Fibrosis, or scarring, is an evolutionarily developed method to rapidly close and repair a wound. Excessive deposition of extracellular matrix and the generation of contractile fibroblasts (myofibroblasts) characterize fibrosis. In the cornea, severe wounds that damage the basement membrane heal using fibrosis. However, in contrast to other tissues where function is maintained, fibrosis in the cornea leaves an opaque cornea that can impair vision. In the past funding period, we made the exciting observation that expression of integrin ?v?6 in the epithelium after wounding appears to play a major role in directing the fibrotic response in the cornea. Integrin ?v?6 binds to ECM, but more importantly also has the ability to activate TGF-?1 (T1) and -?3 (T3). These isoforms of TGF-? have been linked to stimulating (T1) and blunting (T3) fibrosis. Interestingly, our preliminary data indicate that a single application of T3 after wounding almost completely blunts the scarring response. The hypothesis we propose to test is: (1) Corneal epithelium stimulates HCF to undergo fibrosis in the absence of a basement membrane;(2) Integrin ?v?6 upregulation in the epithelium is a key component in the stimulation of a fibrotic response in the stromal cells and (3) Activation of TGF-?3 blunts this fibrotic response. We propose three specific aims to address the following questions. First, does corneal epithelium stimulate a fibrotic response by the stromal fibroblasts in the absence of a basement membrane? Second, is ?v?6 expression required for the stimulation of a fibrotic response? Third, does T3 inhibit fibrosis in corneal wounds? In vivo wound healing models in mice will be used to examine these questions. However, the majority of experiments will make use of an innovative co-culture model that combines human corneal epithelial cells with human corneal fibroblasts that have been stimulated to secrete a stroma-like matrix. Specific components of fibrosis will be examined along with signaling components of fibrosis. Immunofluorescence microscopy, real-time PCR, and TEM will be the major analysis tools. In addition specific inhibitors of ?v?6 expression and function will be used. Scarring processes to be examined include: myofibroblast generation, basement membrane reformation and ECM deposition. Relevance to Public Health - Over 10 million people world-wide suffers from blinding due to corneal scarring. Currently, no therapies are available except for corneal transplantation. Our studies will also evaluate if manipulating ?v?6 or if treating wounds with T3 can reduce corneal scarring in a human model.

Public Health Relevance

Fibrosis, or scarring, is an evolutionarily developed method to rapidly close and repair a wound;however, in corneas, fibrosis leaves the cornea opaque and thus, non-functional. TGF-?1 (T1) has been linked to stimulating fibrosis and TGF-?3 (T3) to blunting it. Integrin ?v?6, which is known to activate both T1 and T3, is upregulated in the migrating epithelium that covers a corneal wound. Therefore, we propose to use a co-culture system consisting of human corneal epithelial cells and fibroblasts, as well as ?v?6-/- mice, to determine if epithelium directs fibrosis in the fibroblasts, if integrin ?v?6 is required to stimulte fibrosis, and if introducing T3 immediately after wounding will blunt the scarring response.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY005665-28
Application #
8371401
Study Section
Special Emphasis Panel (BVS)
Program Officer
Mckie, George Ann
Project Start
1984-12-01
Project End
2013-12-31
Budget Start
2012-09-01
Budget End
2013-12-31
Support Year
28
Fiscal Year
2012
Total Cost
$485,000
Indirect Cost
$235,000
Name
Schepens Eye Research Institute
Department
Type
DUNS #
073826000
City
Boston
State
MA
Country
United States
Zip Code
02114
Karamichos, D; Hutcheon, A E K; Rich, C B et al. (2014) In vitro model suggests oxidative stress involved in keratoconus disease. Sci Rep 4:4608
Stepp, Mary Ann; Zieske, James D; Trinkaus-Randall, Vickery et al. (2014) Wounding the cornea to learn how it heals. Exp Eye Res 121:178-93
Karamichos, Dimitrios; Funderburgh, Martha L; Hutcheon, Audrey E K et al. (2014) A role for topographic cues in the organization of collagenous matrix by corneal fibroblasts and stem cells. PLoS One 9:e86260
Karamichos, D; Hutcheon, A E K; Zieske, J D (2014) Reversal of fibrosis by TGF-?3 in a 3D in vitro model. Exp Eye Res 124:31-6
Karamichos, Dimitrios; Rich, Celeste B; Zareian, Ramin et al. (2013) TGF-*3 stimulates stromal matrix assembly by human corneal keratocyte-like cells. Invest Ophthalmol Vis Sci 54:6612-9
Blanco-Mezquita, Jose Tomas; Hutcheon, Audrey E K; Zieske, James D (2013) Role of thrombospondin-1 in repair of penetrating corneal wounds. Invest Ophthalmol Vis Sci 54:6262-8
Matsuba, Mariko; Hutcheon, Audrey E K; Zieske, James D (2011) Localization of thrombospondin-1 and myofibroblasts during corneal wound repair. Exp Eye Res 93:534-40
Blanco-Mezquita, Jose Tomas; Hutcheon, Audrey E K; Stepp, Mary Ann et al. (2011) *V*6 integrin promotes corneal wound healing. Invest Ophthalmol Vis Sci 52:8505-13
Pal-Ghosh, Sonali; Blanco, Tomas; Tadvalkar, Gauri et al. (2011) MMP9 cleavage of the *4 integrin ectodomain leads to recurrent epithelial erosions in mice. J Cell Sci 124:2666-75
Ren, R; Hutcheon, A E K; Guo, X Q et al. (2008) Human primary corneal fibroblasts synthesize and deposit proteoglycans in long-term 3-D cultures. Dev Dyn 237:2705-15

Showing the most recent 10 out of 36 publications