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Cell adhesion to the extracellular matrix (ECM) provides tissue structure and integrity as well as triggers signals that regulate complex biological processes such as cell cycle progression and tissue-specific cell differentiation. Hence, cell adhesion is critical to numerous physiological and pathological processes, including embryonic development, cancer metastasis, and wound healing, as well as biotechnological applications, such as host responses to implanted devices and integration of tissue-engineered constructs. During the adhesion process, integrin surface receptors bind ECM proteins, cluster, and associate with the actin cytoskeleton. Subsequent strengthening of the integrin/actin cytoskeleton interaction occurs via complexes of proteins known as focal adhesions. Due to the close association between biochemical and biophysical processes within adhesion complexes, mechanical analyses can provide important new insights into structure/function relationships involved in regulating the adhesion process. The objective of this project was to investigate the role of the protein tyrosine kinase FAK in cell adhesion strengthening. Our central hypothesis was that FAK regulates adhesion strengthening by modulating interactions between integrins and FA structural components. Using a novel combination of genetically engineered cells to control the interactions of FAK, a spinning disk adhesion assay with micropatterned substrates to obtain reproducible and sensitive measurements of adhesion strength, and quantitative biochemical assays for analyzing changes in adhesive complexes, we demonstrate that FAK modulates adhesion strengthening via two distinct mechanisms: (1) FAK expression results in elevated integrin activation leading to regulation of strengthening rate and (2) FAK regulates steady-state adhesion strength via vinculin recruitment to focal adhesions. We also show that the autophosphorylation and catalytic sites of FAK are critical to this regulation of adhesion strengthening. This work is significant because it both identifies functional mechanisms of FAK and provides the first evidence that focal adhesion signaling regulates the adhesion strengthening process. Furthermore, this research demonstrates that the dependency of migration on adhesion strength is highly complex and establishes a need for adhesion strengthening metrics in analyzing the functional mechanisms of molecules within adhesion complexes.

Stem cell synthesized extracellular matrix (ECM) may serve as a replacement for current bone grafting techniques. The overall goal of this thesis is to quantify the osteoinductivity of the ECM produced by human amniotic fluid stem cells (AFS cells), compare it to that of human mesenchymal stem cells (MSC), and assess its potential for use in bone tissue engineering therapies. Each stem cell type was cultured in osteogenic media to produce the ECM, which was then decellularized via freeze/thaw cycling and DNase treatment. The success of the decellularization was confirmed with live/dead staining and DNA quantification. A series of in vitro studies were performed to evaluate the characteristics of the ECM relevant to a bone tissue engineering therapy. Reseeded MSCs were able to attach to and proliferate on both ECM types in both 2D and 3D culture. In 2D, cells cultured on both ECM types showed increased levels of calcium deposition. Additionally, cells cultured on the MSC ECM showed increased alkaline phosphatase activity. A synergistic effect on osteogenic differentiation was observed when the osteoinductive factor dexamethasone was added to the culture. In 3D, both ECM types increased the mineralized matrix production of reseeded MSCs. The AFS ECM had a greater effect than the MSC ECM. When ECM was used to treat a rat femoral segmental defect in vivo, it was found that each ECM type increased the rate of bridging of the defect when compared to collagen coated scaffolds. However the ECM did not have a significant effect on the volume of mineralized matrix within the defect site in this study.

Fibrillins are modular, disulphide-rich glycoproteins that assemble into microfibrils in the extracellular matrix (ECM). These microfibrils are critical structural elements of many non-elastic and elastic connective tissues. They also regulate the availability of transforming growth factor-β signalling molecules in the ECM. Defects in microfibrils are associated with acquired and inherited connective tissue disorders. In particular, mutations in the human FBN1 gene, which encodes fibrillin-1, are associated with a spectrum of diseases, including Marfan syndrome (MFS). One of the proposed initial steps in microfibril assembly is the interaction between the N- and C-terminal regions of fibrillin monomers. The minimal regions of human fibrillin-1 required for an interaction in vitro were previously identified: the four N-terminal domains, from the fibrillin unique N-terminal (FUN) domain to the third epidermal growth factor-like (EGF) domain (FUN-EGF3), and the three C-terminal calcium-binding EGF-like (cbEGF) domains (cbEGF41-43). Here, fragments corresponding to these regions were produced and shown to interact in pull-down and surface plasmon resonance assays. In addition, the structure of the FUN-EGF3 fragment was determined using nuclear magnetic resonance spectroscopy. This showed the novel structure of the FUN domain and the interdomain interfaces in this region of fibrillin. Combining structural and sequence conservation data may help to identify regions of FUN-EGF3 important for binding to cbEGF41-43. Here, the interaction was probed by site-directed mutagenesis. However, substituting individual residues in FUN-EGF3 with alanine did not abrogate binding to cbEGF41-43. Three MFS-associated residue substitutions were also introduced into the FUN-EGF3 fragment. While they did not abolish the interaction with cbEGF41-43, they did cause misfolding. Two of these substitutions, N57D and W71R, also resulted in the defective secretion of a larger N-terminal fragment by fibroblast cells, suggesting a potential mechanism of disease pathogenesis. Although specific residues involved in the N-C interaction were not identified here, the FUN-EGF3 structure will be vital for understanding the molecular surfaces involved in microfibril assembly and growth factor binding.

Fibrosis of vital organs remains one of the leading causes of death in the developed world, where it occurs predominantly in soft tissues (liver, lung, kidney, heart) through fibroblast proliferation and deposition of extracellular matrix (ECM). In the process of fibrosis, remodeling and deposition of ECM results in stiffening of cellular microenvironment; cells also respond to these changes in the stiffness through engagement of their cytoskeleton and signaling via cell-ECM contacts. Thus, understanding to what extent the stiffness of the cellular microenvironment changes as a consequence of fibrotic progression, and how cells respond to this change, is critical. In this thesis, we quantitatively measured stiffness of the lung parenchyma and its changes during fibrosis. We find that the average stiffness increases by approximately 10-fold. We then investigated how changes in ECM rigidity affect the cytoskeletal phenotype of lung fibroblasts. We find a complex relation between expression of the glycoprotein Thy-1 (CD90) and ECM rigidity-dependent cytoskeletal phenotype (i.e. “mechanotransduction”). Finally, we investigate a mechanism for the regulation of rigidity sensing by Thy-1 and its involvement in intracellular signaling through cell-ECM contacts. Taken together, this work helps define in vivo parameters critical to the fibrogenesis program and to define unique cellular phenotypes that may respond or contribute to mechanical homeostasis in fibrotic diseases.

The toll skeletal muscle injury, resulting in significant muscle mass loss, has on the patient reaches far more than physical and emotional, as the tolls are financial as well. Approximately more than 3 billion dollars is spent on the initial medical costs and on subsequent disability benefits, following a volumetric muscle loss. Skeletal muscle has a robust capacity for self-repair; this propensity for repair is hindered when skeletal muscle loss is larger than 20% of the total mass of the muscle. Previous work in our lab, has shown functional and morphological improvements following the cellular therapy, with mesenchymal stem cells (MSC), as well as with nerve relocation to the extracellular matrix (ECM). To further observe the regenerative properties of the above treatments, a defect weighing approximately 307 ± 3.7 mg wet weight and measuring approximately 1x 1cm² was removed from the lateral gastrocnemius (LGAS) of male Sprague Dawley rats. Additionally, the medial branch of the plantar nerve was then relocated and implanted to the middle of the ECM. Seven days post injury bone-marrow derived mesenchymal stem cells were injected directly into the implant using a PEGylated Fibrin hydrogel (PEG). Following 56 days of recovery, partial functional restoration was observed in the LGAS ECM seeded with MSC and implanted with the plantar nerve. The LGAS produced 86.3 ± 5.8% of the contralateral LGAS, a value that was significantly higher than ECM implantation alone (p <.05). The implanted ECM seeded with MSC and implanted with the plantar nerve showed significant increases in blood vessel density and myofiber content (p <.05). The data suggest that a volumetric injury can be repaired by neurotization of an implanted muscle-derived ECM seeded with MSCs. / text

Myxomatous mitral valve degeneration (MMVD) is the single most common cardiac disease of the dog, and is analogous to Mitral Valve Prolapse in humans. Very little is known about the aetiopathogenesis of this disease or the changes in valvular interstitial cell populations in diseased valves. The aim of this study was to identify morphological, cellular and molecular changes associated with MMVD. Mitral valve leaflets from both normal and varying grades (Whitney’s 1-4) of diseased dogs were subject to image analysis, immunophenotyping, proteomics and RT-PCR. Image analysis - leaflet thickening due to accumulation of glycosaminoglycan was significant in this disease. MMVD is associated with loss of connective tissue, reduction in cell numbers but no change in cell shape in the overtly myxomatous area. Near the surface, increase in valvular interstitial cells (VIC) towards the damaged endothelium in concert with destruction of collagen and building up of ground substance was manifested during the disease process. Immunophenotyping - activated myofibroblasts were increased and fibroblast-like VICs were reduced without any change in desmin and myosin expression in MMVD compared to clinical normal dogs. In addition, other cell types like macrophage, adipocyte, chondrocyte, mast cell, and stem cell were identified and their possible role in MMVD is discussed. Proteomics - a protein expression profile was established, with 64 proteins being positively identified from dog’s mitral valve using 1-D SDS PAGE LC/MS. Amongst them 44 proteins were differentially expressed comparing normal and severely diseased. Two actin binding proteins, tropomyosin alpha and myosin light chain-2 were found to be differentially expressed in the normal but down regulated in the diseased. RT-PCR was used to assess the expression of 8 genes of interest. Their expression was compared with 3 different housekeeping genes.

Hyaluronidase 2 (HYAL2) is a GPI-linked protein that is proposed to initiate the degradation of hyaluronan (HA), a major extracellular matrix component of many vertebrate tissues. Hyal2 knockout (KO) mice displayed craniofacial abnormalities and severe preweaning lethality. 54% of the surviving KOs developed a grossly dilated left or right atrium, requiring euthanasia, by 3 months of age. We hypothesize that the absence of HYAL2 leads to the accumulation of HA in organs/tissues where HA is normally abundant resulting in developmental defects and organs dysfunction. Molecular and histological analysis of HYAL2 KO hearts demonstrated extracellular accumulation of high molecular mass (HMM) HA in the heart valves, myocardium, serum and lungs which was associated with severe cardiopulmonary dysfunction. Further, structural and functional analyses of Hyal2 KO mouse hearts using high-frequency ultrasound revealed atrial dilation accompanied by diastolic dysfunction that was evident as early as 4 weeks of age, and progressed with age. Further, 50% of HYAL2 KO mice exhibited a triatrial heart (cor-triatriatum). Histological analyses revealed that the atrial dilation was the result of excess tissue, and did not correlate with the presence of cor triatrium. Hyal2 KO mice were found to have increased numbers of mesenchymal cells at early stages of development, presumably due to the presence of excess HA, that lead to cardiac dysfunction. Further examination of HYAL2 distribution in a broad range of mouse tissues, and accumulation of HA in its absence demonstrated that HYAL2 is mainly localized to endothelial cells and some specialized epithelial cells, and plays a major role in HMM-HA degradation. These studies demonstrated that HYAL2 is important for HA degradation and organ development. In the longer term, our findings will be valuable for understanding pathologies associated with the disruption of HA catabolism, and potentially in the identification of HYAL2-deficient patients. / May 2016

Cardiac stem cells (CSCs), isolated from heart tissue explants and expanded via the formation of cardiospheres (Csp), are a promising candidate for cell therapy to prevent heart failure following myocardial infarction. To allow early administration to patients, isolation and expansion of CSCs must be performed in the shortest time possible. Hence, this project aimed to optimize culture conditions and characterize the cardiac explant-derived cells (EDCs), Csp and Csp-derived cells (CDCs) produced. Rat neonatal EDCs contained 4-7% c-kit⁺ cells, measured using flow cytometry. Optimal Csp growth conditions were determined, such that plating 3 x 10^4 EDCs per well of a 24-well plate coated with 16.7 µg/ml poly-D-lysine, in CGM containing 7% serum, improved Csp production and generated 1.5 x 10^7 CDCs in 16 days, a sufficient number for cell therapy. The CDCs expressed the stemness markers; c-kit, Oct3/4, SOX2, and Klf-4, and the cardiac differentiation markers; GATA4 and Nkx2.5. The therapeutic effect of CDCs may be limited by the low, 3 ± 0.1%, c-kit⁺ cell numbers. To increase c-kit⁺ cells in CDCs, an alternate culture method for Csp and different extracellular matrices (ECM) for cell expansion were tested. The hanging drop culture method produced Csp with higher levels of c-kit⁺ cells (9 ± 2%) than poly-D-lysine-coated and low-bind culture dishes. Of five ECM tested, collagen IV was found to enhance EDC migration and CDC proliferation, and produced 11 ± 0.4% c-kit⁺ cells, with Csp cultured in hanging drops. Intramyocardial injection of CDCs improved left ventricular ejection fractions of infarcted rat hearts by 9% and prevented the peri-infarct wall from thinning, measured in vivo using MRI over 16 weeks. To improve cell tracking using MRI, two MR positive contrast agents, gadolinium-DTPA and gadonanotubes were tested. Gd-DTPA had low sensitivity after labelling (1.4 x 10^5 cells/mm2); whereas gadonanotubes did not provide positive contrast at 11.7 T. Thus, neither contrast agent could be used for cell tracking using high magnetic field. In conclusion, CDCs were an effective source of stem cells that could be used for heart repair, although cells could not be tracked using positive MR contrast.

Tissue engineered scaffolds were constructed to mimic the native extracellular matrix (ECM) and promote cell migration of keratinocytes and fibroblasts. Electrospinning technology was used to fabricate these nano-scale matrices that consist of varying compositions and fiber diameters. The purpose of this study was to examine how average fiber diameter and scaffold composition regulate cell migration. Odyssey infrared scanning evaluated this on a macroscopic level, whereas confocal microscopy focused on a more microscopic approach. The expression of proteases released into the culture media was also examined. The results from this study suggest that fiber diameter increases as a function of electrospinning starting concentration. Altering the composition by adding a basement membrane-like material, Matrigel, does not statistically affect the average fiber diameter. Fibroblast migration is greater on collagen scaffolds than gelatin scaffolds based on surface area measurements. Confocal images illustrate a distinct cell polarity and various cell morphologies of fibroblasts on electrospun collagen scaffolds. Cell-matrix interactions are more prominent on intermediate to large scale fibers. However, cell-cell contacts are more prevalent at the smallest fiber diameters, suggesting that this scaffold acts like or as a two-dimensional surface. The expression of matrix metalloproteases (MMPs), specifically MMP-2 and MMP-9, by fibroblasts during in vivo cell migration assays, suggests that the greatest amount of matrix remodeling is at the two extremes of fiber diameters.