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Congratulations on a Successful PhD Defense by Vicky

Dissertation Title: Provisional extracellular matrix citrulination as a stimulus for pathological fibroblast activation.

During Vicky's PhD she was successfully awarded BOTH the NSF GRFP and an NIH F31; a truly amazing feat considering the level of competition for each of these prestigious awards.

Provisional extracellular Matrix Citrullination as a stimulus for pathological fibroblast activation

Activated fibroblasts have been implicated as drivers of disease progression in a variety of conditions including rheumatoid arthritis (RA), fibrotic diseases, and cancer. Cell characteristics such as invasiveness, hyperproliferation, apoptotic resistance, excessive contraction, extracellular matrix (ECM) secretion and remodeling, and enhanced inflammatory cytokine secretion are some of the main factors underlying disease exacerbating capabilities in these cells. Attempts to prevent, cure, or otherwise modulate the pathophysiology of these diseases to date has in part been hindered by a lack of understanding as to how fibroblasts become activated and are able to maintain their activated states, in some cases, even in the absence of immune or inflammatory stimuli.

The central hypothesis of this work is that an inflammation-mediated provisional extracellular matrix (pECM) modification called citrullination may be responsible in whole or in part for activated fibroblast phenotypes. Citrullination is a post-translational protein modification that is known to occur extensively and at very early stages within inflamed RA, fibrotic, and cancer tissues. To explore whether co-localization of activated fibroblasts with citrullinated pECM is merely a phenomenon or is sufficient to drive activated phenotypes, this research aimed to understand how citrullination of pECM alters cellular interactions and signaling at the molecular scale as well as to explore whether exposure to citrullinated pECM is a sufficient stimulus to bring about changes in a variety of fibroblast behaviors.

To investigate the influence of citrullinated pECM at a molecular level, mass spectrometry (MS) analysis was performed on citrullinated fibronectin (Cit Fn); integrin interactions were studied via bio-layer interferometry (BLI), co-immunoprecipitation (Co-IP), and immunocytochemistry (ICC) assays; and finally, downstream signaling and protein products were explored via both Co-IP and ICC assays. MS identified 24 unique citrullination sites, five of which reside in the cell-binding domain of fibronectin (Fn), and result in a preference for α5β1 integrins over αvβ3 integrins on Cit Fn compared to Fn. The consequence of this α5β1 to αvβ3 integrin switch is initiation of mechanotransduction signaling including elevated levels of phospho-focal adhesion kinase (pFAK), phospho-Src (pSRC), phospho-integrin-linked kinase (pILK), vinculin, F-actin, and glycogen synthase kinase (GSK), all of which are integral components of focal adhesions and signaling networks critical in cytoskeletal remodeling and cell motility.

To investigate the ability of Cit-pECM to modulate fibroblast behavior, adhesion, proliferation, metabolic, apoptotic, gel contraction, and migration assays were performed in addition to atomic force microscopy (AFM) to probe cell stiffness and α-actinin and confocal video analysis to probe focal adhesion (FA) turnover. Cit Fn was chosen as the representative pECM protein for these studies due to its prevalence within inflammatory environments as well as known dominance in fibroblast preferential binding among pECM proteins. Fibroblasts were determined to possess decreased adhesion and spreading, increased stiffness, increased FA turnover, and enhanced migration on Cit Fn compared to Fn. No differences in proliferation or metabolism were observed. Apoptotic resistance to oxidative stress was shown to be enhanced in the presence citrullinated fibrin but not on Cit Fn compared to Fn.

Altogether, the findings from these studies indicate that while citrullination of pECM cannot bring about every phenotype associated with fibroblast activation, it is sufficient to change several different fibroblast behaviors relevant to disease progression, especially that of enhanced migration which can contribute to cell invasiveness. Further, the fundamental nature of the α5β1 to αvβ3 integrin switch implies that citrullination has the potential to impact the interaction of many different cells with their ECM leading to potentially diverse phenotypes. Taken together, these results indicate that citrullination within the pECM is has significant consequences on cell function, and it may therefore constitute a viable therapeutic target in helping to prevent or ameliorate inflammation-mediated disease.