Perturbation of hyaluronan synthesis in the trabecular meshwork and the effects on outflow facility

PURPOSE. Hyaluronan (HA) is a major component of the aqueous outflow pathway. However, the contribution of HA to human outflow resistance remains unclear. Three HA synthase genes (HAS1-3) have been identified. Here, we evaluate the contribution of each of the HAS proteins to outflow facility in anterior segment perfusion culture. METHODS. Two methods were used to reduce HA synthesis: 1 mM 4-methylumbelliferone (4MU) was used to inhibit all HAS synthases and shRNA silencing lentivirus was generated to knock down expression of each HAS individually. Quantitative RT-PCR, Western immunoblotting and an HA ELISA assay were used to assess HAS mRNA and protein levels and HA concentration, respectively. The effects of 4MU treatment and HAS gene silencing on outflow facility were assessed in human and porcine perfusion culture. RESULTS. Quantitative RT-PCR and Western immunoblotting showed a reduction of each HAS in response to their respective silencing and 4MU treatment. HA concentration was concomitantly reduced. Treatment with 4MU decreased outflow facility in human anterior segments but increased outflow facility in porcine eyes. Lentiviral delivery of HAS1 and HAS2 silencing vectors caused similar opposite effects on outflow facility. Silencing of HAS3 did not significantly affect outflow resistance in either species. CONCLUSIONS. This is the first conclusive evidence for a significant role of HA in the human outflow pathway. HA chains synthesized by HAS1 and HAS2 contribute to outflow resistance, while hyaluronan produced by HAS3 does not appear to play a significant role. (Invest Ophthalmol Vis Sci. 2012;53:4616-4625) DOI:10.1167/iovs.12-9500 H yaluronan (HA) is a large, negatively charged glycosaminoglycan (GAG) chain that is synthesized by three homologous hyaluronan synthase (HAS) enzymes called HAS1, HAS2 and HAS3. 2 The growing HA chain is extruded directly through the plasma membrane into the extracellular milieu. As a result, HA is not sulfated and does not undergo epimerization. Each HAS produces HA in differing amounts and of different sizes: HAS1 produces low amounts of high molecular weight (HMW) HA, HAS2 produces high amounts of HMW HA, while HAS3 produces high amounts of low MW (LMW) HA. 5 The coumarin derivative, 4-methylumbelliferone (4MU), is a potent inhibitor of HA synthesis and exerts its effects two ways: it decreases HAS2 and HAS3 mRNA levels and also depletes the cellular pool of UDP-glucuronic acid, a building block of HA chains. 6 Subsequent studies suggest that 4MU also decreases HAS1 mRNA levels. 7 Previous studies have shown that HASs are expressed by bovine trabecular meshwork cells and in primate trabecular meshwork tissue. 10 HA distribution in the TM has been assessed using HA binding protein (HAbp). HA was found in the anterior non-filtering portion of the TM and in the juxtacanalicular (JCT) or cribriform region near the inner wall of Schlemm's canal. 14,18 As humans age, there is a decrease in the amount of HA and there is a much greater loss in primary open-angle glaucoma (POAG) TM. 12 Experiments performed in the 1950's demonstrated that treatment of bovine eyes with hyaluronidase to degrade HA decreased outflow resistance and consequently increased outflow facility. Recently, we showed that RNAi silencing of versican, an HA binding protein, reduced outflow facility in human anterior segment perfusion culture

Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings

Involvement of Protein Kinase C in TNF␣ Regulation of Trabecular Matrix Metalloproteinases and TIMPs

PURPOSE. The cytokine TNF␣ is a strong modulator of trabecular meshwork (TM) matrix metalloproteinase (MMP) and tissue inhibitor (TIMP) expression. Studies were conducted to identify signal-transduction pathways involved. METHODS. Porcine TM cells were treated with TNF␣, and MMP and TIMP levels were evaluated by zymography and Western immunoblot. Inhibitors and activators of several signal-transduction pathways were used to select pathways that could be involved. Trabecular protein kinase C (PKC) isoforms were identified and localized by using Western immunoblots and confocal immunohistochemistry. Changes in subcellular distribution of PKC isoforms were evaluated. PKC isoform downregulation and additional inhibition profiles were used to refine the involvement pattern of different isoforms. RESULTS. TNF␣ treatment increased MMP-1, -3, and -9 and TIMP-1 expression, whereas MMP-2 expression was not affected and TIMP-2 expression decreased. Agents that modulate protein kinase A (PKA) or inhibit phosphatidylinositol 3-kinase (PI3K) had minimal effects on trabecular MMP or TIMP induction by TNF␣, whereas several agents that modulate PKC activity were effective. Trabecular cells expressed several PKC isoforms, which exhibited distinctive subcellular localization. TNF␣ treatment triggered some PKC isoform translocations. Exposure of trabecular cells to TNF␣ for 72 hours differentially downregulated several PKC isoforms. Treatment with a phorbol mitogen that stimulates most PKC isoforms produced strong increases in these MMPs. TNF␣'s effects on MMP and TIMP expression were completely blocked by only one PKC inhibitor. CONCLUSIONS. The PKA and PI3K pathways appear not to be involved directly in transducing this TNF␣ signal, but at least one isoform of PKC seems to be required. Based on the inhibitor profiles and the downregulation and translocation studies, PKC appears to be critical in transducing this signal. Unraveling the remaining steps in this and in additional related TM signal-transduction pathways may provide targets for developing improved glaucoma treatments. (Invest Ophthalmol Vis Sci. 2001;42:2831-2838 O ngoing trabecular meshwork (TM) extracellular matrix (ECM) turnover, initiated and controlled at least in part by the matrix metalloproteinase (MMP) family and their tissue inhibitors (TIMPs), is required to maintain normal aqueous humor outflow rates. 1 Increased ECM turnover, initiated by elevated MMP levels in the juxtacanalicular region of the meshwork, may explain the efficacy of laser trabeculoplasty, a common treatment for glaucoma. The MMPs and TIMPs are integrally involved in ECM turnover throughout the body. MMP activity is modulated by extracellular zymogen activation, by TIMP inhibition, and probably by changes in MMP protein interactions and turnover. 9 -11 Intricate and complex transcriptional regulation of MMP and TIMP expression provides an additional level of ECM turnover regulation. The 5Ј-promoter regions of the various MMP and TIMP genes contain a variety of simple and complex enhancer elements, and their expression is modulated by numerous growth factors, cytokines, steroids, integrin ligation, and other extracellular information and conditions

Signaling pathways used in trabecular matrix metalloproteinase response to mechanical

PURPOSE. Trabecular meshwork (TM) matrix metalloproteinase (MMP), and tissue inhibitor (TIMP) changes in response to mechanical stretching appear to be central to intraocular pressure (IOP) homeostasis. Studies were conducted to define the signal transduction pathway responsible for the increases in MMP-2 and -14 that occur in response to mechanical stretching of TM cells. METHODS. Porcine TM cells were subjected to mechanical stretching, and changes in MMP-2 and -14 levels were determined by gelatin zymography and Western immunoblot analysis. Effects of signal transduction pathway inhibitors on MMP levels were analyzed. Phosphospecific antibodies were used to identify phosphorylation changes in select pathway intermediates. In silico secondary structure analysis was conducted on the 5Ј untranslated regions (UTRs) of MMP-2 and -14 mRNAs. RESULTS. The increases in MMP-2 and -14 that occur 24 hours after sustained mechanical stretching of TM cells were blocked by rapamycin. Wortmannin blocked the MMP-2 but not the MMP-14 increase. Protein kinase B (PKB) phosphorylation on S473 and T308 was increased significantly by stretching. Rapamycin-sensitive phosphorylation of T389 in p70/p85 S6 kinase was also increased. The phosphorylations of the translation initiation factor eIF-4E on S209 and of its inhibitory binding protein 4E-BP1 on T70 were both increased by stretch. The calculated free energies of secondary structures of the 5Ј UTRs of the mRNAs for MMP-2 and -14 were negative and relatively large. MMP-2 also had pyrimidine tracts in the extreme 5Ј region of its UTR. CONCLUSIONS. The increases in TM MMP-2 and -14 protein levels in response to mechanical stretching appear to be transduced at least in part by mTOR, the mammalian target of rapamycin (mTOR). The wortmannin sensitivity implicates phosphoinositide 3-kinase as a modulator of the MMP-2 but not the MMP-14 increase. Integrin-linked kinase (ILK), phosphoinositide-dependent kinase (PDK-1), and PKB are implicated in the MMP-2 increase. Translational initiation involving eIF-4E and its inhibitory binding protein 4E-BP1 appear to be involved in both the MMP-2 and -14 increases with stretching and are normally regulated by mTOR. The high degree of secondary structure in the 5Ј UTRs of these transcripts is typically an indicator of genes specifically sensitive to regulation through this pathway. P70/p85 S6 kinase is probably involved downstream from mTOR and PKB in regulating translation of MMP-2, which has pyrimidine tracts in its 5Ј UTR. Manipulation of these transduction pathways may provide new approaches to therapeutic IOP regulation. (Invest Ophthalmol Vis Sci. 2003;44:5174 -5181) DOI:10.1167/iovs.03-0213 T he mechanism that provides normal IOP homeostasis is poorly understood. A relatively effective homeostatic mechanism must exist, because only approximately 3% to 5% of people exhibit pathologic elevations in IOP with subsequent optic nerve damage, even at advanced ages. 1,2 We have hypothesized that trabecular meshwork (TM) cells can adjust the outflow resistance over the time scale of hours to days by modulating TM extracellular matrix (ECM) turnover and subsequent biosynthetic replacement. 3-6 Manipulation of the activity of a family of TM ECM turnover enzymes, the matrix metalloproteinases (MMPs), reversibly modulates outflow facility. 7 Inhibition of the endogenous ECM turnover by these MMPs increases the outflow resistance, thus ongoing ECM turnover must be necessary for normal IOP maintenance

Segmental Versican Expression in the Trabecular Meshwork and Involvement in Outflow Facility

PURPOSE. Versican is a large proteoglycan with numerous chondroitin sulfate (CS) glycosaminoglycan (GAG) side chains attached. To assess versican's potential contributions to aqueous humor outflow resistance, its segmental distribution in the trabecular meshwork (TM) and the effect on outflow facility of silencing the versican gene were evaluated. METHODS. Fluorescent quantum dots (Qdots) were perfused to label outflow pathways of anterior segments. Immunofluorescence with confocal microscopy and quantitative RT-PCR were used to determine versican protein and mRNA distribution relative to Qdot-labeled regions. Lentiviral delivery of shRNAsilencing cassettes to TM cells in perfused anterior segment cultures was used to evaluate the involvement of versican and CS GAG chains in outflow facility. RESULTS. Qdot uptake by TM cells showed considerable segmental variability in both human and porcine outflow pathways. Regional levels of Qdot labeling were inversely related to versican protein and mRNA levels; versican levels were relatively high in sparsely Qdot-labeled regions and low in densely labeled regions. Versican silencing decreased outflow facility in human and increased facility in porcine anterior segments. However, RNAi silencing of ChGn, an enzyme unique to CS GAG biosynthesis, increased outflow facility in both species. The fibrillar pattern of versican immunostaining in the TM juxtacanalicular region was disrupted after versican silencing in perfusion culture. CONCLUSIONS. Versican appears to be a central component of the outflow resistance, where it may organize GAGs and other ECM components to facilitate and control open flow channels in the TM. However, the exact molecular organization of this resistance appears to differ between human and porcine eyes. 1,2 Much of this resistance resides within the trabecular meshwork (TM) putatively within 7 to 14 m of the inner wall of Schlemm's canal in a region known as the juxtacanalicular (JCT) or cribriform region. 1-4 Since the 1950s, involvement of extracellular matrix (ECM) in outflow resistance has commonly been evoked. 1,2,5-10 There is also considerable evidence of a direct contribution to the resistance by some cell populations within this region. 1,3 More recently, synergistic interaction between the JCT and Schlemm's inner wall endothelial cells has been suggested. 2,6,12-14 Although perfusion of GAG-degrading enzymes reduces the outflow resistance in numerous species, in humans and primates, this effect has been controversial. 17-21 The identity of the specific GAGs, proteoglycans, or other ECM components that comprise outflow resistance remains unclear. 2 Both conceptually and based on several observations, versican, with supportive contributions from its attached chondroitin sulfate (CS) GAG chains and hyaluronan (HA) interactions, seems a likely contributor. 2,24 Up to 23 CS GAG side chains can be attached to these two central domains. These CS chains appear to extend away from the core protein in all directions, thus minimizing electrostatic interactions and filling large hydrodynamic volumes. Conceptually, this design is ideal to regulate movement of aqueous humor through the TM. 27 Mechanical stretching or distortion appears to be the mechanism by which TM cells sense elevated IOP and trigger IOP homeostatic responses. 30 -33 TNF␣ and IL-1␣, both of which increase outflow facility, also produce changes in versican mRNA level and isoform distribution similar t

Segmental Versican Expression in the Trabecular Meshwork and Involvement in Outflow Facility

Studies of segmental aqueous humor outflow and versican distributions suggest that this large proteoglycan has a role in outflow resistance. Effects of changing versican levels and glycosaminoglycan side chains on outflow facility support versican as an important component of outflow resistance

Inhibition of hyaluronan synthesis reduces versican and fibronectin levels in trabecular meshwork cells.

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