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SOMSHUVRA BHATTACHARYA, Gudiseva Chandrasekher; Assessing the Origin of Extracellular Matrix Proteins Released from the Cornea in Response to Injury. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3913.
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© ARVO (1962-2015); The Authors (2016-present)
Turnover of corneal stroma extracellular matrix (ECM) protein is very slow, and the keratocytes metabolic activity is quiescent in physiological conditions as requirement for new extracellular protein matrix generation is limited. Our studies showed that the cornea releases different ECM proteins in response to injury (ARVO meeting 2016, abstract no. 4352). The currently undertaken study is to determine the origin of these ECM proteins.
De-epithelialized (injured) and intact porcine corneas were maintained in culture (serum free DMEM/F12) at 37 oC and the cornea organ cultured medium (COCM) was collected at various times (4-24 hours). In separate experiments, corneal stroma extracellular matrix (CSEM) preparation was made by extracting de-epithelialized corneas in a buffer. The presence of multiple extracellular matrix components released in to the COCM and in the CSEM was identified by western immunoblotting. Further, the susceptibility of extracted proteins in CSEM to proteolytic cleavage by the enzymes possibly released into COCM was evaluated.
We have identified in CSEM two different forms of peptides (250 and 140kDa) for both collagen I and collagen IV. The 140 kDa form of both collagen I and collagen IV was also found to be present in COCM collected from injured and uninjured corneas. Further, a low molecular weight (60-80kDa) form of collagen I but not collagen IV was also released from the corneas after injury. Interestingly, a 250 kDa fibronectin that was present in COCM is not identified in the CSEM, instead we found a 100 kDa form. Treatment of the native ECM proteins in CSEM with COCM did not result in the generation of the polypeptides that are identified in the COCM.
Presence of mutiple types of ECM proteins in COCM from intact and de-epithelialized corneas suggests that the stroma component of the cornea is the major site from which they are released. Stromal keratocytes activation that occurs in response to injury leads to the process of healing and remodeling of stroma matrix through proteolytic degradation of damaged proteins and deposition of new ECM proteins. Proteases released from the cornea in response to injury may act on damaged ECM. It is possible that the multiple forms of ECM proteins which appeared in COCM are byproducts of stromal rebuilding process.
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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