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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session B

Poster I12 - Impact of extracellular matrix protein coatings on Fibronectin synthesis and fibril assembly.

Thursday, October 24, 2024
2:45 PM – 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Dysregulation of fibronectin (FN) fibril assembly is involved in many diseases, such as fibrosis. Changes in FN fibril assembly have been shown to be influenced by changes in cell behavior and remodeling of surrounding extracellular matrix (ECM) proteins.
In this project, we investigated how FN fibril assembly changes in different cell types when seeded onto different ECM proteins. Increased FN assembly is a major marker of multiple cell processes such as epithelial-mesenchymal transition (EMT) and fibroblast activation. During EMT, epithelial cells upregulate the production and assembly of ECM proteins, such as collagen (COL) and FN. Fibroblast activation promotes the deposition and remodeling of ECM proteins, and is essential in homeostatic processes like wound healing and tissue regeneration.
The ECM provides cells with structural and biochemical support. ECM composition changes depending on the type of cell. For example, epithelial cells sit on the basement membrane, which includes Collagen Type IV and laminin, while fibroblasts sit in the interstitial matrix, composed of proteins including Collagen Type I (COL I) and FN. We hypothesize that changes in ECM protein coatings will affect FN fibril formation, where we expect to see increased FN fibril formation in response to environments altered from the native ECM. Improving our understanding of FN fibril assembly in response to other ECM proteins across differing cell types is essential to improve our understanding of the mechanisms involved in FN fibril assembly, and its role in driving fibrotic diseases.

Materials and

Methods:  To investigate the effects that different ECM proteins have on FN fibril assembly, we used 2 different cell types: MDCKIIs (canine kidney epithelial cells) and NRK-49Fs (rat kidney fibroblasts). Coverslips were coated with laminin or COL I at concentrations of 250 and 1000 µg/mL. To induce FN fibril assembly, cells were treated with 4 ng/mL of transforming growth factor- beta 1 (TGF-β1) for 24 hours. Cells were fixed and immunofluorescently labeled for actin, cell nuclei, FN, and integrin 𝛼V. Cells were imaged using a 63x objective on a Zeiss AxioObserver. Changes in cell morphology and FN fibril assembly were quantified using a MATLAB image processing code developed by the Lemmon lab. Changes in EMT gene expression were quantified using qRT-PCR.

Results, Conclusions, and Discussions: Immunofluorescence images suggested that fibroblasts seeded on both COL I concentrations produced longer and larger FN fibrils compared to the laminin conditions. Fibroblasts seeded on laminin had shorter FN fibrils that were also found outside of cell boundaries, compared to the longer and fewer fibrils on COL I. The addition of TGF-ꞵ1 showed no significant changes to FN fibril formation in fibroblasts.
Results from epithelial cell studies suggest that increasing COL I concentration induces EMT, indicated by increased formation of actin stress fibers, breakdown of E-Cadherin junctions, and FN fibril assembly when epithelial cells were seeded on 1000 ug/mL COL I concentrations. PCR showed that in MDCKIIs treated with TGFꞵ-1, there was an increase in EMT markers in response to 250 µg/mL COL I and laminin.
Our results suggest that fibroblasts seeded on COL I promote fibronectin fibril assembly into longer, thicker fibrils with limited assembly around cells when compared to laminin conditions. In epithelial cells, we showed that collagen alone begins to induce EMT and increased FN fibril assembly, seen by increased cell size, actin stress fiber formation, and upregulation of EMT gene markers. Future studies will continue to investigate changes in FN fibril assembly in response to different ECM proteins in different cell types.
Understanding how different ECM proteins affect FN fibril assembly in different cell types will help to better understand the progression of diseases like fibrosis and will give insight into novel therapeutic targets.

Acknowledgements (Optional):