The full text of the thesis is currently restricted. / The full text will be available at the end of the embargo period: 1st Nov 2021

Background Hematopoietic stem cells located in the bone marrow interact with a specific microenvironment referred to as the stem cell niche. Data derived from ex vivo co-culture systems using mesenchymal stromal cells as a feeder cell layer suggest that cell-to-cell contact has a significant impact on the expansion, migratory potential and ‘stemness’ of hematopoietic stem cells. Here we investigated in detail the spatial relationship between hematopoietic stem cells and mesenchymal stromal cells during ex vivo expansion. Design and Methods In the co-culture system, we defined three distinct localizations of hematopoietic stem cells relative to the mesenchymal stromal cell layer: (i) those in supernatant (non-adherent cells); (ii) those adhering to the surface of mesenchymal stromal cells (phase-bright cells) and (iii) those beneath the mesenchymal stromal cells (phase-dim cells). Cell cycle, proliferation, cell division and immunophenotype of these three cell fractions were evaluated from day 1 to 7. Results Phase-bright cells contained the highest proportion of cycling progenitors during co-culture. In contrast, phase-dim cells divided much more slowly and retained a more immature phenotype compared to the other cell fractions. The phase-dim compartment was soon enriched for CD34+/CD38− cells. Migration beneath the mesenchymal stromal cell layer could be hampered by inhibiting integrin β1 or CXCR4. Conclusions Our data suggest that the mesenchymal stromal cell surface is the predominant site of proliferation of hematopoietic stem cells, whereas the compartment beneath the mesenchymal stromal cell layer seems to mimic the stem cell niche for more immature cells. The SDF-1/CXCR4 interaction and integrin-mediated cell adhesion play important roles in the distribution of hematopoietic stem cells in the co-culture system.

<p>Integrins are heterodimeric cell surface receptors, composed of an α and a β subunit, mainly binding for extracellular matrix proteins. lntegrin subunit β1 can combine with at least 12 a subunits and thus form the biggest subfamily within the integrin family. In this thesis, functional properties of the splice variant β1Β, and the effects of several mutations in the cytoplasmic tail of integrin subunit β1Α were studied. In addition, the border between the transmembrane and cytoplasmic domains of several integrin subunits was determined.</p><p>The β1Β splice variant has been reported to have a dominant negative effect on functions of β1Α integrins. In this study, it was studied if the expression of β1Β had similar negative effects on the αvβ3 integrin functions since the β3 subunit is structurally similar to β1Α. The β1Β subunit was expressed in an integrin β1-deficient cell line and it was found that the presence of β1Β does not interfere with adhesion or signaling of endogenous αvβ3</p><p>The border between the cytoplasmic domain and the C-terminal end of the transmembrane domain of integrin α and β subunits has been unclear. This question was experimentally addressed for integrin subunits β1, β2, α2 and α5. It was found that integrin subunits contain a positively charged lysine, which is embedded in the membrane in the absence of interacting proteins.</p><p>The functional importance of the lysine in integrin transmembrane domains was investigated by mutating this amino acid to leucine in β1Α. The mutation affected cell spreading and tyrosine phosphorylation of the adapter protein CAS. The activation of focal adhesion kinase and tyrosine phosphorylation of paxillin was not affected. Furthermore, the mutation of two tyrosines to phenylalanines in the β1Α cytoplasmic tail was found to reduce the capability of β1Α integrins to mediate cell spreading and migration. Activation of focal adhesion kinase in response to the later β1Α mutant was shown to be impaired as well as tyrosine phosphorylation of adapter proteins paxillin and tensin whereas overall tyrosine phosphorylation of CAS was unaffected. These data suggests the presence of focal adhesion kinase-dependent and -independent pathways for tyrosine phosphorylation of CAS after integrin β1Α-mediated adhesion. </p>

Integrins are heterodimeric cell surface receptors, composed of an α and a β subunit, mainly binding for extracellular matrix proteins. lntegrin subunit β1 can combine with at least 12 a subunits and thus form the biggest subfamily within the integrin family. In this thesis, functional properties of the splice variant β1Β, and the effects of several mutations in the cytoplasmic tail of integrin subunit β1Α were studied. In addition, the border between the transmembrane and cytoplasmic domains of several integrin subunits was determined. The β1Β splice variant has been reported to have a dominant negative effect on functions of β1Α integrins. In this study, it was studied if the expression of β1Β had similar negative effects on the αvβ3 integrin functions since the β3 subunit is structurally similar to β1Α. The β1Β subunit was expressed in an integrin β1-deficient cell line and it was found that the presence of β1Β does not interfere with adhesion or signaling of endogenous αvβ3 The border between the cytoplasmic domain and the C-terminal end of the transmembrane domain of integrin α and β subunits has been unclear. This question was experimentally addressed for integrin subunits β1, β2, α2 and α5. It was found that integrin subunits contain a positively charged lysine, which is embedded in the membrane in the absence of interacting proteins. The functional importance of the lysine in integrin transmembrane domains was investigated by mutating this amino acid to leucine in β1Α. The mutation affected cell spreading and tyrosine phosphorylation of the adapter protein CAS. The activation of focal adhesion kinase and tyrosine phosphorylation of paxillin was not affected. Furthermore, the mutation of two tyrosines to phenylalanines in the β1Α cytoplasmic tail was found to reduce the capability of β1Α integrins to mediate cell spreading and migration. Activation of focal adhesion kinase in response to the later β1Α mutant was shown to be impaired as well as tyrosine phosphorylation of adapter proteins paxillin and tensin whereas overall tyrosine phosphorylation of CAS was unaffected. These data suggests the presence of focal adhesion kinase-dependent and -independent pathways for tyrosine phosphorylation of CAS after integrin β1Α-mediated adhesion.

yes / Regeneration of a damaged or non-functioning tissue requires adhesion of cells to their extracellular matrix (ECM). Thus the investigation of the level of synthesised cell adhesion molecules (CAMs) in cell culture systems play major roles in cell and tissue engineering. Adhesion of chondrocyte to a collagen type-II rich matrix, is dependent on cell adhesion molecules (CAMs) and integrins and cells adhere to ECM through integrins.

No description available.

Die strahleninduzierte Mucositis enoralis ist eine der bedeutendsten und häufig dosislimitierenden frühen Nebenwirkungen der Strahlentherapie fortgeschrittener Kopf Hals Tumoren. Bis heute hat sich noch kein allgemein gültiges Konzept zur Therapie und Prophylaxe der Mundschleimhautentzündung durchsetzen können. Ein Ansatz zur selektiven, auf der Tumorbiologie beruhenden Beeinflussung der Strahlenempfindlichkeit von Tumoren ist die Blockade des epidermalen Wachstumsfaktor-Rezeptors (EGFR). In Kombination mit Strahlentherapie sollen so die lokale Tumorkontrolle und die Heilungschancen verbessert werden. Die Wirkung der Tyrosinkinase-Inhibitoren BIBX1382BF und Erlotinib auf histomorphologische Parameter in der Mundschleimhaut sowie auf die Expression der als Stammzellmarker diskutierten Proteine p63, Integrin β1 und CD44 wurde in der vorliegenden Arbeit im Vergleich zur alleinigen fraktionierten Bestrahlung untersucht. Für die histologischen Studien erfolgte die zweiwöchige fraktionierte Bestrahlung der Schnauzen von Mäusen des Inzuchtstammes C3H/Neu mit zehn Fraktionen zu je 3 Gy (Tag 0-4, Tag 7-11). Die Versuche gliederten sich in vier Gruppen: • I/A (54 Tiere) und II/A (40 Tiere): fraktionierte Bestrahlung, keine weitere Behandlung • I/B (51 Tiere): fraktionierte Bestrahlung, zusätzlich orale Gabe von BIBX1382BF, 50 mg/kg KG per os, von Tag 0-14 je 30 min nach der Bestrahlung • II/B (35 Tiere): fraktionierte Bestrahlung, zusätzlich orale Gabe von Erlotinib, 50 mg/kg KG per os, von Tag 0-11 je 30 min nach der Bestrahlung. Die Entnahme der Zungen erfolgte im Versuch I bei jeweils drei Tieren pro Tag von Tag 0 bis Tag 17. Im Versuch II wurden an den Tagen 0, 2, 4, 6, 8, 10, 12 und 14 jeweils die Zungen von fünf Tieren entnommen. Anschließend folgten die Fixierung der Zungen in Formalin, die Einbettung in Paraffin und die Anfertigung 3 µm dicker Gewebeschnitte. Die Zungenpräparate wurden für die histologischen Untersuchungen mit Hämatoxylin-Eosin gefärbt. Für die immunhistochemischen Färbungen wurde die ABC-Methode eingesetzt. Das Epithel der Zungenunterseite wurde lichtmikroskopisch hinsichtlich Zellzahl, Schichtdicke und Expression der potentiellen Stammzellmarker p63, Integrin β1 und CD44 ausgewertet. Aufgrund der geringen Gruppengröße (Versuch I: drei Tiere pro Datenpunkt; Versuch II: fünf Tiere pro Datenpunkt) wurde auf eine eingehende statistische Testung verzichtet. Die vorliegende Arbeit beschränkt sich auf eine beschreibende Darstellung des Verlaufs der Einzelparameter über den Gesamtzeitraum. Die Zellzahlen verringerten sich während der ersten Bestrahlungswoche auf 60-70 % der Ausgangswerte, stagnierten in der zweiten Woche und stiegen schließlich bis zum Ende der Nachbeobachtung wieder an. Zwischen den nur bestrahlten und den zusätzlich mit BIBX1382BF behandelten Tieren war kein Unterschied feststellbar. Ein gleichsinniger Verlauf war auch in Versuch II zu beobachten, wobei die Zellzahlen der mit Erlotinib behandelten Tiere in der Funktionsschicht durchgängig höher ausfielen als in Versuchsreihe A. Die Dicke des Gesamtepithels bzw. der einzelnen Epithelschichten zeigte im Versuch I unter Bestrahlung große individuelle Schwankungen. Unter zusätzlicher BIBX1382BF-Gabe wurden oft niedrigere Werte gemessen. Im Versuch II blieb die Dicke des Gesamtepithels unter Fraktionierung konstant. Von Tag 0-12 wurden bei zusätzlicher Erlotinib-Applikation geringere Werte der Gesamtdicke gemessen als unter alleiniger Bestrahlung, ansonsten fielen die Veränderungen der Epitheldicke unabhängig von der Erlotinib-Gabe gering aus. Der kurzzeitigen, mit dem allgemeinen Zellverlust einhergehenden Verringerung der p63-Expression zu Beginn der Bestrahlung folgt bis zum Ende des Beobachtungszeitraumes die Normalisierung der p63-positiven Zellen. Mit EGFR-Blockade sind gegenüber der alleinigen Bestrahlung keine Unterschiede in der p63-Expression festzustellen. Die Integrin β1-Expression nahm im Verlauf der Bestrahlung ab. Unter EGFR-Blockade mit BIBX1382BF zeigte sich an den Tagen 2-9 und 12-16 ein schwächeres Färbesignal als im fraktioniert bestrahlten Epithel, was für eine mögliche Interaktion des EGF-Rezeptors mit Integrin β1 spricht. Im Versuch II waren unabhängig von der Erlotinib-Gabe keine Unterschiede in der Expression von Integrin β1 feststellbar. Die CD44-Expression im Epithel wurde durch Bestrahlung gefördert. Übereinstimmend konnte in der vorliegenden Arbeit in beiden Versuchen eine Steigerung der CD44-Färbeintensität über den jeweiligen Referenzbereich festgestellt werden. Eine Blockade der EGFR-Aktivität durch Erlotinib reduzierte die Expression von CD44, wie in Versuch II/B im initialen Abfall der CD44-Färbeintensität deutlich wurde. Doch schon ab Tag 4 wurden im Versuch II/A und II/B gleich starke Färbesignale für CD44 erfasst. Insgesamt ergaben sich unter EGFR-Inhibition mittels BIBX1382BF oder Erlotinib keine Hinweise auf Veränderungen der untersuchten Parameter während einer zweiwöchigen fraktionierten Bestrahlung. Ob diese Ergebnisse auch auf andere Tyrosinkinase-Inhibitoren bzw. unterschiedliche Wirkstoffklassen (z. B. Anti-EGFR-Antikörper) übertragbar sind, muss in weiteren Studien untersucht werden. / Radiation-induced oral mucositis is one of the most important and often dose limiting early side effects of radiotherapy of advanced tumours in the head-and-neck region. To this day, no general concept for therapy and prophylaxis of the oral mucositis has been established. The inhibition of the epidermal growth factor receptor (EGFR) is one approach to a selective increase of the radiosensitivity of tumours based on the tumour biology. In combination with radiotherapy, application of EGFR-inhibitors is supposed to increase the local tumour control and the chances of cure. The aim of the present study was to investigate the effect of the tyrosine kinase inhibitors BIBX1382BF and Erlotinib on the radiation response of the oral mucosa and on the expression of different proteins that are discussed to be markers of epithelial stem cells. For the histological studies, the snouts of C3H/Neu mice were irradiated with ten daily fractions of 3 Gy over two weeks (on days 0-4, 7-11). The experiments comprised four treatment groups: • I/A (54 animals) and II/A (40 animals): fractionated irradiation, no further treatment • I/B (51 animals): fractionated irradiation, administration of BIBX1382BF, 50 mg/kg per os, once daily (days 0-14) 30 min after the radiation treatment • II/B (35 animals): fractionated irradiation, administration of Erlotinib, 50 mg/kg per os, once daily (days 0-11) 30 min after the radiation treatment. Between day 0 and 17, three animals of the groups I/A and I/B were euthanised per day. In the experimental arms II/A and II/B five mice were killed on day 0, 2, 4, 6, 8, 10, 12 and 14, respectively. The tongues were excised, fixed in formalin, embedded in paraffin and 3 µm thick sections were prepared. Subsequently, the tongue sections were stained with haematoxylin and eosin or with an ABC kit to visualise proteins of interest. The epithelium of the lower tongue was examined by light microscopy regarding the following parameters: cell numbers, thickness of epithelial layers and expression of the potential stem cell markers p63, integrin β1 and CD44. Due to the limited number of animals per data point (experiment I: three mice per data point; experiment II: five mice per data point), a detailed statistical analysis was not performed. The present study is determined to describe the parameter variations over the observation period. Cell numbers decreased to 60-70 % of the pre-treatment control values within the first week of irradiation alone, remained constant in the second week, and then slowly increased until the end of the observation period. There was no difference between radiotherapy alone or combined treatment with BIBX1382BF. In experiment II similar observations were made with higher cell numbers in the functional layer of the epithelium of the Erlotinib treated animals than in the irradiated group. The thickness of the epithelium and its individual layers showed high inter individual differences in experiment I. In treatment group I/B, lower values of thickness were often detected in comparison to group I/A. In experiment II the thickness of the epithelium remained constant under fractionated irradiation. Between day 0 and 12 the Erlotinib treatment slightly decreased the thickness of the whole epithelium in comparison to the irradiated group. Besides, there were only minor changes in the thickness of the different layers. Associated with the general loss of cells, radiation treatment led to a transient decrease in the expression of p63. The number of p63-positive cells recovered until the end of the observation period. A similar expression pattern of p63-positivity was found independent of EGFR inhibition. The expression of integrin β1 decreased during fractionated irradiation. On days 2-9 and 12-16, the changes were more pronounced in combination with BIBX1382BF treatment which indicates a potential interaction of the EGF receptor with integrin β1. In experiment II, no differences between the exclusively irradiated group and the combined treatment with Erlotinib were found for the expression patterns of integrin β1. Irradiation alone resulted in a higher epithelial expression of CD44. Accordingly, a general increase of CD44 staining intensity was observed in both experiments exceeding control values. Due to the EGFR inhibition with Erlotinib, the expression of CD44 initially decreased. However, by day 4 no persisting differences in staining intensity could be observed independent of EGFR inhibition. In summary, EGFR inhibition via BIBX1382BF or Erlotinib did not result in alterations of the analysed parameters during two weeks of fractionated irradiation. Further studies are required to demonstrate if the present findings are transferable to other tyrosine kinase inhibitors or different substance classes (e.g. inhibiting receptor antibodies).

Background Hematopoietic stem cells located in the bone marrow interact with a specific microenvironment referred to as the stem cell niche. Data derived from ex vivo co-culture systems using mesenchymal stromal cells as a feeder cell layer suggest that cell-to-cell contact has a significant impact on the expansion, migratory potential and ‘stemness’ of hematopoietic stem cells. Here we investigated in detail the spatial relationship between hematopoietic stem cells and mesenchymal stromal cells during ex vivo expansion. Design and Methods In the co-culture system, we defined three distinct localizations of hematopoietic stem cells relative to the mesenchymal stromal cell layer: (i) those in supernatant (non-adherent cells); (ii) those adhering to the surface of mesenchymal stromal cells (phase-bright cells) and (iii) those beneath the mesenchymal stromal cells (phase-dim cells). Cell cycle, proliferation, cell division and immunophenotype of these three cell fractions were evaluated from day 1 to 7. Results Phase-bright cells contained the highest proportion of cycling progenitors during co-culture. In contrast, phase-dim cells divided much more slowly and retained a more immature phenotype compared to the other cell fractions. The phase-dim compartment was soon enriched for CD34+/CD38− cells. Migration beneath the mesenchymal stromal cell layer could be hampered by inhibiting integrin β1 or CXCR4. Conclusions Our data suggest that the mesenchymal stromal cell surface is the predominant site of proliferation of hematopoietic stem cells, whereas the compartment beneath the mesenchymal stromal cell layer seems to mimic the stem cell niche for more immature cells. The SDF-1/CXCR4 interaction and integrin-mediated cell adhesion play important roles in the distribution of hematopoietic stem cells in the co-culture system.

Diabetic kidney disease (DKD) is the leading cause of end stage renal disease worldwide and occurs in up to 40% of patients with diabetes. The standard of care for DKD treatment has not kept up with the current health epidemic, which has led to a heavy economic toll and substantial health burden. Targeting either cell surface (cs)GRP78, activated α2-macroglobulin (α2M*) or preventing their interaction may provide a novel anti-fibrotic therapeutic target for the treatment of DKD and potentially non-diabetic chronic kidney disease (CKD) as well. Previously our lab has shown that HG-induced csGRP78 is a mediator of PI3k/Akt signaling and downstream extracellular matrix (ECM) protein production in glomerular mesangial cells (MC). However, the ligand responsible for activating high glucose (HG)-induced csGRP78 had not yet been determined. We have shown thus far that α2M is endogenously produced, secreted, and activated (denoted α2M*) in HG by MC, which leads to its binding to and activation thereof csGRP78. Further, α2M knockdown or α2M* neutralization attenuated Akt activation, the production of the profibrotic cytokine connective growth tissue factor (CTGF) and ECM proteins fibronectin and collagen IV. We have also shown that integrin β1 (Intβ1), a transmembrane receptor, associated with csGRP78 under HG conditions and likely acts as a tether to present csGRP78 completely extracellularly on MC. Interestingly, Intβ1 activation, even in the absence of HG, was sufficient to induce csGRP78 translocation. Further, inhibition of either csGRP78 or Intβ1 prevented synthesis, secretion and signaling of TGFβ1. This data implicates a role for Intβ1 as a required signaling partner for csGRP78-mediated profibrotic signaling. To further our understanding of csGRP78/ α2M*’s role in DKD, we investigated their ability to mediate TGFβ1 signaling through its non-proteolytic activator thrombospondin-1 (TSP1). Here, HG-induced TSP1 expression, ECM deposition, and activation of TGFβ1 was regulated by the PI3k/Akt pathway via csGRP78/α2M* in MC. Furthermore, we assessed whether this csGRP78/ α2M* axis is relevant to promoting profibrotic signaling in other renal cell types, including proximal tubule epithelial cells (PTEC) and renal fibroblasts (RF), that contribute to the pathogenesis of both later stage DKD and non-diabetic CKD. We show evidence here that HG and direct treatment with TGFβ1, a key pathologic regulator of kidney fibrosis, induce GRP78 surface translocation as well as the endogenous production and activation of α2M in both PTEC and RF. Inhibition of either csGRP78 or α2M* prevented TGFβ1 signaling measured as Smad3 activation as well as downstream ECM production. Interestingly, inhibition of this pathway under direct TGFβ1 treatment did not prevent Smad3 activation, implicating a role for Smad-independent TGFβ1 signaling through this axis. We identified the known noncanonical TGFβ1 signaling partners, yes associated protein (YAP) and transcriptional co-activator with PDZ binding motif (TAZ), are mediated by csGRP78 and α2M*. Lastly, we evaluated the potential therapeutic benefit of inhibiting csGRP78/α2M* interaction in the kidney fibrosis model, unilateral ureteral obstruction (UUO). Here, we show evidence that inhibition of this signaling axis using an inhibitory peptide can prevent renal fibrosis. Whether this peptide also prevents fibrosis in DKD is currently being assessed. Together, these studies strongly implicate targeting csGRP78/α2M* interaction as a novel anti-fibrotic therapeutic intervention for early and late stage DKD, as well as a potential role in non-diabetic CKD. / Thesis / Doctor of Philosophy (Medical Science) / Diabetic kidney disease is the leading cause of kidney failure in developed nations. This progressive disease leads to the loss of kidney function due to an accumulation of scar proteins in the kidney over time. High glucose is a major factor that causes this to occur. Our lab studies specific kidney cells called mesangial cells, proximal tubule epithelial cells, and fibroblasts that produce scar proteins in the presence of high glucose. We have shown that when these cells are treated with high glucose, this causes the movement of a protein called GRP78 that normally resides inside the cell to move to the cell’s surface where it can interact with other proteins. My research has established that the proteins alpha 2-macroglobulin (ɑ2M), integrin β1 (Intβ1), and thrombospondin-1 (TSP1) can bind to GRP78 on the cell surface and cause cells to make scar proteins. Preventing ɑ2M or Intβ1 from binding to GRP78 or preventing TSP1 production prevents mesangial cells from making scar proteins when exposed to high glucose. In a mouse model that overproduces these scar proteins, we showed that preventing cell surface GRP78 and α2M interaction prevents scar protein production and is thus a novel potential treatment option for kidney disease.