Methods to Digest and characterize Fetal ECM using MMPs
Jaiganesh, Ajith.
2020
-
Thesis (M.S.)--Tufts
University, 2020.
Submitted to the Dept. of Biomedical Engineering.
Advisor: Lauren Black.
Committee: James Van Deventer, and Madeleine Oudin.
Keyword: Biomedical engineering.
It has been previously shown certain cryptic peptides released from the ECM during remodeling can modulate neonatal cardiomyocyte proliferation. ... read moreAdditionally, we also know that the major drivers of cardiac ECM digestion are the matrix metalloproteases (MMP). We hypothesize that digesting fECM with physiologically relevant proteases may result in more relevant peptides that can influence cell behavior. The purpose of this thesis was to elucidate differences in how fECM gets processed with different proteases. Following the decellularization of fetal Sprague Dawley rat hearts, samples were digested with two different enzymes: MMP1 and pepsin. To explore the effects of the different digests on cell phenotypes, cardiac fibroblasts were treated with both pepsin and MMP digested matrix and assessed for proliferation. Results from these studies indicate there are differences in the peptides derived from fECM when it gets processed with different enzymes. Further, we found that the MMP digested matrix led to a decrease in the number of cardiac fibroblasts as compared to the pepsin digested matrix, leading to a conclusion that there likely exists certain matrikines in the MMP digested ECM that are affecting fibroblast growth. To develop a more efficient pipeline for characterizing matrikine effects on cell phenotype, we also went on to develop an in-silico method to model the potential mechanism of action of matrikines using a matrikine (F1R1) previously identified by the lab. This matrikine has been implicated in cardiomyocyte proliferation. We have modelling to try and identify a potential binding partner for this peptide. Overall, we have developed a standardized method to digest, assess, and understand matrikines generated from protease digestion of the ECM which could serve as a platform to create solubilized therapeutics for fibrosis.read less - ID:
- 3b591p95d
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