Heparan sulfate (HS) is a polysaccharide of glycosaminoglycan type composed of alternating hexuronic acid [either glucuronic acid (GlcA) or iduronic acid (IdoA)] and glucosamine (GlcN) units that can be sulfated in various positions. HS binds to a large number of proteins and these interactions promote many biological processes, including cell adhesion and growth factor signaling. This thesis deals with the structural analysis of short heparan sulfate sequences that mediate binding to fibroblast growth factors FGF1 and FGF2, their receptor FGFR4, and the angiogenesis inhibitor endostatin. Both FGF1 and FGF2 were shown to interact with N-sulfated hexa- and octasaccharide fragments isolated from HS. A pool of HS fragments depleted for FGF1 binding retained the ability to bind FGF2. Changes in 6-O sulfation affected binding to FGF1 but not FGF2, indicating that these proteins bind to distinct HS sequences. All octasaccharides with high affinity for FGF1 contained an internal IdoA2S-GlcNS6S-IdoA2S trisaccharide motif as shown by exoenzyme-based sequence analysis. FGF2 bound to a mono-O-sulfated hexasaccharide with an internal IdoA2S unit, although the affinity was higher for a di-O-sulfated octasaccharide displaying an IdoA2S-GlcNS-IdoA2S trisaccharide motif. FGFR4 was shown to bind the HS analogue heparin with a KD value of 0.3 μM. The interaction between FGFR4 and HS depends on both IdoA2S and GlcNS6S units. Sequence analysis suggested that the number but not the precise location of 6-O-sulfate groups determines affinity. The HS-binding site of endostatin was identified through alanine scanning. Endostatin mutants with reduced affinity for HS were unable to counteract angiogenesis induced by FGF2. The predominant HS motif recognized by endostatin was shown to consist of two N-sulfated domains separated by N-acetylglucosamine units.

In the healing of cutaneous wounds, paracrine communication between keratinocytes and fibroblasts regulates cell differentiation, proliferation and synthesis of extracellular matrix. Deficient epidermal coverage, as seen in burn-wounds, frequently results in hypertrophic scars. Previous studies suggest that keratinocytes downregulate the production of collagen and profibrotic factors in fibroblasts. We hypothesized that keratinocytes downregulate the expression of the profibrotic factor connective tissue growth factor (CTGF) in fibroblasts, and regulate fibroblast expression of genes important to wound healing. In keratinocyte-fibroblast cocultures, keratinocytes downregulated CTGF mRNA and protein in fibroblasts, through the secretion of interleukin-1 (IL-1) α. Using Affymetrix DNA microarrays, it was demonstrated that factors from keratinocytes regulate the expression of 69 genes important to wound healing. The regulation of 16 of these genes was confirmed by Northern blotting, and IL-1α from keratinocytes regulated all the 16 genes examined. IL-1-mediated CTGF gene regulation was further investigated. Both IL-1 isoforms, α and β, suppressed CTGF expression through an inhibition of CTGF promoter activity. Interestingly, transforming growth factor-β-stimulated Smad phosphorylation was not affected by IL-1. Finally, we hypothesized that CTGF is downregulated in burn wound by split-thickness skin grafting and that the expression of CTGF is suppressed during reepithelialization. The expression of CTGF protein was decreased in successfully skin-grafted wound areas, and increased in open, granulating burn wounds. Moreover, CTGF protein expression was absent beneath the migrating edge of reepithelialization ex vivo. In conclusion, we demonstrate that, in in vitro models, keratinocyte-derived IL-1α regulates the expression of CTGF and other genes with importance to wound healing. Furthermore, it is shown that CTGF expression is suppressed by epidermal wound coverage i burn wounds. These findings may have implications for the understanding of keratinocyte-fibroblast interplay during wound healing and in hypertrophic scar pathogenesis.

Heparan sulfate (HS) is an unbranched and negatively charged polysaccharide of the glycosaminoglycan family, based on the repeated (GlcNAcα1-4GlcAβ1-4) disaccharide structure. The HS backbone is modified by epimerization and sulfation in various positions. HS chains are composed of N-sulfated (NS) domains – predominant locations for further modification steps –, the poorly modified N-acetylated (NA) domains and the alternating NA/NS-domains. HS is present at the cell surface and in the extra-cellular matrix and interacts at these sites with various proteins involved in numerous biological processes, such as angiogenesis. Both pro- and anti-angiogenic proteins can interact with HS and this study was focused on how HS binds to the anti-angiogenic proteins endostatin (ES) and histidine-rich glycoprotein (HRGP) and to pro-angiogenic fibroblast growth factors (FGFs). Here we show that ES recognizes NS-domains in HS spaced by NA-disaccharides, and that binding to ES is abolish through cleavage at these NA-disaccharides. HRGP335, a peptide derived from the His/Pro-rich domain of HRGP is shown to bind to heparin and HS to the same extent as full-size HRGP, in a Zn2+-dependent manner. Moreover, the ability of HRGP to inhibit endothelial cell migration is located to the same region of the protein. We analyzed HS structure in respect to binding to HRGP335 and FGF-2, and show that the ability of HS to bind to those proteins depends on chain length and composition. Finally, the role of HS in FGF–HS–FGF receptor ternary complexes is evaluated using biosynthetic analogs of NS-domains. For stabilization of such complexes the overall sulfation degree of HS seems to play a more pronounced role than the exact distribution of sulfate groups. The results presented in this thesis contribute to a greater understanding of the role of HS in angiogenesis and may provide valuable information for the development of cures against angiogenesis-related disorders.

Pericytes are cells of mesenchymal origin located on the abluminal side, juxtapositioned to the endothelial cells in capillaries, venules and small arterioles. They are important for maintaining vessel integrity in resting tissues as well as the formation and stabilization of new vessels. They have been suggested to function as mesenchymal stem cells thereby contributing to the connective tissue cell population in reactive tissues. In this thesis the role of pericytes as progenitors for fibroblasts was further defined both in vitro and in vivo. In the first study connective tissue cells of mesenchymal origin were investigated based on their marker expression and relation to the microvasculature. The expression of alpha smooth muscle actin (α-SMA), a marker for myofibroblasts, was compared to the expression of certain integrins in three reactive conditions in human tissues. There was a co-localization of α-SMA and α1β1 integrins, indicating that α1 integrin was important for acquiring the α-SMA myofibroblast phenotype. To further investigate this, two animal models for carcinoma growth and wound healing using α1 deficient mice were employed. Reduction/lack of α-SMA expressing myofibroblasts substantiated or findings in human tissues, strengthening the hypothesis that the α1 integrin is important for the differentiation of α-SMA expressing myofibroblasts. In study two the effects of the HDAC inhibitor valproic acid (VPA) on pericyte function in vitro was investigated. This revealed that VPA had an inhibitory effect on pericyte proliferation, migration and differentiation into collagen type I producing fibroblasts. In addition qPCR array studies on angiogenesis related gene expression identified an up-regulation of genes involved in vessel stabilization in VPA treated pericytes. This suggests that VPA promotes a pericyte phenotype favoring vessel stability. In study three the differentiation from early mesenchymal stem cell like pericyte to fully differentiated fibroblast was further defined by flow cytometry marker analysis. By isolating pericytes from human placenta with a phenotype resembling the in vivo phenotype the differentiation pathway could be defined in five consecutive steps. The five steps were defined by their marker expression and their ability to give rise to the other cell populations in the differentiation lineage, as well as their slow cycling characteristics. A better understanding of how connective tissue cells are derived in fibrotic conditions may be beneficial in trying to modulate the outcome of the healing process towards optimal tissue regeneration with minimal fibrosis.

Augmented Soft Tissue Mobilization, a massage therapy which uses a solid instrument rather than human fingers to treat musculoskeletal injuries, has been successful in treating tendinitis. Davidson et al. studied the functional and morphological affects of ASTM on collagenase induced Achilles tendinitis in Sprague-Dawley rats. Morphological observations showed a significant increase in the number and activation of fibroblasts in the ASTM treated Groups. The authors suggested that the physical force of ASTM may promote tendon healing via increased fibroblast recruitment. An important, but unexplained, question is how ASTM would affect the fibroblasts of native, noncollagenase injured, tendons. Studies have shown that mechanical forces can alter cellular functions. The purpose of this study was to examine the morphological changes in native Sprague-Dawley rat Achilles tendons after ASTM therapy using different application pressures.Three animal Groups were randomly established: A) control Group with no ASTM; B) light ASTM with 1 Newton of pressure; and C) heavy ASTM with 3 Newtons of pressure. Upon completion of the therapy, the Achilles tendons of each Group were examined with light and electron microscopy techniques to assess fibroblast number, tendon morphology, and the presence of type I and type III collagen. Fibroblast counts from each Group were compared using a two-way ANOVA, multiple regression, and curvilinear regression analysis. Morphological differences were shown between the three Groups, especially between the non force Group and the two force Groups. The ASTM Group treated with one Newton demonstrated the greatest mean fibroblast count (165.1+/-55.8&160.7+/-49.8). Electron microscopy revealed the presence of activated fibroblasts in the tendons of the two force Groups, ASTM Groups. Polarizing microscopy showed a dramatic increase in the amount of Type III collagen in the two force Groups compared to the non force Group.Ball State UniversityMuncie, IN 47306

Heparin binding (HB) proteins mediates a wide range of important cellular processes, which makes this class of proteins biopharmaceutically important. Engineering HB proteins could bring many advantages, but it necessitates cost effective and efficient purification methodologies compared to the currently available methods. One of the most important classes of heparin binding protein is the fibroblast growth factors (FGFs) and its receptors (FGFRs). In this study, we report an efficient off-column purification of FGF-1 from soluble fractions and purification of the D2 domain of FGFR from insoluble inclusion bodies, using a weak amberlite cation (IRC) exchanger. This approach is an alternative to conventional affinity column chromatography, which exhibit several disadvantages, including time-consuming experimental procedures and regeneration and results in high cost for production of recombinant proteins. Authenticity of the purified proteins was verified by SDS-PAGE and MALDI mass spectrum analysis. Results of the heparin binding chromatography and steady state fluorescence experiments showed that the FGF-1 and the D2 are in a native biologically active conformation. The findings of this study will not only aid an in-depth investigation of this class of proteins but will also provide avenues for inexpensive and efficient purification of other important biological macromolecules.

FGFR4 and β-Klotho in Metastatic Prostate Cancer by Derek LaMar Shenefelt Fibroblast growth factors and fibroblast growth factor receptors have been associated with the aggressiveness and progression of Prostate Cancer (PCa). Also, β-Klotho is a known co-receptor with FGFR4 for FGF19 in the liver however, the role of this co-receptor pair remains unclear in the setting of PCa. I demonstrated that FGFR4 and KLB mRNA and protein are highly expressed in PCa cells when compared to bone marrow stromal cells, a common site of metastasis. I also provide support for the association of FGFR4 and KLB in PCa, suggesting a functional co-receptor pair capable of altering cellular signaling. FGFR4-KLb may also provide some level of protection to PCa cells from chemotherapeutics. This analysis of FGFR4 and KLB expression and signaling in PCa has provided novel insights into phenotypic alterations during PCa progression while also providing new avenues of study to further explore the role and importance of this exciting co-receptor complex.

The cell surface is an active area where extracellular molecules meet their receptors and affect the cellular fate by inducing for example cell proliferation and adhesion. Syndecans and integrins are two transmembrane molecules that have been suggested to fine-tune these activities, possibly in cooperation. Syndecans are proteoglycans, i.e. proteins with specific types of carbohydrate chains attached. These chains are glycosaminoglycans and either heparan sulfate (HS) or chondroitin sulfate (CS). Syndecans are known to influence cell adhesion and signaling. Integrins in turn, are important adhesion molecules that connect the extracellular matrix with the cytoskeleton, and hence can regulate cell motility. In an attempt to study how the two types of glycosaminoglycans attached to syndecan-1 can interact with integrins, a cell based model system was used and functional motility assays were performed. The results showed that HS, but not CS, on the cell surface was capable of regulating integrin-mediated cell motility. Regulation of intracellular signaling is crucial to prevent abnormal cellular behavior. In the second part of this thesis, the aim was to see how the presentation of glycosaminoglycan chains to the FGF signaling complex could affect the cellular response. When attached to the plasma membrane via syndecan-1, CS chains could support the intracellular signaling, although not promoting as strong signals as HS. When glycosaminoglycans were attached to free ectodomains of syndecan-1, both types of chains sequestered FGF2 from the receptors to the same extent, pointing towards functional overlap between CS and HS. To further study the interplay between HS and CS, their roles in the formation of pharyngeal cartilage in zebrafish were established. HS was important during chondrocyte intercalation and CS in the formation of the surrounding extracellular matrix. Further, the balance between the biosynthetic enzymes determined the ratio of HS and CS, and HS biosynthesis was prioritized over CS biosynthesis. The results presented in this thesis provide further insight into the regulation of HS biosynthesis, as well as the roles of both HS and CS on the cell surface. It is evident, that in certain situations there is a strict requirement for a certain HS structure, albeit in other situations there is a functional overlap between HS and CS.

<p>Development requires a delicate balance of proliferation and differentiation. Too little proliferation can result in dysfunctional tissues, while prolonged or heightened proliferation can result in tumor formation. This is clearly seen with the granule neuron precursors (GNPs) of the cerebellum. Too little proliferation of these cells during development results in ataxia, whereas too much proliferation results in the cerebellar tumor medulloblastoma. While these cells are known to proliferate in response to Shh, it is not clear what controls the differentiation of these cells in vivo.</p><p> Previous work from our lab has identified basic fibroblast growth factor (bFGF) as a candidate differentiation factor for these cells. In this thesis, I characterize some of the cellular and molecular mechanisms involved in FGF-mediated inhibition (FMI) of Shh-induced GNP proliferation. In addition, I employ FGFR knockouts and a bFGF gain-of-function mouse to determine whether FGF signaling is necessary and/or sufficient for differentiation of GNPs during cerebellar development. Finally, the question of whether bFGF can be effective as a therapeutic agent for in vivo tumor treatment is tested in a transplant model.</p><p> These experiments indicate that FGF signaling is neither necessary nor sufficient for GNP differentiation during cerebellar development. However, transplanted tumors are potently inhibited by bFGF treatment. Furthermore, FMI is shown to occur around the level of Gli2 processing in the Shh pathway, implying that such a treatment has promise to be widely effective in treatment of Shh-dependent medulloblastomas.</p> / Dissertation

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