Struktur-Funktions-Korrelationen in PEG-DA- und Hyaluronsaeure-Hydrogelen

Die Erforschung von Struktur-Funktions-Korrelationen in biologischen Geweben ist seit vielen Jahren ein grosses Forschungsgebiet und dient nicht nur der grundlagenorientierten Wissenschaft, sondern auch der Entwicklung von Konzepten zur Herstellung neuartiger Biomaterialien. In dieser Arbeit wurden neuartige bio-inspirierte Vernetzer, die durch das Quervernetzermolekuel im Elastin, das sogenannte Desmosin, inspiriert sind, mit langkettigen thiolierten Hyaluronsaeuremolekuelen zu dreidimensionalen Hydrogelen vernetzt und charakterisiert. Durch Variation des Vernetzungsgrads und auch der Ladungsdichte des Polymernetzwerks wurden mithilfe von mechanischen Analysen Struktur-Funktions-Korrelationen herausgearbeitet. Dabei wurde herausgefunden, dass eine positive Ladung auf dem Vernetzermolekuel, wie sie auch das Desmosin-Molekuel besitzt, die mechanischen Eigenschaften und Schwellraten der Gele signifikant und charakteristisch beeinflusst. Zur Strukturanalyse der Gele wurden theoretische Netzwerkmodelle herangezogen. Das Spannungs-Deformations-Verhalten bei starker Deformation wurde charakterisiert und bewertet. Durch die Analyse der Transportprozesse groeßendefinierter Testmolekuele in den Gelen wurden weitere Strukturuntersuchungen durchgefuehrt, das Diffusionsverhalten charakterisiert und dieses mit Diffusionsmodellen analysiert. In einem vollsynthetischen Modellsystem aus PEG-DA-Hydrogelen wurden weitere Diffusions- und Permeabilitaetsstudien durchgefuehrt und mithilfe von Modellanalysen bewertet. Dabei hat sich gezeigt, dass Wechselwirkungen zwischen den diffundierenden Molekuelen (FITC-Dextranen) und dem PEG-DA-Polymernetzwerk eine grosse Rolle spielen. Diese konnten durch ein empirisches Diffusionsmodell mit einem Wechselwirkungsterm bei unterschiedliche Schwellraten und auch bei unterschiedlichen Temperaturen praezise beschrieben werden

Mapping the mechanics and macromolecular organization of hyaluronan-rich cell coats

The hyaluronan (HA)-rich pericellular coat (PCC) enveloping most mammalian cells plays a vital role in biological processes such as cell adhesion, proliferation, motility and embryogenesis. In particular its presence on chondrocytes, which live in the load-bearing cartilage, has a wide range of implications in diseases such as osteoarthritis, highlighting its mechanical role in living organisms. Despite its significance, the macromolecular organization of the cell coat remains speculative. In order to obtain a more detailed spatial picture of highly hydrated PCCs, we present two independent but complementary non-invasive techniques for the position-resolved analysis of the cell coat's mechanical and structural properties. Position-dependent microrheology provides a micromechanical map of the PCC that reveals a gradient of increasing elastic stiffness towards the plasma membrane on model rat chondrocyte cells (RCJ-P). This gradient can be correlated with the relative distribution of HA, which is inferred using an eGFP-labelled neurocan-binding domain, a small fluorescent molecule that binds to HA. The spatial variation of the HA concentration profile is consistent with the position-dependent elasticity. Combining these approaches sheds light on the molecular architecture of the PCC

Inhibition of PI3K p110α impairs GBM cell proliferation and anchorage-independent growth.

<p>(A) Cell proliferation of GBM cells in the presence of class I_A PI3K isoform-specific inhibitors (72 h). (B) Cell proliferation of GBM cells transiently transfected with siRNA targeting class I_A PI3K isoforms p110α (PIK3CA), p110β (PIK3CB), or p110δ (PIK3CD) 48 h post transfection. TOX and SCR siRNAs were used as positive and negative, non-targeting controls, respectively. (C) Anchorage-independent growth (colony formation in soft agar) of GBM cells treated with PI3K p110α-specific inhibitors YM024, A66, PIK75, or PI3K p110β-specific inhibitor TGX221 (28 d). (D) Cell proliferation and anchorage-independent growth of GBM cells in the presence of the dual PI3K/mTOR inhibitor BEZ235 (72 h and 28 d, respectively). Curves and bars represent the means of three individual experiments ± standard deviation; single experiment for soft agar assay with BEZ235; *: p≤0.05, **: p≤0.01, ***: p≤0.001 compared to 0.0 μM inhibitor or SCR non-targeting siRNA control as determined by two-sided, one-sample Student’s <i>t</i>-tests.</p

Expression analysis of proteins in the PI3K/Akt/mTOR signaling pathway in glioma and GBM samples.

<p>(A) IHC staining of glioma tissue microarray demonstrating increased expression of all antigens shown in GBM (WHO grade IV) compared to pilocytic astrocytoma (WHO grade I). Bars represent 100 μm. (B) Multivariable Cox-regression of IHC factors in 74 glioma patients by individually adding the immunostaining status to the clinical factors age, gender, and WHO grade. Results for p-S6 (S235/236) and S6 are shown in the upper and lower panel, respectively. HR: hazard ratio, LCL/UCL: lower/upper boundary of 95% confidence interval for HR. (C) Western blot analysis of protein levels of EGFR, PTEN, class I_A PI3K isoforms, and downstream signaling proteins Akt and S6 in human GBM cell lines and <i>ex vivo</i> cultures. Normal human brain and cerebellum tissue as well as non-transformed type II human pneumocytes were used as controls.</p

PI3K p110α-specific inhibition of T98G tumors formed on the chick chorioallantoic membrane impairs tumor growth.

<p>(A) Representative pictures of T98G tumors formed on the CAM three days post cell application (10 × magnification). Tumors were treated with PIK75 as indicated for four consecutive days. (B) Quantification of changes in tumor volume before and after PIK75 or control treatment. Lines indicate the mean of each group. *: p = 0.02 compared to control treatment as determined by two-sided, two-sample Student’s <i>t</i>-test.</p

Class I_A PI3K isoforms p110α and p110β play a role in GBM cell migration.

<p>(A) Analysis of the migratory potential of GBM cells by means of wound healing assays in the presence of PI3K p110α or PI3K p110β-specific inhibitors (18 h). Bars represent the means of three individual experiments ± standard deviation; *: p≤0.05, **: p≤0.01, ***: p≤0.001 compared to 0.0 μM inhibitor as determined by two-sided, one-sample Student’s <i>t</i>-tests. (B) Migration speed of T98G cells in a random migration experiment following downregulation of class I_A PI3K isoforms by siRNA. *: p≤0.05 compared to SCR non-targeting siRNA as determined by two-sided, one-sample Student’s <i>t</i>-test. (C) Distance of migration in a random migration experiment of T98G cells transfected with non-targeting control (SCR) or PI3K p110β-targeting siRNA (PIK3CB). (D) Migration speed of T98G cells in a random migration experiment following downregulation of PI3K/Akt signaling pathway molecules by siRNA. *: p≤0.05, **: p≤0.01 compared to SCR non-targeting siRNA as determined by two-sided, two-sample Student’s <i>t</i>-tests.</p

Pharmacological inhibition of PI3K p110α and PI3K p110β impairs downstream signaling.

<p>(A) Western blot analysis of basal Akt/mTOR signaling activation by detection of phosphorylated downstream proteins Akt and S6 in GBM cells following treatment with increasing concentrations of PI3K p110α-specific inhibitors YM024, PIK75, or A66 (6 h). (B) Western blot analysis of phosphorylated downstream proteins Akt and S6 protein of GBM cells following treatment with increasing concentrations of the PI3K p110β-specific inhibitor TGX221 (6 h). (C) Growth factor-induced PI3K/Akt signaling activation after pretreatment of T98G and EV7 cells with PI3K p110α-specific inhibitor YM024. (D) Growth factor-induced PI3K/Akt signaling activation after pretreatment of T98G cells with PI3K p110β-specific inhibitor TGX221.</p

Inhibition of the PI3K isoform p110α with isoform-specific inhibitor PIK75 induces apoptosis.

<p>(A) Western blot analysis of apoptosis markers of GBM cells following treatment with increasing concentrations of PI3K p110α-specific inhibitors YM024 or PIK75 (6 h). (B) Flow cytometry analysis of T98G cells after treatment with PI3K p110α-specific inhibitor PIK75 (8 h) stained with Annexin-V and PI. (C) Quantification of Annexin-V-positive/PI-negative T98G cells after treatment with PI3K p110α-specific inhibitors YM024, A66, PIK75, or PI3K p110β-specific inhibitor TGX221 (8 h). Bars represent the means of three or two individual experiments ± standard deviation for PI3K p110α- or PI3K p110β-specific inhibitors, respectively; ***: p≤0.001 compared to 0.0 μM inhibitor as determined by two-sided, one-sample Student’s <i>t</i>-tests.</p