Browsing by Subject "rho-Associated Kinases"
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Item Differentiation of Normal and Cystic Fibrosis Human Lung Epithelial Cells in a Decellularized and Reconstituted Mouse Lung(2017-04-14) LaRanger, Ryan; Hsieh, Jenny; Garcia, Christine K.; Pasare, Chandrashekhar; Lu, Christopher Y.; Shay, Jerry W.; Wright, Woodring E.Engineered lung tissue may eventually address the chronic shortage of transplantable lung tissue and permit modeling of lung disease in a controlled ex vivo environment. However, there are presently no sources of primary lung stem cells which can be expanded at sufficient scale to permit engineering multiple lungs from a single donor. I have developed a method for conditionally reprogramming primary human bronchial epithelial cells in culture to extend their functional lifespan, and have used these cells to reconstitute lung epithelium in a decellularized lung matrix. For conditional reprogramming, I cultured primary human bronchial epithelial cells derived from patients with or without cystic fibrosis with a small molecule Rho-associated coiled kinase inhibitor and co-cultured it with irradiated J2 3T3 fibroblasts. I determined the ability of the human bronchial epithelial cells to differentiate after 40 population doublings by culture at an air liquid interface for 35 days as confirmed by transepithelial electrical resistance measurement, histology, Ussing chamber analysis, and immunofluorescence staining of differentiation factors. I also found that this conditional reprogramming method permits cloning of human bronchial epithelial cells, and that these cells can support genetic modification by CRISPR. Next, I developed a method for decellularizing and reconstituting murine lungs in a bioreactor with vascular perfusion and simulated breathing. Lungs reconstituted with the conditionally reprogrammed human bronchial epithelial cells formed both upper and lower airway structures after only 12 days of culture. I confirmed the formation of a bronchial pseudostratified epithelium and alveolar formation in the reconstituted lungs by histology, western blotting, and immunofluorescence staining. To develop an eventual universal donor paradigm for engineered tissue, I developed an in vivo luciferase rejection screen in mice using luciferase expressing life-extended primary skin fibroblasts transplanted intradermally. The methods developed for long-term culture of primary lung epithelial cells permits rapid scale-up of patient derived human bronchial epithelial cells and clonal selection without the need for genetic manipulation; facilitating the study of lung diseases and optimization of organ reconstitution in tissue engineered models.Item Investigation of Cell Morphology and Cell-Induced 3-D Matrix Reorganization(2008-05-13) Kim, Areum; Petroll, W. MatthewThe overall goal is to develop and apply quantification techniques for assessing the underlying pattern of cytoskeletal organization and cell-matrix mechanical interactions in corneal fibroblasts at the sub-cellular level. To specifically study how Rho and Rac regulate sub-cellular mechanical behavior, cells were plated inside 3-D matrices and incubated with activators and/or inhibitors of Rho and Rac and 3-D optical section images were collected simultaneously. Cell morphology, collagen density and orientation were quantitatively studied. The first important finding is that Rho kinase-dependent contractile force generation leads to co-alignment of cells. This process contributes to global matrix contraction and thus may play a central role in cell transformation and force generation during wound healing. In contrast, activation of Rac using PDGF induces dramatic cell elongation without significant matrix reorganization. PDGF may play a role in cell migration during wound healing process since migration requires protrusion of cell extensions, but not necessarily large contractile force. In order to obtain more detailed understanding of how cells reorganize matrices over time, 4-D imaging techniques were used. Cells were plated inside 3-D matrices and time-lapse DIC and LSCM imaging was performed while disrupting cytoskeletal proteins in the presence or absence of the Rho kinase inhibitor. Addition of nocodazole induced rapid microtubule disruption which resulted in Rho activation and cellular contraction. The matrix was pulled inward by retracting pseudopodial processes, and focal adhesions appeared to mediate this process. When Rho-kinase was inhibited, disruption of microtubules resulted in retraction of dendritic cell processes, and rapid formation and extension of lamellipodial processes at random locations along the cell body, eventually leading to a convoluted, disorganized cell shape. These data suggest that microtubules modulate both cellular contractility and local collagen matrix reorganization via regulation of Rho/Rho kinase activity. In addition, microtubules appear to play a central role in dynamic regulation of cell spreading mechanics, morphology and polarity in 3-D culture. Taken together, these experiments demonstrate that quantitative static and dynamic imaging of cells in 3-D matrices is capable of providing unique insights into the role of specific signaling pathways on the underlying pattern of cytoskeletal organization and cell-matrix mechanical interactions.Item Proteomic Discovery of Functionally Important Pathways in Myocardial Ischemia-Reperfusion Injury(2016-04-01) Ahmed, Kamran; Hill, Joseph A.; Rosenzweig, Anthony; Munshi, Nikhil; Sadek, Hesham A.BACKGROUND: Coronary heart disease, a source of myocardial ischemia-reperfusion injury (IRI), is the world's leading cause of death and disability. Insulin-like growth factor 1 (IGF1) transgenic (Tg) mouse hearts are protected from IRI, whereas Akt-Tg mouse hearts recover poorly from IRI. Surprisingly, Akt is a downstream component of IGF1 signaling. The Akt-Tg phenotype can be rescued by cardiac gene transfer of activated PI3-kinase (PI3K), another component of the IGF1 pathway, suggesting that PI3K-dependent but Akt-independent pathways are key determinants of IRI. To discern such pathways, we analyzed the proteomic and phosphoproteomic changes in wild-type (WT) mouse hearts subjected to IRI ex vivo, and IGF1-TG and Akt-Tg mouse hearts in order to identify 20 differentially regulated candidates as potential modifiers of IRI, and began testing their functional roles in an in vitro model. OBJECTIVE: We hypothesize that the cardioprotection observed in IGF1 overexpression is a result of PI3K-dependent but Akt-independent signaling pathways. METHODS: WT hearts were collected at 4 time points of ex-vivo Langendorff IRI and analyzed with liquid chromatography-tandem mass spectrometry to determine protein abundance and phosphorylation changes. IGF1-Tg and Akt-Tg hearts were analyzed at baseline. Protein network analysis was performed using Cytoscape software. The functional effects of candidates with abundance or phosphorylation differences ≥2-fold were assessed in rat neonatal ventricular myocytes using in vitro redox-based viability assays and cell proliferation studies. RESULTS: In the WT IRI studies, 6403 proteins and 22833 phosphopeptides were quantified. During IRI, no proteins changed in abundance, 10 phosphopeptides were upregulated, and 330 phosphopeptides were downregulated. In the IGF1-Tg and Akt-Tg hearts, 6700 proteins and 23000 phosphopeptides were quantified. Out of the significantly regulated proteins, in vitro knockdown of rho-associated protein kinase 2 (ROCK2) increased the viability signal by 17% in normoxia and 33% in simulated IRI (p<0.05) and increased EdU incorporation from 28.9% to 40.15% (p<0.00001). Network analysis of Akt-Tg hearts revealed significant downregulation of 24 out of 45 subunits of Complex I of the electron transport chain (p<0.05). CONCLUSION: Dephosphorylation of the cardiac phosphoproteome is the dominant pattern in IRI, which may reflect phosphatase activation or reduced ATP levels inhibiting kinase activity. ROCK2 knockdown increased the viability signal by stimulating proliferation in vitro. Whether ROCK2 is involved in cardiomyogenesis in the adult heart will be addressed in future studies. Akt-Tg hearts may be susceptible to IRI due to a reduced ATP reserve caused by Complex I downregulation.