FGFR 3 is one of a family of four high affinity receptors for FGF ligands. Activating mutations in FGFR 3 result in skeletal dysplasias that vary in severity from undetectable to neonatal lethal. Mice with congenital deficiency of FGFR3 develop severe kyphosis and skeletal overgrowth. FGFR3 is also expressed in calvarial pre-osteoblasts, osteoblast and articular chondrocytes, although it biological role in these cells remains undefined. By changing the genetic background of the Fgfr3-/- mice we were able to extend their lifespan and examine its impact on post-natal skeletal growth. To investigate the implication of FGFR 3 in post-natal cartilage and bone metabolism we used a combination of imaging, classic histology, molecular biology and biomechanical testing. The results demonstrated that the synovial joints of young adult Fgfr3-/- mice revealed a progressive deterioration, loss of the joint space width and changes in the subchondral bone. These alterations were accompanied by an increase of cartilage matrix degradation. Increased aggrecan and collagen type II degradation products, generated by MMPs were detected with DIAPEN and COL2-3/4C antibodies. Increased collagen type X, cellular hypertrophy and loss of proteoglycan at the articular surface were also demonstrated. A novel micro-mechanical indentation protocol revealed that the humeral heads of Fgfr3-/- mice were less stiff than those of wild type littermates. On the other hand, young adult Fgfr3-/- mice are osteopenic due to reduced cortical bone thickness and defective trabecular bone mineralization. The reduction in mineralized bone and lack of trabecular connectivity observed by micro-computed tomography were confirmed by histological and histomorphometric analyses, which revealed a significant decrease in calcein labeling of mineralizing surfaces and a significant increase in osteoid in the long bones of 4-month-old Fgfr3-/- mice. Primary cultures of adherent bone marrow-derived cells from Fgfr3-/- mice expressed markers of differentiated osteoblasts but developed fewer mineralized nodules than Fgfr3+/+ cultures of the same age. These data point to a major role for FGFR3 signaling in development and homeostatic maintenance of cartilage and bone post-natally and identify FGFR3 as a potential target for intervention in degenerative disorders of cartilage, osteopenia and those associated with defective bone mineralization.

The role of Indian hedgehog (Ihh) signalling in the regulation of endochondral bone formation is well established. Ihh controls the rate of bone growth by negatively regulating differentiation and positively regulating growth plate chondrocyte proliferation. It has been well documented also that mutations resulting in constitutive activation of signalling through FGFR3 in chondrodysplasia, lead to a significant decrease in this important signalling factor accompanied by reduced proliferation of the chondrocytes and a dwarf phenotype. / In an attempt to rescue the chondrodysplasia phenotype hedgehog agonist Hh-Ag 1.4 was injected subcutaneously into mice with achondroplasia (ACH) or with severe achondroplasia with developmental delay and acanthosis negricans (SADDAN) with mixed results. / Administration of a hedgehog agonist in SADDAN mice led to a significant up-regulation of both Ptch and Gli1, as measured by quantitative PCR, indicating that Hh-Ag 1.4 does indeed stimulate hedgehog signalling in vivo. Also, in situ hybridization for Ihh seems to show a down regulation of native Ihh expression in pre-hypertrophic chondrocytes, possibly due to the activation of the negative PTHrP feedback loop. In our study, Hh-Ag 1.4 treatment resulted in an increased growth plate length and reduced size of the hypertrophic zone. The cortical bone flanking the growth plate in mice injected with Hh-Ag 1.4 was 2-3 times thicker than in control mice, which may be attributed to the positive effect of increased Ihh signalling in osteoblastogenesis. Contrary to our expectations, there was also a noticeable reduction in chondrocyte proliferation in mice treated with the agonist. / Overall, the effect on the growth of long bones was not beneficial and the treatment with high doses of Hh-Ag 1.4 did not result in an amelioration of the chondrodysplastic phenotype.

The tumour-associated stroma, consisting of fibroblasts, inflammatory cells, vasculature and extracellular matrix proteins, plays a critical role in tumour growth, but how it regulates cancer cell migration and metastasis is poorly understood. The Wnt-planar cell polarity (PCP) pathway regulates convergent extension movements in vertebrate development. However, it is unclear whether this pathway also functions in cancer cell migration. In addition, the factors that mobilize long-range signalling of Wnt morphogens, which are tightly associated with the plasma membrane, have yet to be completely characterized. Here, I show that fibroblasts secrete membrane microvesicles of endocytic origin, termed exosomes, which promote tumour cell protrusive activity, motility and metastasis via the exosome component Cd81. In addition, I demonstrate that fibroblast exosomes activate autocrine Wnt-PCP signalling in breast cancer cells as detected by the association of Wnt with Fzd receptors and the asymmetric distribution of Fzd-Dvl and Vangl-Pk complexes in exosome-stimulated cancer cell protrusive structures. Moreover, I show that Pk expression in breast cancer cells is essential for fibroblast-stimulated cancer cell metastasis. Lastly, I reveal that trafficking in cancer cells promotes tethering of autocrine Wnt11 to fibroblast exosomes. These studies further our understanding of the role of the tumour-associated stroma in cancer metastasis and bring us closer to a more targeted approach for the treatment of cancer spread.

The tumour-associated stroma, consisting of fibroblasts, inflammatory cells, vasculature and extracellular matrix proteins, plays a critical role in tumour growth, but how it regulates cancer cell migration and metastasis is poorly understood. The Wnt-planar cell polarity (PCP) pathway regulates convergent extension movements in vertebrate development. However, it is unclear whether this pathway also functions in cancer cell migration. In addition, the factors that mobilize long-range signalling of Wnt morphogens, which are tightly associated with the plasma membrane, have yet to be completely characterized. Here, I show that fibroblasts secrete membrane microvesicles of endocytic origin, termed exosomes, which promote tumour cell protrusive activity, motility and metastasis via the exosome component Cd81. In addition, I demonstrate that fibroblast exosomes activate autocrine Wnt-PCP signalling in breast cancer cells as detected by the association of Wnt with Fzd receptors and the asymmetric distribution of Fzd-Dvl and Vangl-Pk complexes in exosome-stimulated cancer cell protrusive structures. Moreover, I show that Pk expression in breast cancer cells is essential for fibroblast-stimulated cancer cell metastasis. Lastly, I reveal that trafficking in cancer cells promotes tethering of autocrine Wnt11 to fibroblast exosomes. These studies further our understanding of the role of the tumour-associated stroma in cancer metastasis and bring us closer to a more targeted approach for the treatment of cancer spread.

Human physiology is dependent on an accurate phosphate (Pi) homeostasis. Defective Pi regulation causes hyper- or hypophosphatemia, which are associated with ectopic calcification or impaired bone mineralization, and a shortened life span. Current endocrine models of Pi homeostasis are incomplete. However, studies of acquired and hereditary disorders of Pi homeostasis have revealed new potential Pi regulating hormones, Phosphatonin(s). One of these is fibroblast growth factor-23 (FGF23). FGF23 is produced in bone and is secreted into the circulation. Mutations in FGF23 causes disturbed Pi regulation, without the appropriate counter-regulatory actions of parathyroid hormone or vitamin D. By the generation of FGF23 transgenic mice, which display phenotypic similarities to patients with hypophosphatemic disorders, we show that FGF23 exerts endocrine actions in the kidney and causes osteomalacia. Renal FGF23 actions severely decrease Pi reabsorption and expression of Klotho, a suggested age suppressor gene, known to be crucial in FGF23 receptor binding and activation. In bone, our transgenic model displays impaired osteoclast polarization, which should be detrimental to osteoclastic bone resorption in osteomalacia. However, in our model osteoclasts efficiently participate in bone matrix degradation. Furthermore, we investigated a large population-based cohort in order to elucidate the role of FGF23 in normal physiology. Importantly, we were able to demonstrate an association of FGF23 to parathyroid hormone, renal function and bone mineral density and we found a correlation of FGF23 to weight and body fat mass. The studies on which this thesis is based, demonstrate that FGF23 has phosphatonin-like properties and that the skeleton functions as an endocrine organ. In addition, the results indicate that FGF23 has a role in bone mineral and lipid metabolism, and that FGF23 is a possible diagnostic marker and therapeutic target for the future.

This thesis demonstrates the use of numerical simulation in predicting the behavior of proteins in a flow environment. A novel convection-diffusion-reaction computational model is first introduced to simulate fibroblast growth factor (FGF-2) binding to its receptor (FGFR) on cell surfaces and regulated by heparan sulfate proteoglycan (HSPG) under flow in a bioreactor. The model includes three parts: (1) the flow of medium using incompressible Navier-Stokes equations; (2) the mass transport of FGF-2 using convection-diffusion equations; and (3) the cell surface binding using chemical kinetics. The model consists of a set of coupled nonlinear partial differential equations (PDEs) for flow and mass transport, and a set of coupled nonlinear ordinary differential equations (ODEs) for binding kinetics. To handle pulsatile flow, several assumptions are made including neglecting the entrance effects and an approximate analytical solution for axial velocity within the fibers is obtained. To solve the time-dependent mass transport PDEs, the second order implicit Euler method by finite volume discretization is used. The binding kinetics ODEs are stiff and solved by an ODE solver (CVODE) using Newton’s backward differencing formula. To obtain a reasonable accuracy of the biochemical reactions on cell surfaces, a uniform mesh is used. This basic model can be used to simulate any growth factor-receptor binding on cell surfaces on the wall of fibers in a bioreactor, simply by replacing binding kinetics ODEs. Circulation is an important delivery method for natural and synthetic molecules, but microenvironment interactions, regulated by endothelial cells and critical to the molecule’s fate, are difficult to interpret using traditional approaches. Growth factor capture under flow is analyzed and predicted using computer modeling mentioned above and a three-dimensional experimental approach that includes pertinent circulation characteristics such as pulsatile flow, competing binding interactions, and limited bioavailability. An understanding of the controlling features of this process is desired. The experimental module consists of a bioreactor with synthetic endotheliallined hollow fibers under flow. The physical design of the system is incorporated into the model parameters. FGF-2 is used for both the experiments and simulations. The computational model is based on the flow and reactions within a single hollow fiber and is scaled linearly by the total number of fibers for comparison with experimental results. The model predicts, and experiments confirm, that removal of heparan sulfate (HS) from the system will result in a dramatic loss of binding by heparin-binding proteins, but not by proteins that do not bind heparin. The model further predicts a significant loss of bound protein at flow rates only slightly higher than average capillary flow rates, corroborated experimentally, suggesting that the probability of capture in a single pass at high flow rates is extremely low. Several other key parameters are investigated with the coupling between receptors and proteoglycans shown to have a critical impact on successful capture. The combined system offers opportunities to examine circulation capture in a straightforward quantitative manner that should prove advantageous for biological or drug delivery investigations. For some complicated binding systems, where there are more growth factors or proteins with competing binding among them moving through hollow fibers of a bioreactor coupled with biochemical reactions on cell surfaces on the wall of fibers, a complex model is deduced from the basic model mentioned above. The fluid flow is also modeled by incompressible Navier-Stokes equations as mentioned in the basic model, the biochemical reactions in the fluid and on the cell surfaces are modeled by two distinctive sets of coupled nonlinear ordinary differential equations, and the mass transports of different growth factors or complexes are modeled separately by different sets of coupled nonlinear partial differential equations. To solve this computationally intensive system, parallel algorithms are devised, in which all the numerical computations are solved in parallel, including the discretization of mass transport equations and the linear system solver Stone’s Implicit Procedure (SIP). A parallel SIP solver is designed, in which pipeline technique is used for LU factorization and an overlapped Jacobi iteration technique is chosen for forward and backward substitutions. For solving binding equations ODEs in the fluid and on cell surfaces, a parallel scheme combined with a sequential CVODE solver is used. The simulation results are obtained to demonstrate the computational efficiency of the algorithms and further experiments need to be conducted to verify the predictions.

Cardiac myofibroblasts participate in post-myocardial infarct (MI) wound healing, infarct scar formation, and remodeling of the ventricle remote to the site of infarction. The role of intracellular calcium handling in cardiac myofibroblasts as a modulator of cellular motility, contractile responses, and proliferation is largely unexplored. We have investigated the role of sodium calcium exchange (Na Ca exchange or NCX1.1) and non-selective cation channels (NSCCs) in regulation of myofibroblast function using a pharmacological inhibitor approach in vitro. Primary myofibroblasts were stimulated with PDGF-BB and cellular chemotaxis, contraction and proliferative responses were characterized using standard bioassays (Costar Transwell apparatuses, pre-formed collagen type I gel deformation assays, and 3H-thymidine incorporation). Stimulated cellular responses were compared to those in the presence of AG1296 (PDGFβR inhibitor), KB-R7943 (NCX inhibitor), gadolinium, nifedipine or ML-7. Immunofluorescence was used to determine localized expression of αSMA, SMemb, NCX1.1, and Cav1.2a in cultured myofibroblasts. Motility of myofibroblasts in the presence of PDGF-BB was blocked with AG1296 treatment. Immunoblotting and immunocytochemical studies revealed expression of NCX1.1 in fibroblasts and myofibroblasts. Motility (in the presence of either PDGF-BB or CT-1), contraction (in the presence of either PDGF-BB or TGFβ1), and proliferation (in the presence of PDGF-BB) were sensitive to KB-R7943 treatment of cells (7.5 and 10 μM for motility, 5 and 10 μM for contractility, and 10 μM for proliferation). Proliferation (in the presence of PDGF-BB), and contractility (in the presence of either PDGF-BB or TGFβ1) but not motility (in the presence of PDGF-BB) are sensitive to nifedipine treatment, while gadolinium treatment was associated only with decreased motility of cells (in the presence of either PDGF-BB, CT-1, or LoFGF-2). We found that ML-7 treatment inhibited cellular chemotaxis, and contraction. Thus cellular chemotaxis, contractile, and proliferation responses were sensitive to different pharmacologic treatment. Regulation of transplasmalemmal calcium movements may be important in cytokine and growth factor receptor-mediated cardiac myofibroblast motility, contractility, and proliferation. Furthermore, our results support the hypothesis that activation of specific calcium transport proteins is an important determinant of physiologic responses.

The development of bioengineered cartilage for replacement of damaged articular cartilage has gained momentum in recent years. One such approach has been developed in the Kandel lab, whereby cartilage is formed by seeding primary articular chondrocytes on the top surface of a porous biodegradable calcium polyphosphate (CPP) bone substitute, permitting anchorage of the tissue within the pores of the substrate; however, the interfacial shear properties of the tissue-substrate interface of these biphasic constructs are 1 to 2 orders of magnitude lower than the native cartilage-subchondral bone interface. To overcome this limitation, a strategy was devised to generate a zone of calcified cartilage (ZCC), thereby mimicking the native architecture of the osteochondral junction; however, the ZCC was located slightly above the cartilage-CPP interface. Thus, it was hypothesized that polyphosphate released from the CPP substrate and accumulating in the tissue inhibits the formation of the ZCC at the tissue-substrate interface. Based on this information, a strategy was devised to generate biphasic constructs incorporating a properly located ZCC. This approach involved the application of a thin calcium phosphate film to the surfaces of porous CPP via a sol-gel procedure, thereby limiting the accumulation of polyphosphate in the cartilaginous tissue. This modification to the substrate surface did not negatively impact the quality of the in vitro-formed cartilage tissue or the ZCC. Interfacial shear testing of biphasic constructs demonstrated significantly improved interfacial shear properties in the presence of a properly located ZCC. These studies also led to the observation that chondrocytes produce endogenous polyphosphate and that its levels in deep zone cartilage appear inversely related to mineral deposition within the tissue. Using an in vitro model of cartilage calcification, it was demonstrated that polyphosphate levels are modulated in part by the inhibitory effects of fibroblast growth factor 18 on exopolyphosphatase activity in the tissue. Polyphosphate also appears to act in a feedback loop to control exopolyphosphatase activity. Interestingly, polyphosphate also exhibits positive effects on cartilage matrix accumulation. The potential implication of polyphosphate in the maintenance of articular cartilage homeostasis is intriguing and must be investigated further.

The development of bioengineered cartilage for replacement of damaged articular cartilage has gained momentum in recent years. One such approach has been developed in the Kandel lab, whereby cartilage is formed by seeding primary articular chondrocytes on the top surface of a porous biodegradable calcium polyphosphate (CPP) bone substitute, permitting anchorage of the tissue within the pores of the substrate; however, the interfacial shear properties of the tissue-substrate interface of these biphasic constructs are 1 to 2 orders of magnitude lower than the native cartilage-subchondral bone interface. To overcome this limitation, a strategy was devised to generate a zone of calcified cartilage (ZCC), thereby mimicking the native architecture of the osteochondral junction; however, the ZCC was located slightly above the cartilage-CPP interface. Thus, it was hypothesized that polyphosphate released from the CPP substrate and accumulating in the tissue inhibits the formation of the ZCC at the tissue-substrate interface. Based on this information, a strategy was devised to generate biphasic constructs incorporating a properly located ZCC. This approach involved the application of a thin calcium phosphate film to the surfaces of porous CPP via a sol-gel procedure, thereby limiting the accumulation of polyphosphate in the cartilaginous tissue. This modification to the substrate surface did not negatively impact the quality of the in vitro-formed cartilage tissue or the ZCC. Interfacial shear testing of biphasic constructs demonstrated significantly improved interfacial shear properties in the presence of a properly located ZCC. These studies also led to the observation that chondrocytes produce endogenous polyphosphate and that its levels in deep zone cartilage appear inversely related to mineral deposition within the tissue. Using an in vitro model of cartilage calcification, it was demonstrated that polyphosphate levels are modulated in part by the inhibitory effects of fibroblast growth factor 18 on exopolyphosphatase activity in the tissue. Polyphosphate also appears to act in a feedback loop to control exopolyphosphatase activity. Interestingly, polyphosphate also exhibits positive effects on cartilage matrix accumulation. The potential implication of polyphosphate in the maintenance of articular cartilage homeostasis is intriguing and must be investigated further.

Cardiac myofibroblasts participate in post-myocardial infarct (MI) wound healing, infarct scar formation, and remodeling of the ventricle remote to the site of infarction. The role of intracellular calcium handling in cardiac myofibroblasts as a modulator of cellular motility, contractile responses, and proliferation is largely unexplored. We have investigated the role of sodium calcium exchange (Na Ca exchange or NCX1.1) and non-selective cation channels (NSCCs) in regulation of myofibroblast function using a pharmacological inhibitor approach in vitro. Primary myofibroblasts were stimulated with PDGF-BB and cellular chemotaxis, contraction and proliferative responses were characterized using standard bioassays (Costar Transwell apparatuses, pre-formed collagen type I gel deformation assays, and 3H-thymidine incorporation). Stimulated cellular responses were compared to those in the presence of AG1296 (PDGFβR inhibitor), KB-R7943 (NCX inhibitor), gadolinium, nifedipine or ML-7. Immunofluorescence was used to determine localized expression of αSMA, SMemb, NCX1.1, and Cav1.2a in cultured myofibroblasts. Motility of myofibroblasts in the presence of PDGF-BB was blocked with AG1296 treatment. Immunoblotting and immunocytochemical studies revealed expression of NCX1.1 in fibroblasts and myofibroblasts. Motility (in the presence of either PDGF-BB or CT-1), contraction (in the presence of either PDGF-BB or TGFβ1), and proliferation (in the presence of PDGF-BB) were sensitive to KB-R7943 treatment of cells (7.5 and 10 μM for motility, 5 and 10 μM for contractility, and 10 μM for proliferation). Proliferation (in the presence of PDGF-BB), and contractility (in the presence of either PDGF-BB or TGFβ1) but not motility (in the presence of PDGF-BB) are sensitive to nifedipine treatment, while gadolinium treatment was associated only with decreased motility of cells (in the presence of either PDGF-BB, CT-1, or LoFGF-2). We found that ML-7 treatment inhibited cellular chemotaxis, and contraction. Thus cellular chemotaxis, contractile, and proliferation responses were sensitive to different pharmacologic treatment. Regulation of transplasmalemmal calcium movements may be important in cytokine and growth factor receptor-mediated cardiac myofibroblast motility, contractility, and proliferation. Furthermore, our results support the hypothesis that activation of specific calcium transport proteins is an important determinant of physiologic responses.