Indiana University-Purdue University Indianapolis (IUPUI) / Cell shape changes and signaling pathways are essential for the development and function of the lens. During lens development proliferating epithelial cells will migrate down to the equator of the lens, differentiate into lens fiber cells, and begin to elongate along the lens capsule. The Fibroblast Growth Factor (FGF) signaling pathway has been extensively studied for its role in lens fiber cell differentiation and elongation. However, the main mediators of FGF stimulated lens fiber cell elongation have not been identified. Adaptor proteins Crk and CrkL are SH2- and SH3-containing proteins that transduce signals from upstream tyrosine phosphorylated proteins to downstream effectors, including Ras, Rac1 and Rap1, which are important for cell proliferation, adhesion and migration. Underlying their diverse function, these two adaptor proteins have been implicated in receptor tyrosine kinase signaling, focal adhesion assembly, and cell shape. To explore the role of Crk and CrkL in FGF signaling-dependent lens development and fiber elongation, we employed Cre/LoxP system to generate a lens specific knockout of Crk/CrkL. This led to extracellular matrix defects, disorganization of the lens fiber cells, and a defect in lens fiber cell elongation. Deletion of Crk and CrkL in the lens also mitigated the gain-of-function phenotype caused by overexpression of FGF3, indicating an epistatic relationship between Crk/CrkL and FGF signaling during lens fiber cell elongation. Further studies, revealed that the activity of Crk and CrkL in FGF signaling is controlled by the phosphatase Shp2 and the defect observed in lens fiber cell elongation can be rescued by constitutive activation of the GTPases Ras and Rac1 in the Crk and CrkL mutant lens. Interestingly, the deletion of the GTPases Rap1 in the lens showed no obvious phenotype pertaining to lens fiber cell elongation. These findings suggest that Crk and CrkL play an important role in integrating FGF signaling and mediating lens fiber cell elongation during lens development.

Indiana University-Purdue University Indianapolis (IUPUI) / Fibroblast growth factor-23 (FGF23) is a hormone produced in bone that acts upon the kidney to control blood phosphate and 1,25-(OH)2 vitamin D concentrations. Chronic kidney disease-mineral bone disorder (CKD-MBD) is a major public health problem, affecting 1 in 8 individuals. These patients can have markedly elevated FGF23 at end stage disease which is associated with metabolic bone anomalies, left ventricular hypertrophy, as well as increased mortality (>6-fold). The FGF23 co-receptor αKlotho (αKL) is a membrane-bound protein (mKL) that forms heteromeric complexes with FGF receptors (FGFRs) to initiate intracellular signaling. It also circulates as a cleavage product of mKL (‘cleaved’, or cKL). Previously, a patient with increased plasma cKL from a balanced translocation between chromosomes 9 and 13 in the KLOTHO gene presented with metabolic bone disease and a complex endocrine profile, despite hypophosphatemia. The lack of a reliable cell model in which to study potential FGF23-cKL interactions is a major hurdle for the field of phosphate metabolism. The goal of the present studies was to test and characterize bone cell lines that may respond to FGF23 and/or cKL, permitting study of novel aspects of phosphate handling and control of FGF23 expression. It was confirmed that stable delivery of cKL via AAV2/8 to wild type (WT) and KL-KO mice resulted in highly elevated bone FGF23 mRNA. MC3T3 (mouse) and ROS (rat) osteoblastic cell lines were tested for p-ERK1/2 responses to control FGFs, as well as FGF23 and cKL, alone or in combination. Importantly, both cell lines demonstrated responsiveness to FGF23+cKL only, and not the individual factors. To test responsiveness at the cell level, EGR1 mRNA was tested as an index of FGFR activity and showed modest increases with the same treatments, supporting that other factors may be required for full transcriptional effects. The present studies show that MC3T3 have FGF-dependent signaling capabilities, and that the combination of FGF23+cKL is required for efficient MAPK signaling. These results demonstrated that cKL provision is permissive for efficient FGF23 signaling in bone, and revealed important implications for the regulation of FGF23 and cKL in Mendelian, and common, genetic disorders of phosphate handling and biomineralization.

Cultured bovine aortic endothelial (BAE) cells were found to synthesize and secrete heparan sulfate proteoglycans (HSPG), which bound basic fibrobalst growth factor (bFGF). bFGF is a known mitogen for vascular smooth muscle cells, and is indicated to have a role in some proliferative vascular disorders. In the present study, we have purified proteoglycans from BAE cells conditioned media (BAE PG), and further separated the PG into two fractions, PG-I and PG-II, by ion exchange chromatography on a Q-Sepharose column using a linear salt gradient (0.15 M to 1.2 M). PG-I and PG-II elute at 0.85M salt and 0.1M salt respectively. BAE PG is primarily composed of heparan sulfate, which is accessible to the digestion of Heparinase I/III and nitrous acid treatment; and a small amount of chondroitin sulfate, which can be digested by Chondroitinase ABC. Gel filtration chromatography (Sepharose CL-2B and CL-4B columns) showed that BAE PG consisted of two different sized peaks, and had an average molecular weight of approximately 5 x 10⁵ Da. SDS-PAGE with silver staining indicated that BAE PG had two core proteins with estimated sizes of 300kDa and 320kDa, which corresponded to the core protein of PG-I and PG-II respectively. Western blotting with anti-perlecan primary antibody recognized the core proteins of BAE PG. Size exclusion chromatography (Sepharose CL-6B column) following β-elimination showed that BAE PG had GAG chains with an estimated size less than 2 x 10⁵ Da. A protocol to investigate the cell free binding of bFGF with purified BAE PG was established using the BioRad Bio-Dot apparatus - the cationic filtration assay (CAFAS). Using a simple monovalent binding model, we obtained values for the equilibrium dissociation constant, K_D, of (1.6 ± 0.8) x 10⁻¹⁰ M; the dissociation rate constant, k_r, of 0.01 min⁻¹; the association rate constant, k_f, of 6.2 x 10⁷ M⁻¹min⁻¹ and the total binding sites of the proteoglycan, R_T, of 0.1~0.2 (# of site)/(molecule of PG). The comparison of experimental data with model predictions indicates that when the number of binding sites provided by the PG is similar or greater than that of bFGF, the monovalent binding model is valid. When the number of binding sites is less than that of bFGF, one possibility is that the binding might not be the described simple monovalent reaction, and bFGF might bind to the PG as dimers or oligomers. In addition, a model is proposed for BAE PG, in which 5 ~ 10 BAE PG molecules form a high affinity binding site for bFGF. Experimentally we find that exogenous heparan sulfate competes with BAE PG for binding with bFGF, while chondroitin sulfate seems to facilitate the binding. This result may be a useful consideration when we want to design possible pharmaceutical compounds. / Master of Science

Comprehensive genomic profiling has an emerging role in cancer therapeutics. As treatment options remain needed for advanced cancers, patients are relying increasingly more on tumor genomic alterations as possible targets for cancer treatment. Frequent tumor fibroblast growth factor receptor (FGFR) alterations are seen in many cancers, and include genetic amplifications, mutations, rearrangements and fusions. FGFR inhibitors target these receptor alterations and show promise as a drug class. Currently 2 medications are currently FDA approved: erdafitinib and pemigatinib. Through the FDA accelerated approval process, erdafitinib is indicated to treat metastatic urothelial carcinoma with FGFR2 and FGFR3 alterations, whereas pemigatinib is indicated to treat unresectable cholangiocarcinoma with FGFR2 alterations. Despite growing knowledge about such advanced cancers, treatment is usually palliative. With multiple FGFR inhibitors in the pipeline, further FDA approvals are possible, and it is likely their role in therapy will extend to other cancer types. This review outlines erdafitinib, pemigatinib, their role in cancer, as well as outlining the possible future use of other FGFR inhibitors in urothelial carcinoma, cholangiocarcinoma, and other malignancies.

Indiana University-Purdue University Indianapolis (IUPUI) / Objective: To determine whether specific mutations within the fibroblast growth factor receptor 2 (FGFR2) gene associated with Crouzon syndrome cause a phenotype with extreme variability of expression suggesting non-penetrance in clinically normal appearing individuals. Methods: Most mutations responsible for Crouzon syndrome occur in exons IIIa(U) or IIIc(B) of the FGFR2 gene, facilitating allelotyping by using polymerase chain reaction to mediate mutation analysis. Once a specific mutation is identified in the index case, remaining affected family members and clinically normal first-degree relatives are screened in order to correlate genotype with phenotype. Results: A novel missense mutation, a G to T transversion, involving the first base of codon 362 (Ala362Ser), was identified following DNA sequencing of exon IIIc, and a specific restriction fragment length polymorphism following BstNI enzyme digestion was found in all Crouzon-affected family members and in one clinically normal-appearing parent. Pattern profile analysis demonstrated a consistent collection of abnormal cephalometric measurements in the Crouzon affected family members, and to a lesser degree, in the clinically normal parent. Conclusion: We have identified a novel missense mutation in the FGFR2 gene predicting an Ala362Ser substitution that is shared by all family members affected by Crouzon syndrome, and a clinically normal appearing father. These data support non-penetrance of Crouzon syndrome.

Device anchoring of artificial cornea implants, through tissue integration of stromal tissue, is necessary to ensure long-term success. In this work, the delivery of basic fibroblast growth factor (FGF-2), a key modulator in corneal wound healing, via heparin modified materials was investigated as a means of sustained, soluble growth factor delivery for stimulation of device anchorage. Two materials types, commonly used for ophthalmic applications and currently under investigation for use in artificial cornea applications, were utilized. Poly (dimethyl siloxane) (PDMS) is currently under investigation as the base material for keratoprosthetic devices; dendrimer crosslinked collagen has been examined as the basis for use as a tissue engineered corneal equivalent. PDMS surfaces were modified directly or indirectly, through a poly (ethylene oxide) (PEO) spacer, to contain functionalized reactive NSC groups capable of binding heparin and FGF-2 Surface modifications were characterized with attenuated total reflection Fourier transform infrared spectrophotometer (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and water contact angles. Heparin coverage was assessed with metachromatic and bioactivity assays. Heparinized collagen gels were crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and polypropyleneimine octaamine G2 dendrimers. Gel integrity was assessed with water uptake, differential sr::anning calorimetry, and heparin and dendrimer stability. Both materials were exposed to radiolabelled FGF-2 and growth factor immobilization and delivery were quantified. Heparinized PDMS surfaces were capable of binding on average 100 ng/cm2 ofFGF-2, while heparinized collagen gels had higher FGF-2 immobilization, 300 ng, likely attributed to their higher heparin densities and the fact that the bulk gel rather than the surface only was modified. Delivery of FGF-2 from the heparinized materials revealed a first order release profile, with an initial burst of FGF-2, followed by gradual growth factor release. Release rates, over a 2 week period, reached 6.5% and 50%, for 1 day and 3 day FGF-2 exposed heparinized PDMS modified surfaces, while hepruinized dendrimer crosslinked collagen gels released 40%. / Thesis / Master of Applied Science (MASc)

Distraction Osteogenesis (DO) is a technique for bone lengthening and filling of bone defects following trauma, infection or resection of tumors. DO consists of an osteotomy of the bone to be lengthened, followed by controlled distraction of the bone segments with an external fixator until the desired lengthening is obtained (distraction phase). This is followed by the consolidation phase, during which the external fixator is kept in place until the newly formed bone in the distracted zone consolidates. This phase is long and may cause numerous problems. Ongoing research aims at finding a method to accelerate the consolidation of the newly formed bone. / Fibroblast Growth Factors (FGF) play a significant role in bone development and repair. FGF 18 has been shown to be the only FGF member to be expressed throughout both the distraction and the consolidation phases of DO. It was also reported that FGF18 is the physiological ligand of FGFR3. Therefore, we hypothesized that FGF18 and FGFR3 may have an important role in DO. / To test this hypothesis, we investigated DO in FGFR3 deficient mice (FGFR3-/-). (FGF18 deficient mice are not viable). A miniaturized DO apparatus was applied to the tibia followed by an osteotomy. Distraction began after a 5-day latency period at a rate of 0.2 mm/12 hours for 12 days. / Samples were collected at 3 time points comparing the mutants (FGFR3-/-) to their wild type litter' sates: end of distraction (17 days post-surgery), mid-consolidation (34 days post-surgery), and end of consolidation (51 days post surgery). The samples were analyzed using X-ray, DEXA, microCT, histology, biomechanical testing and Real-Time PCR. / Our results revealed that FGFR3 deficient mice showed accelerated bone formation compared to the W.T. littermates at mid-consolidation where the parameters measured revealed increased bone mineral density, bone mineral content and trabecular number in the mutant tibial samples. The newly regenerated bone consolidated faster in the FGFR3 knock-out mice and the bone was of better quality as revealed by biomechanical tests in which more force was needed to break the mutant bone because it exhibited higher resistance than the age matched wild-type sample. The marker gene expression patterns revealed an up-regulation of chondrogenic markers that suggest that the knock-out mice follow the endochondral ossification pathway during DO. All results were statistically significant. / These results show that signaling through FGFR3 acts to decrease bone formation during DO. Consequently, blocking FGFR3 may lead to accelerated bone formation in DO. This may have important clinical implications in attempts to improve the functional outcome of DO by decreasing the long duration that the external fixator has to be kept on.

No description available.

The progressively increasing prevalence of the Metabolic Syndrome (MetS) has emerged as a major global health concern since the MetS is associated with an increased risk for cardiovascular morbidity and mortality. Central obesity represents a key feature of the MetS and is strongly related to all MetS comorbidities. Dysregulation of adipose tissue-derived proteins, so called adipokines, has been implied to partially contribute to these effects. Recently, fibroblast growth factor-21 (FGF-21) has been introduced as a novel insulin sensitizing and weight reducing adipokine with potential therapeutic properties. However, data on FGF-21 elimination are rather limited. Therefore, FGF-21 regulation in relation to renal function has been investigated in a patient population with chronic kidney disease (CKD, study population 1), as well as one with acute kidney impairment (study population 2). In study population 1 (n = 499), patients were distributed into five CKD subgroups according to estimated glomerular filtration rate (eGFR). Median FGF-21 serum concentrations progressively increased from CKD stage 1 to stage 5 and highest values of FGF-21 were detected in stage 5 (1: 86.4 ng/l; 2: 206.4 ng/l; 3: 289.8 ng/l; 4: 591.3 ng/l; 5: 1918.1 ng/l). Furthermore, eGFR remained the strongest predictor for FGF-21 levels in multivariate analysis. For study population 2 (n = 32), blood samples were obtained before elective unilateral partial or total nephrectomy, as well as within 30 hours after surgery. In this population FGF-21 levels significantly increased after surgery (325.0 ng/l) as compared to before surgery (255.5 ng/l). Furthermore, relative changes of FGF-21 were independently and positively predicted by relative changes of creatinine in this cohort. These results are in accordance with the hypothesis that FGF-21 is eliminated by the kidneys and that the extent of kidney dysfunction substantially contributes to serum FGF-21 levels. However, additional animal experiments and prospective clinical studies are needed to further elucidate the role of the kidneys in FGF-21 physiology.

In der vorliegenden Arbeit wurde der Einfluss des Fibroblast Growth Factor 9 (FGF-9) auf die Remyelinisierung in einem Modell der Multiplen Sklerose untersucht. Dafür wurde ein Mausmodell benutzt, bei dem es durch Fütterung von Cuprizon zu Oligodendrozytentod und Demyelinisierung kommt sowie nach Absetzen des Kupferchelators zu Remyelinisierung. FGF-9 wurde zum Zeitpunkt des Absetzens des toxischen Futters stereotaktisch in den Balken der Tiere injiziert. Der Fortschritt der Remyelinisierung wurde an zwei Zeitpunkten analysiert, weshalb eine Hälfte der Tiere nach 3 Tagen und die andere Hälfte nach 6 Tagen perfundiert und histologisch untersucht wurde. Die Beeinflussung von Remyelinisierung durch FGF-9 wurde auf 3 Ebenen beurteilt. (1) Die lichtmikroskopische Analyse der Remyelinisierung zeigte keinen Einfluss einer Behandlung mit FGF-9. (2) Auch in der elektronenmikroskopischen Untersuchung zeigte sich keine Beeinflussung der Anzahl remyelinisierter Fasern durch eine FGF-9- Behandlung. (3) Durch die Antikörper-vermittelte Anfärbung und Quantifizierung verschiedener Oligodendroglia-Populationen konnten folgende Beobachtungen gemacht werden: Während fortlaufender Remyelinisierung kam es zu einer Vermehrung von Oligodendrozyten-Vorläuferzellen. Dies zeigte sich an einer signifikant höheren Zahl von NG-2-markierten Zellen in Tieren, die 6 Tage nach Absetzen von Cuprizon untersucht wurden. Außerdem konnte ein Einfluss von FGF-9 auf die Population myelinbildender Zellen nachgewiesen werden. Die Dichte von Zellen, die das Myelinprotein PLP exprimierten, war bei FGF-9-behandelten Präparaten signifikant niedriger. Die Dichte reifer, Nogo-A-exprimierender Zellen war in der FGF-9- behandelten Versuchsgruppe an Tag 6, jedoch nicht an Tag 3 erniedrigt. Dass FGF-9 einen Einfluss auf Oligodendroglia in vitro hat, ist vorbeschrieben. Dabei gibt es jedoch keine übereinstimmende Aussage, ob der Wachstumsfakor proliferationshemmend oder -fördernd auf die Oligodendrogliagenese wirkt. Zudem fehlt der Nachweis, ob dies auch die Remyelinisierung beeinflusst. In dem hier durchgeführten in-vivo-Experiment konnte dieser Schritt ebenfalls nicht nachgewiesen werden. Die Zellzählungen lassen jedoch vermuten, dass FGF-9 einen hemmenden Einfluss auf die Entwicklung von Oligodendrozyten zu myelinbildenen Oligodendrozyten hat. Somit ist FGF-9 ein möglicher Induktor eines Differenzierungsblockes. In der Literatur wird eine solche Hemmung der Ausreifung von Oligodendrozyten als Ursache für die bei MS-Patienten unvollständig ablaufende Remyelinisierung postuliert. Unzureichende Remyelinisierung gilt als Korrelat der nicht vollständigen Kompensation von Behinderungen, die im Verlauf der autoimmunen Entzündungsschübe bei Multipler Sklerose entstehen.