Einleitung: Die Wundheilung kornealer Endothelzellen erfolgt hauptsächlich durch Migration benachbarter Zellen. Die Endothelzellen sind in vivo ständig in Kontakt mit Kammerwasser (KW). TGF-ß2 und Ascorbin- säure (AS) sind in hoher Konzentration im KW enthalten. Der Einfluß von humanem KW, TGF-ß2 und AS auf Proliferation und Migration boviner kornealer Endothelzellen (BCEC) in Kultur wurde untersucht. Methoden: Proliferation: BCEC der 1.Passage wurden zu 1,5x104 Zellen/Well ausgesät. Mit frischem Medium und Zusatz von humanem KW (10% und 100%), TGF-ß2 (0,1; 1 und 10 ng/ml) und AS (50, 100 und 200 µg/ml) wurden die Kulturen für 72 oder 96 h inkubiert und anschließend gezählt. Migration: Konfluente Zellkulturen der 1.Passage wurden verwendet. Mit einem modifizierten Trepan (Durchmesser 5,5 mm) wurde eine zentrale, zirkuläre "Wunde" gesetzt. Die Kulturen wurden mit frischem Medium, das humanes KW, TGF-ß2 und AS in den o. g. Konzentrationen enthielt, für 72 oder 96 h inkubiert. Zur Auswertung wurden die in den Wundbereich migrierten Zellen an 5 verschiedenen Stellen, ausgehend vom Wundrand, gezählt. Ergebnisse: Die Proliferation der BCEC wurde durch humanes KW (unverdünntes KW: nahezu 100%; 10%iges KW: 30%) und Ascorbinsäure (ca. 90% bei AS 200 µg/ml) gehemmt. TGF-ß2 stimulierte die Proliferation bis zu einem 3fachen Wert der Kontrollen. Die Migration wurde durch humanes KW (unverdünntes KW: nahezu 100%; 10%iges KW: 30%) und TGF-ß2 (ca. 50% bei allen Konzentrationen) gehemmt. AS hatte einen stimulierenden Einfluß (20-50%) auf die Migration. Schlußfolgerung: Noch ist unklar, welche Substanzen im KW für den proliferationshemmenden Einfluß auf korneale Endothelzellen verantwortlich sind. Sowohl AS als auch TGF-ß2 zeigten einen gegensätzlichen Effekt auf Proliferation und Migration kultivierter BCEC. Die Bedeutung dieser Beobachtungen für die Wundheilung humaner kornealer Endothelzellen in vivo ist Gegenstand weiterer Untersuchungen. / Purpose: Corneal endothelial cells are non-proliferating, wound healing primarily occurs by migration of adjacent cells. Endothelial cells in vivo are constantly exposed to aqueous humor (AH). Elevated concentrations of TGF-ß2 and ascorbic acid (AA) are present in aqueous humor. The influence of human AH, TGF- ß2 and AA on proliferation and migration of bovine corneal endothelial cells in vitro was investigated. Methods: Proliferation assays: BCEC at first passage were seeded at 1.5x104 cells/well. Fresh medium containing human AH (10% and 100%), TGF-ß2 (0,1;1 and 10 ng/ml) and AA (50, 100 and 200 µg/ml) was added to the cells. 72 or 96 hours later the cells were counted. Migration assays: BCEC at first passage were grown to confluency. A central, circular "wound" was made with an especially designed trephine (diameter 5.5 mm). The cultures were incubated with fresh medium containing human AH, TGF-ß2 and AA in the same concentrations for 72 or 96 hours. The cells were then counted in five randomly chosen sections from the wound edge. Results: Proliferation of BCE cells was inhibited by human AH (pure AH: nearly 100%, 10% AH: 30%) and AA (about 90% at 200 µg/ml). TGF-ß2 stimulated the proliferation up to 3 fold compared to the controls. Migration was inhibited by human AH (pure AH: nearly 100%, 10% AH: 30%) and TGF-ß2 (about 50% at all concentrations). AA had a stimulatory effect (20-50%) on migration. Conclusion: Presently, it remains unknown which substances in AH are responsible for the inhibiting effect on corneal endothelial proliferation. Both TGF-ß2 and AA showed to have a differential effect on proliferation and migration of cultured BCE cells. The significance of these observations for wound healing of human corneal endothelial cells in vivo has to be investigated.

<p>Michelle N. Ingle. M.S., Purdue University, December 2019. Computational Modeling of Transforming Growth Factor-β2 Receptor Complex Assembly. Major Professor: David M. Umulis.</p> <p> </p> <p> Transforming growth factor (TGF)-β1, TGF-β2, and TGF-β3 are secreted signaling proteins that play an essential role in tissue development, immune response, and physiological homeostasis. TGF-β ligands signal through a tetrameric complex made up of two type I receptors (TβRI) and two type II receptors (TβRII). Dysregulation of TGF-β signaling has been linked to uncontrolled cell proliferation and cancer metastasis. An accurate understanding of TGF-β’s receptor complex assembly pathway may allow for pharmacological intervention and/or preservation of proper TGF-β signaling.</p> <p> Amongst the ligand types, TGF-β1 and TGF-β3 are efficient signalers, presumably by strong binding to both type I and II receptors. However, TGF-β2 has a very weak affinity for TβRII and requires an additional membrane-bound protein called betaglycan (BG) to achieve similar levels of downstream signaling. While computational modeling has been performed on the signaling pathway of the TGF-β system, to date no computational modeling has aimed to decipher BG’s role in the potentiation of TGF-β2 signal. To determine the role of BG in selectively facilitating signaling by TGF-β2, we developed computational models with different assumptions based on the levels of cooperativity between receptor subtypes and types of BG behavior (No Receptor Recruitment model, Single-stage Receptor Recruitment model, and Two-stage Receptor Recruitment model). </p> <p> With each of the receptor recruitment models we hypothesized that BG uses two domains to successfully enhance TGF-β2 signaling. This model was first proposed in Villarreal et al., 2016 and is further investigated in this work using a two-step computational approach. First, a root mean square error (RMSE) calculation was performed between our computational models with no BG present and published experimental signaling data in cell lines with no BG present. Lower RMSE values indicate the simulated data is more representative of experimental signaling behavior when no BG is present. The second round of model validation was performed by adding BG into the simulations and comparing its behavior to experimentally determined and hypothesized behaviors of BG. </p> <p> In summary, the simulations indicate there may be more cooperative receptor recruitment present in the system then stated in literature. Furthermore, it appears that BG binding to TGF-β2 ligand through two domains provides an effective transfer mechanism that can be tuned to control differential signaling between TGF-β ligand subtypes. Experiments were then suggested in order to support or refute one of the models offered in this thesis. For the purpose of uncovering how BG enhances TGF-β2 signaling, the computational work performed in this thesis highlights the areas where researchers should focus their experimental efforts and provides a baseline model for further computational work in the TGF-β system.</p>