trabecular meshwork ECM composition and organization is required to maintain the aqueous humor outflow resistance and intraocular pressure homeostasis in the eye, coordinated coupling of HA levels and several of its ECM binding partners should facilitate this process.
Highlights
Trabecular meshwork (TM) cells are a specialized endothelial cell type in the anterior segment of the eye. [1] TM cells have two main functions: One is to phagocytose debris present in aqueous humor as it passes through the filter-like tissue of the TM and drains into Schlemm’s canal. [2,3] The other main function of TM cells is to modulate the flow resistance to aqueous humor in order to maintain intraocular pressure (IOP). [4,5] The TM cells responsible for IOP homeostasis reside deep within the TM in an area closest to Schlemm’s canal, a region known as the juxtacanalicular (JCT) or cribriform region
Effects of 4MU on Versican 4MU is an inhibitor of HA synthesis. [22,23,24] Previously, we found that 4MU treatment decreased HA synthesis by 60–75% in cell lysates and media of porcine TM cells
The V3 (74 kDa) isoform was not detected. This data is consistent with the relative levels of isoform-specific transcripts detected by Quantitative RT-PCR (qRT-PCR) in TM cells. [8,27] Densitometry was used to quantify the intensity of the V1 isoform bands in the media immunoblot
Summary
Trabecular meshwork (TM) cells are a specialized endothelial cell type in the anterior segment of the eye. [1] TM cells have two main functions: One is to phagocytose debris present in aqueous humor as it passes through the filter-like tissue of the TM and drains into Schlemm’s canal. [2,3] The other main function of TM cells is to modulate the flow resistance to aqueous humor in order to maintain intraocular pressure (IOP). [4,5] The TM cells responsible for IOP homeostasis reside deep within the TM in an area closest to Schlemm’s canal, a region known as the juxtacanalicular (JCT) or cribriform region. [2,3] The other main function of TM cells is to modulate the flow resistance to aqueous humor in order to maintain intraocular pressure (IOP). Modification of outflow resistance appears to involve cellular recognition of elevated IOP as a stretch/ distortion, focal proteinase degradation of the existing extracellular matrix (ECM) and biosynthesis of replacement components. [8] This newly deposited ECM alters the resistance to aqueous outflow Dysregulation of this tightly regulated balance of degradation and synthesis can result in elevated IOP. The effects of 4-methylumbelliferone (4MU), an inhibitor of HA synthesis, [22,23,24] and RNAi silencing of each individual HAS gene on outflow facility in an ocular perfusion culture model of aqueous humor outflow was reported. [25] Inhibition of HA synthesis by both methods increased outflow resistance in human eyes
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