June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Matricellular Protein Cysteine-Rich Angiogenic Inducer 61 (CCN1/CYR61) Attenuates Fibrogenic Response In Trabecular Meshwork Cells
Author Affiliations & Notes
  • Ishita Gupta
    Undergraduate Studies, Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, United States
    Department of Ophthalmology, Case Western Reserve University, Cleveland, Ohio, United States
  • Padmanabhan P Pattabiraman
    Department of Ophthalmology, Case Western Reserve University, Cleveland, Ohio, United States
  • Footnotes
    Commercial Relationships   Ishita Gupta, None; Padmanabhan Pattabiraman, None
  • Footnotes
    Support  Eversight Eye and Vision Research Awarded to PPP and RPB Departmental Grant
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3492. doi:
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      Ishita Gupta, Padmanabhan P Pattabiraman; Matricellular Protein Cysteine-Rich Angiogenic Inducer 61 (CCN1/CYR61) Attenuates Fibrogenic Response In Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3492.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Well-documented corroborations link actin cytoskeleton, extracellular matrix (ECM) proteins in trabecular outflow pathway and intraocular pressure (IOP). This study investigates the role of CCN1, a secreted matricellular protein, that binds to and signal via integrins, in the regulation of trabecular meshwork (TM) contractile properties and aqueous humor outflow homeostasis.

Methods : Using immunofluorescence and immunoblotting analyses, we assessed - a) the cellular distribution, basal expression and secretion of CCN1 in primary human TM (HTM) cells and in culture media, b) the regulation of CCN1 by lysophosphatidic acid (LPA), dexamethasone, transforming growth factor-β2 (TGFβ2), and vascular endothelial growth factor (VEGF), c) the effects of - recombinant human CCN1 (rhCCN1)-induced- and lentiviral shRNA mediated knockdown of CCN1 using shCCN1- on changes in actin cytoskeletal organization and fibrogenic markers including α-smooth muscle actin (α-SMA) and collagen1A. All experiments described had sample sizes greater than three. Results before and after treatments were compared by paired Student's t-test. Results were significant if p<0.05.

Results : Immunoblotting analysis confirmed CCN1 (40kDa) expression in HTM cells and its secretion in conditioned media. Cellular distribution of CCN1 assessed by immunofluorescence showed predominantly cytoplasmic localization. Serum-starved HTM cells when treated with TGFβ2 (10ng/ml) and VEGF (20ng/ml) significantly increased intracellular CCN1 expression (≥1.5 folds) within 5h whereas LPA (10μM) for 24h and dexamethasone (500nM) for 5 days did not. Further, treatment with rhCCN1 (20ng/ml) for 5h led to decreased actin stress fibers and expression of the fibrotic markers - α-SMA and collagen1A, as visualized by immunofluorescence; and a 2.6 fold decrease in α-SMA compared to 0.1% BSA treated control HTM cells as detected by immunoblotting. Additionally, TGFβ2-mediated induction of α-SMA was significantly decreased (1.6 fold) by rhCCN1 treatment. On the other hand, knockdown of CCN1 using shCCN1 led to a significant increase in α-SMA levels (1.7 fold).

Conclusions : Taken together, it is evident that CCN1 decreases TM cytoskeletal integrity and plays an important role in regulating the fibrogenic responses in TM. This suggests that CCN1 can decrease outflow resistance making it a potential therapeutic target.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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