Min Hyung Kang

Coordinated regulation of extracellular matrix synthesis by the microRNA-29 family in the trabecular meshwork.

PURPOSE The microRNA-29 (miR-29) family has emerged, in various tissues, as a key modulator of extracellular matrix (ECM) homeostasis. In this study, the authors investigate the role of the miR-29 family in the regulation of ECM synthesis in the trabecular meshwork (TM) under basal and TGF-β2 stimulatory conditions. METHODS Human TM cells were incubated with 2.5 ng/mL activated, recombinant human TGF-β2 for 24, 48, and 72 hours. A specific pharmacologic inhibitor was used to block SMAD3 function in the context of TGF-β2 stimulation. Changes in the expression of the miR-29 family were assessed by real-time PCR. The effect of miR-29 molecules and inhibitors on ECM levels was determined by immunoblot analysis. RESULTS All three members of the miR-29 family were expressed in cultured TM cells. Although the incubation of TM cells with TGF-β2 induced miR-29a and suppressed miR-29b levels, no significant effect was observed on miR-29c expression. Additional studies revealed that SMAD3 modulates miR-29b expression under basal and TGF-β2 conditions. Subsequent gain- and loss-of-function experiments demonstrated that the miR-29 family functions as a critical suppressor of various ECM proteins under basal and TGF-β2 stimulatory conditions. CONCLUSIONS The findings derived from this study identify the miR-29 family as a critical regulator of ECM expression in the TM and suggest that its modulation by TGF-β2 may be important in controlling ECM synthesis. Together, these data provide further insight into the complex regulatory mechanisms mediating TGF-β2 signaling and ECM production in the TM.

SPARC-null mice exhibit lower intraocular pressures.

PURPOSE SPARC is a matricellular protein that is highly expressed in remodeling tissues, including the trabecular meshwork and ciliary body. The hypothesis for the study was that SPARC contributes to the regulation of intraocular pressure (IOP). The IOPs of SPARC-null mice, their corresponding wild-type (WT), and heterozygous animals were compared. METHODS Diurnal and nocturnal IOPs of C57Bl/6x129SvJ WT, SPARC-null, and heterozygous mice were measured. Fluorophotometric measurements were made to assess aqueous turnover. Central corneal thickness (CCT) was measured using histology, ultrasound biomicroscopy, and optical coherence tomography. Iridocorneal angles were examined using light microscopy (LM). RESULTS During the day, the mean IOP of SPARC-null mice (n = 142, 16.9 +/- 2.4 mm Hg) was lower than that of both WT mice (n = 104, 19.9 +/- 2.9 mm Hg; P < 10(-12)), and heterozygotes (n = 38, 19.3 +/- 2.5 mm Hg; P < 10(-4)). At night, SPARC-null mice also exhibited a blunted increase in IOP in comparison to WT and heterozygous mice. CCTs were not significantly different between WT and SPARC-null mice. Heterozygous mice tended to have thicker corneas (3.4%). Fluorophotometric measurements suggest that aqueous turnover rates in SPARC-null mice are equal to if not greater than rates in WT mice. LM of the SPARC-null iridocorneal angle revealed morphology that is indistinguishable from WT. CONCLUSIONS SPARC-null mice have lower IOPs than do their WT counterparts with equal CCTs. The rate of aqueous turnover suggests that the mechanism is enhanced outflow resistance.

Matricellular proteins in the trabecular meshwork

The trabecular meshwork is one of the primary tissues of interest in the normal regulation and dysregulation of intraocular pressure (IOP) that is a causative risk factor for primary open-angle glaucoma. Matricellular proteins generally function to allow cells to modulate their attachments with and alter the characteristics of their surrounding extracellular matrix (ECM). In non-ocular tissues, matricellular proteins generally increase fibrosis. Since ECM turnover is very important to the outflow facility, matricellular proteins may have a significant role in the regulation of IOP. The formalized study of matricellular proteins in trabecular meshwork is in its infancy. SPARC, thrombospondins-1 and -2, and tenascins-C and -X, and osteopontin have been localized to varying areas within the trabecular meshwork. Preliminary evidence indicates that SPARC and thrombospondin-1 play a role in the regulation of IOP and possibly the pathophysiology of glaucoma. These data show promise that matricellular proteins are involved in IOP dysregulation and are potential therapeutic targets. Further study is needed to clarify these roles.

Canonical wnt signaling regulates extracellular matrix expression in the trabecular meshwork.

PURPOSE Canonical Wnt signaling has emerged as a critical regulator of aqueous outflow facility and intraocular pressure (IOP). In this study, we examine the role of canonical Wnt signaling on extracellular matrix (ECM) expression in the trabecular meshwork (TM) and explore the molecular mechanisms involved. METHODS β-catenin localization in human TM tissue was examined using immunofluorescent staining. Primary human TM cells were incubated with lithium chloride (LiCl) and the effect on active β-catenin expression was assessed by immunoblot. Adenovirus expressing a dominant-negative TCF4 mutant that lacks a β-catenin binding domain was used. Changes in the levels of the microRNA-29 (miR-29) family and ECM proteins were determined by real-time quantitative PCR and immunoblot analysis, respectively. RESULTS β-catenin was expressed throughout the TM, with localization primarily to the plasma membrane. Incubation of TM cells with lithium chloride increased the expression of active β-catenin. Lithium chloride treatment upregulated miR-29b expression, and suppressed the levels of various ECM proteins under both basal and TGF-β2 stimulatory conditions. Infection of TM cells with a dominant-negative TCF4 mutant induced ECM levels without a significant change in the expression of the miR-29 family. CONCLUSIONS Collectively, our data identify the canonical Wnt signaling pathway as an important modulator of ECM expression in the TM and provide a mechanistic framework for its regulation of outflow facility and IOP.

The effects of tenascin C knockdown on trabecular meshwork outflow resistance

PURPOSE Tenascin C (TNC) is a matricellular glycoprotein whose expression in adult tissue is indicative of tissue remodeling. The purpose of the current study was to determine the localization of TNC in trabecular meshwork (TM) tissue and to analyze the effects of TNC on intraocular pressure (IOP). METHODS Human TM frontal sections were immunostained with anti-TNC and imaged by confocal microscopy. TNC mRNA and protein levels were quantitated in anterior segments perfused at physiological and elevated pressure. Short, hairpin RNA (shRNA) silencing lentivirus targeting full-length TNC (shTNC) was applied to anterior segment perfusion organ cultures. The IOPs and central corneal thickness (CCT) of wild-type, TNC(-/-), and tenascin X (TNX(-/-)) knockout mice were measured. RESULTS TNC was distributed in the juxtacanalicular (JCT) region of adult human TM, predominantly in the basement membrane underlying the inner wall of Schlemms canal. Application of shTNC lentivirus to human and porcine anterior segments in perfusion culture did not significantly affect outflow rate. Although TNC was upregulated in response to pressure, there was no difference in outflow rate when shTNC-silenced anterior segments were subjected to elevated pressure. Furthermore, IOPs and CCTs were not significantly different between TNC(-/-) or TNX(-/-) and wild-type mice. CONCLUSIONS TNC does not appear to contribute directly to outflow resistance. However, TNC immunolocalization in the JCT of adult human eyes suggests that certain areas of the TM are being continuously remodeled with or without an IOP increase.

AMP-activated protein kinase regulates intraocular pressure, extracellular matrix, and cytoskeleton in trabecular meshwork

PURPOSE In this study, we investigate how adenosine monophosphate-activated protein kinase (AMPK) affects extracellular matrix (ECM) and cellular tone in the trabecular meshwork (TM), and examine how deletion of its catalytic α2 subunit affects IOP and aqueous humor clearance in mice. METHODS Human TM tissue was examined for expression of AMPKα1 and AMPKα2, genomically distinct isoforms of the AMPK catalytic subunit. Primary cultured human TM cells were treated for 24 hours with the AMPK activator 5-amino-1-β-Dffff-ribofuranosyl-imidazole-4-carboxamide (AICAR), under basal or TGF-β2 stimulatory conditions. Conditioned media (CM) was probed for secreted protein acidic and rich in cysteine (SPARC), thrombospondin-1 (TSP-1), and ECM proteins, and cells were stained for F-actin. Cells underwent adenoviral infection with a dominant negative AMPKα subunit (ad.DN.AMPKα) and were similarly analyzed. Intraocular pressure, central corneal thickness (CCT), and aqueous clearance were measured in AMPKα2-null and wild-type (WT) mice. RESULTS Both AMPKα1 and AMPKα2 are expressed in TM. AICAR activated AMPKα and suppressed the expression of various ECM proteins under basal and TGF-β2 stimulatory conditions. AICAR decreased F-actin staining and increased the phospho-total RhoA ratio (Ser188). Transforming growth factor-β2 transiently dephosphorylated AMPKα. Infection with ad.DN.AMPKα upregulated various ECM proteins, decreased the phospho-total RhoA ratio, and increased F-actin staining. AMPKα2-null mice exhibited 6% higher IOP and decreased aqueous clearance compared with WT mice, without significant differences in CCT or angle morphology. CONCLUSIONS Collectively, our data identify AMPK as a critical regulator of ECM homeostasis and cytoskeletal arrangement in the TM. Mice that are AMPKα2-null exhibit higher IOPs and decreased aqueous clearance than their WT counterparts.