Differential effects of caveolin-1 and -2 knockdown on aqueous outflow and altered extracellular matrix turnover in caveolin-silenced trabecular meshwork cells

Mini Aga, John M. Bradley, Rohan Wanchu, Yong Feng Yang, Ted Acott, Kate Keller

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

PURPOSE. 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 a-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.

Original languageEnglish (US)
Pages (from-to)5497-5509
Number of pages13
JournalInvestigative Ophthalmology and Visual Science
Volume55
Issue number9
DOIs
StatePublished - 2014

Fingerprint

Caveolin 2
Caveolins
Trabecular Meshwork
Caveolin 1
Extracellular Matrix
Disintegrins
Lentivirus
Perfusion
Metalloproteases
Endocytosis
Glaucoma
Actins
Cortactin
Stress Fibers
Chromosomes, Human, Pair 7
Extracellular Matrix Proteins
Matrix Metalloproteinase 2
Enzyme Assays
Matrix Metalloproteinases
Fibronectins

Keywords

  • Endocytosis
  • Extracellular matrix
  • Glaucoma anterior segment
  • Matrix metalloproteinases
  • Trabecular meshwork

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience
  • Medicine(all)

Cite this

Differential effects of caveolin-1 and -2 knockdown on aqueous outflow and altered extracellular matrix turnover in caveolin-silenced trabecular meshwork cells. / Aga, Mini; Bradley, John M.; Wanchu, Rohan; Yang, Yong Feng; Acott, Ted; Keller, Kate.

In: Investigative Ophthalmology and Visual Science, Vol. 55, No. 9, 2014, p. 5497-5509.

Research output: Contribution to journalArticle

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abstract = "PURPOSE. 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 a-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.",
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T1 - Differential effects of caveolin-1 and -2 knockdown on aqueous outflow and altered extracellular matrix turnover in caveolin-silenced trabecular meshwork cells

AU - Aga, Mini

AU - Bradley, John M.

AU - Wanchu, Rohan

AU - Yang, Yong Feng

AU - Acott, Ted

AU - Keller, Kate

PY - 2014

Y1 - 2014

N2 - PURPOSE. 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 a-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.

AB - PURPOSE. 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 a-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.

KW - Endocytosis

KW - Extracellular matrix

KW - Glaucoma anterior segment

KW - Matrix metalloproteinases

KW - Trabecular meshwork

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