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

ABSTRACT: 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 ?2 subunit affects IOP and aqueous humor clearance in mice.Human TM tissue was examined for expression of AMPK?1 and AMPK?2, 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-?-Dffff-ribofuranosyl-imidazole-4-carboxamide (AICAR), under basal or TGF-?2 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 AMPK? subunit (ad.DN.AMPK?) and were similarly analyzed. Intraocular pressure, central corneal thickness (CCT), and aqueous clearance were measured in AMPK?2-null and wild-type (WT) mice.Both AMPK?1 and AMPK?2 are expressed in TM. AICAR activated AMPK? and suppressed the expression of various ECM proteins under basal and TGF-?2 stimulatory conditions. AICAR decreased F-actin staining and increased the phospho-total RhoA ratio (Ser188). Transforming growth factor-?2 transiently dephosphorylated AMPK?. Infection with ad.DN.AMPK? upregulated various ECM proteins, decreased the phospho-total RhoA ratio, and increased F-actin staining. AMPK?2-null mice exhibited 6% higher IOP and decreased aqueous clearance compared with WT mice, without significant differences in CCT or angle morphology.Collectively, our data identify AMPK as a critical regulator of ECM homeostasis and cytoskeletal arrangement in the TM. Mice that are AMPK?2-null exhibit higher IOPs and decreased aqueous clearance than their WT counterparts.

SUBMITTER: Chatterjee A

PROVIDER: S-EPMC4023495 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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

Chatterjee Ayan A Villarreal Guadalupe G Oh Dong-Jin DJ Kang Min Hyung MH Rhee Douglas J DJ

Investigative ophthalmology & visual science 20140408 5

<h4>Purpose</h4>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.<h4>Methods</h4>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 t ...[more]

PMID: 24713487

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Project description:PurposeBiophysical and biochemical attributes of the extracellular matrix are major determinants of cell fate in homeostasis and disease. Ocular hypertension and glaucoma are diseases where the trabecular meshwork tissue responsible for aqueous humor egress becomes stiffer accompanied by changes in its matrisome in a segmental manner with regions of high or low flow. Prior studies demonstrate these alterations in the matrix are dynamic in response to age and pressure changes. The underlying reason for segmentation or differential response to pressure and stiffening are unknown. This is largely due to a lack of appropriate models (in vitro or ex vivo) to study this phenomena.MethodsPrimary trabecular meshwork cells were isolated from segmental flow regions, and cells were cultured for 4 weeks in the presence or absence or dexamethasone to obtain cell derived matrices (CDM). The biomechanical attributes of the CDM, composition of the matrisome, and incidence of crosslinks were determined by atomic force microscopy and mass spectrometry.ResultsData demonstrate that matrix deposited by cells from low flow regions are stiffer and exhibit a greater number of immature and mature crosslinks, and that these are exacerbated in the presence of steroid. We also show a differential response of high or low flow cells to steroid via changes observed in the matrix composition. However, no correlations were observed between elastic moduli and presence or absence of mature and immature crosslinks in the CDMs.ConclusionRegardless of a direct correlation between matrix stiffness and crosslinks, we observed distinct differences in the composition and mechanics of the matrices deposited by segmental flow cells. These results suggest distinct differences in cellular identify and likely a basis for mechanical memory post isolation and culture. Nevertheless, we conclude that although a mechanistic basis for matrix stiffness was undetermined in this study, it is a viable tool to study cell-matrix interactions and further our understanding of trabecular meshwork pathobiology.

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

Publications

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

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