title: Differential effects of caveolin-1 and -2 knockdown on aqueous outflow and altered extracellular matrix turnover in caveolin-silenced trabecular meshwork cells.
abstract: OBJECTIVE
  A single nucleotide polymorphism (SNP) identified between caveolin-1 (CAV1) and caveolin-2 (CAV2) on chromosome 7 is associated with glaucoma. One function of CAVs is endocytosis and recycling of extracellular matrix (ECM) components. Here, we generated CAV-silencing lentivirus to evaluate the effects on ECM turnover by trabecular meshwork (TM) cells and to measure the effect on outflow facility in anterior segment perfusion culture.
  
  
  METHODS
  Short hairpin CAV1 and CAV2 silencing and control lentivirus were generated, characterized, and applied to anterior segments in perfusion culture. Colocalization of CAVs with various ECM molecules in TM cells was investigated using immunofluorescence and confocal microscopy. Western immunoblotting and fluorogenic-based enzyme activity assays were used to investigate ECM protein levels and degradation, respectively.
  
  
  RESULTS
  Endogenous CAVs colocalized with cortactin at podosome- or invadopodia-like structures (PILS), which are areas of focal ECM degradation. In perfusion culture, outflow rates increased significantly in CAV1-silenced anterior segments, whereas outflow significantly decreased in CAV2-silenced anterior segments. Matrix metalloproteinase (MMP)2 and MMP14, and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS4) colocalized with both CAVs in TM cells. Protein levels and enzyme activities of MMP/ADAMTS4, fibronectin protein levels, actin stress fibers, and α-smooth muscle actin were all increased in CAV-silenced cells.
  
  
  CONCLUSIONS
  Caveolin-mediated endocytosis is one mechanism by which TM cells can alter the physiological catabolism of ECM in order to change the composition of the outflow channels in the TM to regulate aqueous outflow resistance. Dysregulation of CAV function could contribute to the pathological changes in ECM that are observed in glaucoma.
authors.affiliation: Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States.
authors.forename: Mini
authors.initials: M
authors.lastname: Aga
authors.affiliation: Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States.
authors.forename: John M
authors.initials: JM
authors.lastname: Bradley
authors.affiliation: Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States.
authors.forename: Rohan
authors.initials: R
authors.lastname: Wanchu
authors.affiliation: Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States.
authors.forename: Yong-feng
authors.initials: YF
authors.lastname: Yang
authors.affiliation: Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States.
authors.forename: Ted S
authors.initials: TS
authors.lastname: Acott
authors.affiliation: Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States.
authors.forename: Kate E
authors.initials: KE
authors.lastname: Keller
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism; D000071121:ADAMTS4 Protein; D000704:Analysis of Variance; D000869:Anterior Eye Segment / Q000503:physiopathology; D015415:Biomarkers / Q000378:metabolism; D051242:Caveolin 1 / Q000235:genetics / Q000502:physiology*; D051243:Caveolin 2 / Q000235:genetics / Q000502:physiology*; D005109:Extracellular Matrix / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D020868:Gene Silencing; D005902:Glaucoma, Open-Angle / Q000503:physiopathology; D006801:Humans; D020781:Matrix Metalloproteinase 1 / Q000378:metabolism; D011348:Procollagen N-Endopeptidase / Q000378:metabolism; D014129:Trabecular Meshwork / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D051242:Caveolin 1; D051243:Caveolin 2; D005353:Fibronectins; D051722:ADAM Proteins; D011348:Procollagen N-Endopeptidase; D020781:Matrix Metalloproteinase 1; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human
keywords: endocytosis; extracellular matrix; glaucoma anterior segment; matrix metalloproteinases; trabecular meshwork
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