The human body consists of a number of very complex, highly specialised organs which perform a variety of functions that are essential to life and health. One of the main functions of the skin, the largest of the human organs, is to maintain the integrity of the body. It does this by acting as a physical barrier, preventing micro-organisms and other potentially harmful substances from entering the body. When the integrity of the skin is damaged through injury, a self-protective mechanism is triggered and the reparative wound healing process begins. Under normal circumstances the wound healing process culminates in the skin recuperating its normal characteristics and functions at the site of the injury, with only a small visible mark being left behind. However, in some cases the wound healing process may become altered leading to the production of abnormal scars, such as keloids. Keloid scars are formed from scar tissue at the site of an injury, as a result of excessive tissue repair that extends beyond the boundaries of the original wound. These scars are characterised by excess collagen deposition produced during the wound healing process. It is estimated that as many as 20% of the black and Hispanic population are affected by keloid scarring. In addition to the aesthetic aspect, keloid scars can also be painful, itchy and prone to become infected. Keloid scar formation can be triggered by skin injuries caused by, for example, acne, wounds, shaving, burns, and surgical incisions. The mechanism by which keloid scars form is currently not well understood and consequently no effective treatments exist to date.This thesis describes an investigation into the mechanical properties of single keloid and normal skin fibroblast cells for the purpose of establishing if there is a quantitative difference between the two types of cells. This information will be of benefit to researchers looking for a better understanding of the keloid formation mechanism and for those seeking improved treatments. An atomic force microscope (AFM) was employed to indent single Keloid and normal skin fibroblast cells taken from five patients. Values for the apparent Young's modulus of the cells were then calculated by fitting the experimental data using Hertz's model. Apparent Young's modulus values were then compared. The findings of the analysis indicate that statistically, there is a significant difference in the Young's modulus values of normal and keloid cells, with keloid cells exhibiting substantially greater stiffness than normal skin fibroblast cells. To enable the keloid and normal skin fibroblast cells to be studied in as close to their natural, physiological environment as is possible the AFM experiments described in this thesis were undertaken in a phosphate buffered saline (PBS) solution. In such cases the use of a fluid medium presents additional complexities, not least of which is the introduction of a hydrodynamic drag force due to viscous friction of the cantilever with the liquid which can affect the experimental data obtained and consequently any material properties calculated as a result of using these data. In order to investigate the effect of dragging force on the experimental data obtained from the AFM a novel integrated finite element based model was developed. The model, described in this thesis, provides quantification of the drag force in AFM measurements of soft specimens in fluids, consequently enabling more accurate interpretation of the data obtained from AFM experimentation. The model is validated using extensive data obtained from AFM experimentation undertaken in a number of fluids of different viscosities, at a variety of tip velocities and platform-tip separations and by comparison with an existing analytical model. The novel model is shown to accurately account for drag forces in AFM in fluid media without the need for extrapolation of experimental data and can be employed over the range of tip geometries and velocities typically utilised in AFM experimentation.The work described in this thesis demonstrates that the AFM is a valuable tool that can be used to successfully investigate the mechanical properties of biological samples in fluids. It was shown that increased accuracy in the interpretation of data obtained from AFM experimentation can be obtained by taking into account the hydrodynamic drag force due to viscous friction of the cantilever with the liquid. The investigation into the mechanical behaviour of keloid cells described in this thesis significantly adds to the yet small body of research undertaken on keloid cells to date. The findings of the investigation will provide valuable information that will be of benefit in the future to researchers looking to develop effective treatments for the prevention, reduction or removal of keloid scars.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13386号 / 論医博第2218号 / 新制||医||1048(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 妻木 範行, 教授 安達 泰治 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23409号 / 医博第4754号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 武藤 学, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

For healthy individuals, wound healing mainly occurs without medical intervention, yet for the growing elderly, diabetic, or obese populations, as well as for those recovering from surgery, disregulated wound healing poses a serious health risk. Therefore, understanding the cellular processes regulating wound healing and correcting them when they go awry is essential for meeting these population’s healthcare needs. Wound healing is a complex process consisting of a suite of injury repair programs executed by cells in the injured tissue. While several of these programs have been previously described, there are many possible cellular signalling pathways that can mediate a given repair program, and its unclear which pathway mediates a specific process. In this work, we aimed to identify the key cellular signaling pathway that regulates the injury contraction process in a dermal microtissue on a chip model. We found that a balance of tissue forces generated via RhoA activation is critical for injury contraction, and that spatially localized RhoA activation can recruit new cells to participate in injury contraction. During our experiments, we also discovered and characterized a novel actin cytoskeleton-plasma membrane topology present in human dermal fibroblasts at the extreme end of cellular contractility. We also developed several technical advances: the real-time imaging and manipulation of calcium in 3D microtissues, the development of a reporter for smooth muscle actin and a labeled cellular fibronectin fusion protein, and the optimization of Forster Resonance Energy Transfer sensors. Taken together, our experimental results demonstrate the importance of RhoA-mediated force balance during injury contraction, which also has implications for scarring wound pathologies, while the tools we developed provide support for future investigations into the cellular signaling mediating injury repair programs.

Cardiac fibroblasts play an important role in myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix proteins. Estrogen has been reported to have a number of cardioprotective properties. However, it is unclear whether estrogen affects cardiac fibroblast differentiation. The purpose of the present study was to investigate the effect of estrogen on angiotensin II-induced cardiac fibroblast proliferation and differentiation. Cardiac fibroblasts were stimulated with angiotensin II (1 μM) in the presence or absence of 17β-estradiol (100 nM). Pretreatment of cardiac fibroblasts with 17β-estradiol significantly inhibited angiotensin II-induced cardiac fibroblast proliferation and differentiation (indicated by a reduction in alpha-smooth muscle actin (α-SMA) expression) by 25% and 20%. Pretreatment of 17β-estradiol significantly reduced angiotensin II-increased levels of phospho-p38 mitogen-activated protein kinase (MAPK) by 40% and nuclear factor-κB (NF-κB) binding activity in cardiac fibroblasts by 55%. Our data suggests estrogen could have an anti-fibrotic effect through limiting cardiac fibroblast proliferation and differentiation, which are the critical steps in the pathogenesis of cardiac fibrosis.

The prevalence of cardiovascular disease in the United States dramatically increases with age. A hallmark feature of the aged myocardium is increased fibrosis resulting in diastolic dysfunction. Moreover, the survival of patients subsequent to a myocardial infarction is inversely related to age because of a certain extent to maladaptive remodeling mediated by cardiac fibroblasts. Our hypothesis is that cardiac fibroblast (CF) dysfunction results in overexpressed TGF-β1 leading to increased cardiac collagen content in the aged population. TGF-β1 stimulates the synthesis of the extracellular matrix proteins, including collagen in the cardiac tissues. The RT-PCR analysis of mRNA expression of TGF-β1 of the CF was increased by 43% in the aged mice as compared to the younger. The stiffness of the left ventricle is expressed with the slope of the end-diastolic pressure-volume relationship parameter, β (mmHg/μL). In a mouse model, we demonstrated that β was 0.30 ± 0.05 in the young as compared to 0.52 ± 0.10 in the aged (p < .05). The ventricular stiffness was associated with the myocardial collagen content; namely, young versus the aged was 9.5 ± 4.0 as compared to 16.4 ± 2.3% of total protein, respectively (p < .05). In conclusion, the gene structure-function relationships support our hypothesis that cardiac fibroblast disregulation contributes to diastolic filling dysfunction in elderly persons. These data provide a potential contributory mechanism for diastolic dysfunction that may be vital in caring for the aged open-heart surgical patient.

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.

β-adrenergic receptor (β-AR) stimulation increases extracellular levels of ubiquitin (UB), and exogenous UB plays an important role in β-AR-stimulated myocardial remodeling with effects on heart function, fibrosis and myocyte apoptosis. Cardiac fibroblasts are vital for maintaining the normal function of the heart, and in the structural remodeling of the heart in response to injury. Here we hypothesized that extracellular UB modulates cardiac fibroblast phenotype and function via its interaction with CXC chemokine receptor type 4 (CXCR4). Main methods: Serum starved adult cardiac fibroblasts were used to identify CXCR4 as a receptor for UB. Fluorescent microscopy, co-immunoprecipitation, western blot, proliferation, migration and collagen contraction assays were performed to investigate the role of UB/CXCR4 axis on cell signaling, and modulation of fibroblast phenotype and function. Key findings: Using fluorescent microscopy and co-immunoprecipitation assay, we provide evidence that extracellular UB interacts with CXCR4. CXCR4 antagonist, AMD3100, inhibited interaction of UB with CXCR4. UB activated ERK1/2, not Akt. It enhanced VEGF-A expression, while decreasing β3 integrins expression. Two mutated UB proteins (V70A and F4A; unable to interact with CXCR4) failed to affect the expression of VEGF-A and β3 integrins. UB treatment inhibited migration of cells into the wound and FBS-stimulated cell proliferation. UB enhanced expression of α-smooth muscle actin (marker of myofibroblast differentiation) and contraction of fibroblast-populated collagen gel pads. Most of the effects of UB were negated by AMD3100. Significance: The data presented here suggest that UB interacts with CXCR4, and UB/CXCR4 interaction affects intracellular signaling, and modulates fibroblast phenotype and function.

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.