Proteinase activated receptors (PARs) comprise a novel family of cell membrane proteins that are potent mediators of tissue inflammation, pain, and repair. PARs are activated by serine proteases such as thrombin, trypsin, and tryptase that are released in injured or inflamed tissues. We have recently reported that the cornea expresses two of the principal proteinase activated receptors: PAR-1 and PAR-2. The long-term goal of this project is to determine the role of serine proteases and PARs to mediate a variety of corneal epithelial cell wound healing and inflammatory responses. This application will test the hypothesis that serine proteases such as thrombin and tryptase are potent modulators of corneal epithelial cell inflammatory and wound healing responses that are mediated by the activation of specific corneal epithelial cell PARs. In this proposal, we will undertake the following specific Aims:
SPECIFIC AIM #1 : To examine the effect of seine proteases on human corneal epithelial cell biological activities critical for corneal inflammatory and wound healing responses;
SPECIFIC AIM #2 : To determine if corneal epithelial cell responses to serine proteases are mediated by the activation of specific functional protease activated receptors (PARs);
SPECIFIC AIM #3 : To determine the role of serine protease activation of PARs in mediating in vivo corneal inflammatory responses. To conduct the proposed studies, human corneal epithelial cells and human corneal tissue will be utilized for in vitro studies and the proposed in vivo studies will utilize both normal mice as well as PAR-1 and PAR-2 knockout mice. The ability of serine proteases to influence human corneal epithelial cell proliferation, differentiation, migration, MMP/TIMP expression, and proinflammatory protein production will be determined in vitro. The specific role of PAR-1 and PAR-2 in these serine protease induced human corneal epithelial cell responses will be assessed using serine proteases and I specific native peptide derived PAR-1 and PAR-2 agonists and antagonists. Additionally, preliminary """"""""proof of principle"""""""" in vivo studies will be undertaken to determine the capacity of serine proteases and specific PAR-1 and PAR-2 agonists to induce corneal inflammatory responses in wild type mice in comparison to PAR-I(-/-) and I PAR-2 (-/-) animals. Proteases and PARs may be important mediators of corneal inflammation and wound healing and therefore could prove to be novel therapeutic targets for a wide range of ocular diseases.
