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PhD Thesis Defense Seminar

Sloan Auditorium, Room 101, Robert B. Goergen Hall

“Mechanosignaling from Extracellular Matrix Fibronectin Mediates Endothelial Cell Responses to Flow”

Presented by: William Okech
Supervised by: Prof. Ingrid Sarelius

The endothelium is constantly exposed to various hemodynamic forces including blood pressure and fluid shear stress which aid in regulating vascular development and physiology. Fluid shear stress acts in a tangential direction on the apical surface of endothelial cells (EC) and both in vivo and in vitro studies have demonstrated that EC are responsive to this mechanical stimulus. One approach to determine the mechanosensitivity of the endothelium is to measure changes in cell morphology and cytoskeletal realignment in the direction of blood flow. The existing paradigm holds that this realignment is signaled directly from changes in perceived wall shear stress by cellular mechanotransducers linked to the cytoskeleton. Components of the glycocalyx, cell-matrix adhesions and cell-cell junctions have all been implicated as potential candidates in the process of mechanotransduction. The studies presented in this thesis sought to establish whether non-cellular components such as ECM fibronectin contributed to the observed mechanoresponses. In response to mechanical force, type III repeats (FNIII) within fibronectin can unfold and expose cryptic binding sites. Previous studies have shown that the biological activity of ECM fibronectin is localized, in part, to a matricryptic, heparin-binding site located within its 1st type III repeat (FNIII1H). A series of engineered fibronectin matrix mimetics that mimic the effects of ECM fibronectin on cells have been developed using recombinant protein production and purification. These mimetics couple the matricryptic, heparin-binding site (FNIII1H) to variants of the cell-binding domain (FNIII8-10) and provide us with the ability to study the effects of cryptic site signaling and various fibronectin conformations on endothelial cell responses to flow. Therefore, the overall goal of this work was to determine the role of ECM fibronectin (FN) and specific matricryptic signaling elements within the 1st type III repeat in EC responses to flow.

We subjected human umbilical vein endothelial cells seeded on various fibronectin matrix mimetics to flow in perfusable microslides and measured the short-term (t ≤ 8 hrs.) shear stress-induced changes in endothelial cell morphology and stress fiber and cell alignment. In the 1st part of the study, we show that ECM fibronectin via a matricryptic, heparin-binding site within its 1st type III repeat mediates endothelial cell mechanosensitive responses when flow is used as mechanical stimulus. Specifically, the matricryptic, heparin-binding site (FNIII1H) is required for stress fiber realignment when cells are adhered via the integrin α5β1 but is not necessary when endothelial cells are adhered via the integrin αvβ3. In the 2nd part of the study, we show that endothelial cell-cell junctions are not required for FNIII-1H mediated stress fiber realignment but they are necessary when endothelial cell adhesion is via the integrin αvβ3. In subconfluence, we also demonstrate that cell, but not stress fiber alignment is independent of matricryptic site signaling. Lastly, we show that subconfluent endothelial cells utilize the matricryptic site and the integrin αvβ3 to mediate different morphological responses to flow. In summary, the studies presented in this thesis identify a pivotal role for fibronectin conformation and cryptic site signaling in mediating endothelial cell mechanosensitive responses to mechanical stimuli.