September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Suppression of Integrin-Linked Kinase in Human Trabecular Meshwork Alters SPARC’s Effect on Extracellular Matrix and Intraocular Pressure
Author Affiliations & Notes
  • Emily Niloufar Ahadizadeh
    School of Medicine, Case Western Reserve University, Cleveland Heights, Ohio, United States
    Ophthalmology , University Hospitals Case Medical Center, Cleveland, Ohio, United States
  • Min Hyung Kang
    Ophthalmology , University Hospitals Case Medical Center, Cleveland, Ohio, United States
    School of Medicine, Case Western Reserve University, Cleveland Heights, Ohio, United States
  • Douglas J Rhee
    Ophthalmology , University Hospitals Case Medical Center, Cleveland, Ohio, United States
    School of Medicine, Case Western Reserve University, Cleveland Heights, Ohio, United States
  • Footnotes
    Commercial Relationships   Emily Ahadizadeh, None; Min Hyung Kang, None; Douglas Rhee, None
  • Footnotes
    Support  NIH R01 EY 019654-01 (DJR)
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 5626. doi:
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      Emily Niloufar Ahadizadeh, Min Hyung Kang, Douglas J Rhee; Suppression of Integrin-Linked Kinase in Human Trabecular Meshwork Alters SPARC’s Effect on Extracellular Matrix and Intraocular Pressure. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5626.

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

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Abstract

Purpose : Intraocular pressure (IOP) is a major risk-factor for primary open-angle glaucoma (POAG).[1] Secreted Protein, Acidic and Rich in Cysteine (SPARC) is a matricellular protein which has been shown to elevate intraocular pressure (IOP), and increase components of the extracellular matrix (ECM), including collagen I, IV and VI, laminin, fibronectin, and matrix metalloproteases (MMPs) in trabecular meshwork (TM).[2,3] Integrin-linked kinase (ILK) is a serine/threonine kinase that regulates ECM organization through signaling cascades and interacts with SPARC.[4] We hypothesize that SPARC regulates ECM proteins, in part, through ILK in TM.

Methods : Human TM cells were treated with an adenovirus carrying cDNA of human SPARC (Ad-SPARC) and a lentivirus carrying short-hairpin RNA targeting human ILK (Lenti-shILK). The adenovirus was constructed using the Ad5 vector system (Ad-SPARC), and the lentivirus was constructed using the pLKO.1 vector system. Ad-SPARC was used to overexpress SPARC in human TM cells at the multiplicity of infections (MOI) 50, and Lenti-shILK was used to inhibit ILK at MOI 5. ECM proteins from cell lysates and conditioned media were analyzed by immunoblotting. Human anterior chamber perfusion culture (ex vivo) was treated with Ad-SPARC and subsequently with Lenti-shILK 48 hours later, and IOP measurement was taken.

Results : We observed that SPARC was overexpressed with Ad-SPARC (6.6 ± 9.8, p=0.13) compared to Ad-control, and induced elevated collagen IV (5.7 ± 10.3, p=0.03) and fibronectin (3.14 ± 3.8, p=0.04) in TM cells. The Lenti-shILK suppressed ILK (0.9 ± 1.4, p=0.18) and attenuated the effect of SPARCs on collagen IV (0.75 ± 0.23, p=0.067) and fibronectin (1.3 ± 1.34, p=0.32), in addition to significantly suppressing the elevation of IOP in the human anterior perfusion cultures (-25.4 ± 3.95%, p=0.07, n=2 at 222hrs).

Conclusions : The effect of SPARC on collagen IV and fibronectin and the regulation of IOP are through ILK signaling.
1. Nemesure B, et.al. Ophthalmology. 2007;114:1810–1815.
2. Haddadin, RI, et al. IOVS. 2009;50(8):3771-3777.
3. Oh D-J, et al. IOVS. 2013;54:3309–3319.
4. Barker TH, et al. J Bio Chem. 2005;280:36483-36493.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

Figure: Western blot results from TM cultures treated with Ad-SPARC and Lenti-shILK.

Figure: Western blot results from TM cultures treated with Ad-SPARC and Lenti-shILK.

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