Cochlin, Intraocular Pressure Regulation and Mechanosensing
Publication Date
April 04, 2012
Journal
PLOS ONE
Authors
Manik Goel, Adam E. Sienkiewicz, Renata Picciani, Jianhua Wang, et al
Volume
7
Issue
4
Pages
e34309
DOI
https://dx.plos.org/10.1371/journal.pone.0034309
Publisher URL
http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0034309
PubMed
http://www.ncbi.nlm.nih.gov/pubmed/22496787
PubMed Central
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3319572
Europe PMC
http://europepmc.org/abstract/MED/22496787
Web of Science
000304855200038
Scopus
84859301157
Mendeley
http://www.mendeley.com/research/cochlin-intraocular-pressure-regulation-mechanosensing
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Mendeley | Further Information

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Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/337931", "https://ndownloader.figshare.com/files/337986", "https://ndownloader.figshare.com/files/338080"], "description"=>"<div><p>Fluid shear modulates many biological properties. How shear mechanosensing occurs in the extracellular matrix (ECM) and is transduced into cytoskeletal change remains unknown. Cochlin is an ECM protein of unknown function. Our investigation using a comprehensive spectrum of cutting-edge techniques has resulted in following major findings: (1) over-expression and down-regulation of cochlin increase and decrease intraocular pressure (IOP), respectively. The overexpression was achieved in DBA/2J-Gpnmb<sup>+</sup>/SjJ using lentiviral vectors, down-regulation was achieved in glaucomatous DBA/2J mice using targeted disruption (cochlin-null mice) and also using lentiviral vector mediated shRNA against cochlin coding region; (2) reintroduction of cochlin in cochlin-null mice increases IOP; (3) injection of exogenous cochlin also increased IOP; (4) increasing perfusion rates increased cochlin multimerization, which reduced the rate of cochlin proteolysis by trypsin and proteinase K; The cochlin multimerization in response to shear stress suggests its potential mechanosensing. Taken together with previous studies, we show cochlin is involved in regulation of intraocular pressure in DBA/2J potentially through mechanosensing of the shear stress.</p> </div>", "links"=>[], "tags"=>["intraocular", "mechanosensing"], "article_id"=>126792, "categories"=>["Biochemistry", "Medicine", "Physiology", "Biotechnology", "Cell Biology", "Biophysics"], "users"=>["Manik Goel", "Adam E. Sienkiewicz", "Renata Picciani", "Jianhua Wang", "Richard K. Lee", "Sanjoy K. Bhattacharya"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0034309.s001", "https://dx.doi.org/10.1371/journal.pone.0034309.s002", "https://dx.doi.org/10.1371/journal.pone.0034309.s003"], "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Cochlin_Intraocular_Pressure_Regulation_and_Mechanosensing/126792", "title"=>"Cochlin, Intraocular Pressure Regulation and Mechanosensing", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-04-04 01:53:12"}
  • {"files"=>["https://ndownloader.figshare.com/files/657354"], "description"=>"<p>(A) DBA/2J-Gpnmb<sup>+</sup>/SjJ mice (n = 42–48 for each vector at each time point, as indicated by the symbols) at six months of age were injected with a lentiviral vector bearing the COCH-GFP transgene or GFP alone (sham) or human serum albumin (HSA) in the anterior chamber (all under the control of a CMV promoter). IOP was recorded at the indicated time periods. (B) C57BL/6J mice at six months of age were injected with a lentiviral vector bearing the <i>COCH</i>-<i>GFP</i> transgene (n = 20) or <i>GFP</i> alone (n = 20) in the anterior chamber. The mice were followed and IOP recorded at the indicated time periods. (C) Representative immunohistochemical analysis of DBA/2J-Gpnmb<sup>+</sup>/SjJ mice eyes injected with <i>COCH-GFP</i> transgene or GFP alone. Top panel shows the anatomy of the anterior chamber stained with haematoxylin and eosin. SC = Schlemm's canal, TM = trabecular meshwork; Bar = 125 µm Bottom panels show eyes injected with COCH-GFP or GFP alone as indicated. The brightfield and antibody probing for cochlin (magenta), GFP (green) and merge image has been shown as indicated. Scale bar = 100 µm (D) Western analysis of the TM protein extract of the DBA/2J-Gpnmb<sup>+</sup>/SjJ mice injected with GFP alone or COCH transgene with IRES mediated GFP expression (COCH-GFP). GAPDH has been shown as a loading control. (E) Western analysis of the TM extracts of DBA/2J mice (8 months old) un-injected control or injected with cochlin shRNA. Coomassie blue stained gel shows total protein loading. (F) Downregulation of cochlin in DBA/2J cochlin<sup>+/+</sup> mice decreases the mean IOP. DBA/2J cochlin<sup>+/+</sup> mice (n = 38–45 for each vector at each point) at six months of age were injected with a lentiviral vector bearing cochlin shRNA or GFP alone in the anterior chamber. IOP was recorded at the indicated time periods. (G) Level of cochlin expression in the TM of DBA/2J cochlin<sup>+/+</sup> mice correlates with the IOP. The level of cochlin expression was quantified at different ages (n = 10 at each age group) in vivo using the spectral domain OCT (experimental procedures) and IOP are represented by hollow and solid symbols respectively. (H) Representative SD OCT image of the anterior chamber angle a 6 month old DBA/2J cochlin<sup>+/+</sup> mice before (left) and after (right) injection of infra-red (IR-800) dye coupled anti-cochlin antibody. The arrow indicates the region undergoing a change in intensity of signal before and after injection. The images obtained before and after injection were digitally subtracted for the region of interest (inset). Error bars (A, B, F and G) depict ± standard deviation.</p>", "links"=>[], "tags"=>["recombinant", "cochlin", "mice", "tm", "concomitant", "iop"], "article_id"=>327833, "categories"=>["Biochemistry", "Medicine", "Physiology", "Biotechnology", "Cell Biology", "Biophysics"], "users"=>["Manik Goel", "Adam E. Sienkiewicz", "Renata Picciani", "Jianhua Wang", "Richard K. Lee", "Sanjoy K. Bhattacharya"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0034309.g001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Elevated_recombinant_cochlin_expression_in_mice_TM_is_concomitant_with_the_IOP_elevation_/327833", "title"=>"Elevated recombinant cochlin expression in mice TM is concomitant with the IOP elevation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-04 02:10:33"}
  • {"files"=>["https://ndownloader.figshare.com/files/657612"], "description"=>"<p>(A) Western analysis of purified recombinant cochlin (*) using anti-cochlin antibody before after sheer stress, revealing shear stress-induced mulitmers. Cochlin was subjected to fluid shear of 3 µl/min for 150 cycles. (B) Comparison of proteolytic digestion (in percentage) of native (left side) and multimerized (right side) cochlin by trypsin (open bars) and proteinase K (filled bars) showing slower digestion of multimerized cochlin. Digestion of multimerized and native cochlin (10 µg) was performed using 0.01 µg/10 µg Trypsin or 0.05 µg/10 µg Proteinase K. After incubation at room temperature, the samples were boiled using 1 mM DTT at 100°C for 1 minute and analyzed on SDS-PAGE. Densitometric analyses data from three independent experiments (mean± standard deviation) has been presented. (C) A representative Western analysis of native and multimerized cochlin subjected to subcatalytic amounts of trypsin and proteinase K digestion for 10 minutes at room temperature. Purified recombinant cochlin (*), native monomeric cochlin and multimeric cochlin (initial amount 1 µg) has been depicted as Mo and Mu respectively. Digestion was performed by 0. 1 µg/10 µg Trypsin or 0.5 µg/10 µg Proteinase K. Digestion was stopped by heating at 100°C, separated on a 10% reducing SDS-PAGE and probed with chicken polyclonal antibody against cochlin. Proposed model of cochlin mechanosensing and associated global change in TM. (D) Illustration depicting that the cochlin constitutively secreted by the normal TM cells is degraded by the proteases (dashed lines denoting degraded proteins). (E) Cochlin in the presence of fluctuating shear stress (or elevated divalent cations), forms multimers which are resistant to proteolysis. Multimerized cochlin may potentially interact with the transmembrane proteins TREK-1 or slc44a2 either directly, functionally, or indirectly leading to events that may result in cytoskeletal reorganization. (F) Confocal microscopy image showing the sieve like structure of TM (arrow). A representative area of TM with rectangular arrangement of cells is highlighted by a yellow box. SC = Schlemm's canal; TM = trabecular meshwork. The magnified view of rectangular arrangement of cells highlighted by a yellow box also has been shown. (G) The line diagram depicts the filter like structure of the TM. A magnified view mimicking one such rectangle as in (F) with an additional cell in the middle of the rectangle has been shown. Notice the change in orientation of the middle cell opens up interstitial space for additional fluid flow indicated by dashed arrows. (H) When incremental space opening is insufficient, larger orientation changes by cells in the middle and those forming rectangle are necessary to increase fluid flow. Newly opened space is indicated by arrow head. The dark lines and dark dashed lines are to indicate orientation changes. (I) Cartoon illustrating concerted orientation changes in several individual cells (dashed lines) leading to a global change in the structure of the sieve like TM. (J) Confocal microscopy image of cochlin transfected TM cells (left; i) expressing cochlin (pink) and tiny aggregates of cochlin (arrows) secreted into the media. These small cochlin aggregates coalesce (arrow) to form larger deposits as shown in human glaucomatous TM probed for cochlin (green; right; ii). Scale bar = 25 µm (left) and 50 µm (right). (K) Confocal microscopy image of trilayer of cochlin transfected TM cells on a PVDF membrane exposed to continuous fluctuation in fluid shear stress in an Ussing type chamber showing the development of cochlin deposits (red; arrows).</p>", "links"=>[], "tags"=>["mechanosensing", "tm"], "article_id"=>328090, "categories"=>["Biochemistry", "Medicine", "Physiology", "Biotechnology", "Cell Biology", "Biophysics"], "users"=>["Manik Goel", "Adam E. Sienkiewicz", "Renata Picciani", "Jianhua Wang", "Richard K. Lee", "Sanjoy K. Bhattacharya"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0034309.g003", "stats"=>{"downloads"=>4, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cochlin_mechanosensing_and_proposed_model_of_potential_associated_TM_changes_/328090", "title"=>"Cochlin mechanosensing and proposed model of potential associated TM changes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-04 02:14:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/657481"], "description"=>"<p>(A) Agarose gel (1.2%) showing the absence of PCR products for exons 8, 10 and 11 in the DBA/2J cochlin<sup>−/−</sup> mice. These exons are present in DBA/2J cochlin<sup>+/+</sup> and cochlin<sup>+/−</sup> mice. DNA was isolated from mice tail and subjected to PCR amplification for the indicated exons. (B) Western blot analysis of TM extract of DBA/2J cochlin<sup>+/+</sup>, DBA/2J cochlin<sup>+/−</sup> and DBA/2J cochlin<sup>−/−</sup> mice showing the complete absence of cochlin in DBA/2J cochlin<sup>−/−</sup> mice. Coomassie blue stained gel shows equal loading of protein in all the lanes. (C) Intraocular pressure (IOP in mmHg) in different mice strains at indicated ages (n = 35–45 for each strain at each age group). The DBA/2J cochlin<sup>+/+</sup> mice show a higher mean IOP as compared to DBA/2J-Gpnmb<sup>+</sup>/SjJ, DBA/2J cochlin<sup>+/−</sup> and DBA/2J cochlin−/− mice at 6 months and at 8 months of age. The difference is most marked at 8 months of age. By 12 months, the mean IOP is similar for the different strains. (D) Western blot analysis of the TM protein extract from the DBA/2J cochlin<sup>+/+</sup> and DBA/2J-Gpnmb<sup>+</sup>/SjJ at the indicated ages, probed for cochlin. GAPDH has been shown as a loading control. (E) Generation 10 (Gen 10) DBA/2J cochlin<sup>−/−</sup> at six months of age were injected with a lentiviral vector bearing the COCH-GFP transgene (n = 20) or GFP alone (n = 20) in the anterior chamber. The mice were followed and IOP recorded at the indicated time periods. Error bars (C and E) depict ± standard deviation.</p>", "links"=>[], "tags"=>["disruption", "cochlin", "mice", "tm", "commensurate"], "article_id"=>327959, "categories"=>["Biochemistry", "Medicine", "Physiology", "Biotechnology", "Cell Biology", "Biophysics"], "users"=>["Manik Goel", "Adam E. Sienkiewicz", "Renata Picciani", "Jianhua Wang", "Richard K. Lee", "Sanjoy K. Bhattacharya"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0034309.g002", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Down_regulation_and_or_disruption_of_cochlin_expression_in_mice_TM_is_commensurate_with_reduction_in_mean_IOP_/327959", "title"=>"Down-regulation and/or disruption of cochlin expression in mice TM is commensurate with reduction in mean IOP.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-04 02:12:39"}

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