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Myostatin is a secreted growth factor of the transforming growth-factor $\beta$ (TGF$\beta$) superfamily, and a powerful negative regulator of muscle mass in vertebrates. As such, there is considerable interest in developing pharmacological agents which inhibit myostatin signalling in order to stimulate muscle growth in the context of pathological muscle wasting. Like other TGF$\beta$ family proteins, myostatin is biosynthesised as an inactive (latent) precursor protein which requires proteolytic processing to liberate the mature bioactive growth factor. To examine the molecular basis of pro-myostatin latency and the mechanism by which it is activated in the extracellular space, I have determined the crystal structure of unprocessed human pro-myostatin and studied the properties of the protein at various stages of activation. Crystallographic analysis of pro-myostatin reveals a unique domain-swapped dimeric structure, with an open V-shaped conformation distinct from the prototypical family member, TGF$\beta$1. Following cleavage of the prodomains by furin, pro-myostatin persists as a stable non-covalent complex which is resistant to the natural inhibitor follistatin and exhibits significantly weaker bioactivity than the mature growth factor. A number of distinct structural features combine to stabilise the interaction between pro and mature domains and in doing so confer latency to the pro-complex. This facilitates a controlled, step-wise process of activation in the extracellular space and contributes to a complex network of regulatory control. The results presented here provide a structural basis for understanding the effect of natural polymorphisms on myostatin function and a starting point for structure-guided development of next generation myostatin inhibitors. As a proof-of-concept, I present preliminary data on prodomain derived stapled peptides as inhibitors of myostatin signalling.

Introduction: Prostate Cancer (PC) is the most common cancer in men and the second biggest cause of cancer-related death of men in the Western world. There are significant ethnic differences in the incidence of PC between African Americans, Caucasians and Asians. At the present time, the only method used for PC screening is Prostate Specific Antigen (PSA). Prostate is an androgen regulated gland and it can also affect the concentration of growth hormone by activation of androgen receptor. The aim of this research was to investigate ethnic group-specific relationship between PC incidence, concentration of circulating hormones and polymorphisms in genes that are involved in regulation of androgen metabolism. Specific objectives were: (i) to determine whether circulating concentration of androgens testosterone, dihydrotestosterone (DHT) and a growth hormone insulin-like growth factor-I (IGF-I) differ between Bristol Caucasian (BC) and Malaysian Chinese (MC) men; (ii) to investigate whether variations in concentration of circulating androgens in control and PC cohorts are ethnic group-specific; (iii) to determine whether frequency of DNA polymorphisms in CYP17, SRD5A2 and androgen receptor (AR) genes are ethnic group-specific; (iv) to investigate the relationship between genetic polymorphisms, concentration of circulating androgens and incidence of PC in BC and MC groups. Material and Method: Blood sample were obtained from newly diagnosed PC patients from Caucasian and MC ethnic groups whose diagnosis was confirmed by biopsy. Control group consisted of patients with confirmed Benign Prostatic Hyperplasia with low risk of PC. The studied populations consisted of 50 Caucasian cancer cases, 50 Caucasian controls, 50 MC cancer cases and 49 MC control participants. Analysis of testosterone, DHT and IGF-I concentrations was performed on serum using ELISA kits. DNA polymorphisms were determined using genomic DNA isolated from whole blood (by PCR amplification) and follow-on DNA sequencing. The obtained DNA sequences were aligned with appropriate DNA sequences of the reference genes. The software "Geneious" was used to identify DNA SNPs and amino acid substitutions in translated protein. Statistical analysis was performed using SPSS version 20.0. Results: This study established ethnic group-specific differences in circulating androgens between control and PC groups. In particular, testosterone concentration was significantly lower, and DHT/T was significantly higher in the MC cancer patients when compared to Caucasians. IGF-I concentration was significantly higher in the Caucasian cancer and controls than the MC sub-groups. A1/A2 and A2 + A1/A2 alleles of CYP17 gene were associated with a higher risk of PC in both ethnic groups. LL and L/V genotypes of SRD5A2 were prevalent in MC cancer and control group when compared to Bristol Caucasian PC and controls. These genotypes, when present, were associated with increased risk of PC in Caucasians but not in MC group. No relationship was found between a number of CAG-repeats in AR gene and PC in Caucasian or MC populations. No significant relationship was found between SNPs in CYP17, SRD5A2 and AR genes and circulating concentration of androgens and IGF-I in Caucasian and MC populations. The only exception was a positive association between the SRD5A2 polymorphism in the position 261C→G and IGF-I concentration in Caucasian control group. Conclusions: This study demonstrated positive relationship between the concentration of circulating androgens, namely testosterone, IGF-I growth factor and the severity of the PC in Bristol Caucasians and MC groups. It also established ethnic-group specific polymorphisms in CYP17 and SRD5A2 genes, but not in the AR gene. It was suggested that the CYP17 and SRD5A2 polymorphisms might contribute to the risk of PC development in Caucasians and MC. Further evaluation of the results on a larger cohort and a wider range of ethnic groups are required in order to make definite conclusions about CYP17, SRD5A2 as ethnic-group specific biomarkers for PC.

Retinitis pigmentosa (RP) is a genetically and phenotypically heterogeneous condition that affects approximately 1 in 4000 individuals worldwide. The most common presentation of RP is a rod-cone dystrophy, where the degeneration of cone photoreceptors occurs secondary to advanced rod loss, leading to a significant decline in central vision and a corresponding reduction in patient quality of life. The mechanisms underlying secondary cone loss are poorly understood, particularly in disorders where the gene defect is unknown or manifest only in rod photoreceptors. Consequently, the thesis presented herein proceeds on several fronts. First, in the long term a greater understanding of the causes underlying cone loss in RP is likely to be beneficial, and so in chapter one a dominant cone degeneration is characterized using intrinsically fluorescent cone photoreceptors to track the degenerative process. Second, as we develop a greater understanding of the genetic etiology underlying RP it is likely that the number of large genes identified as being causative will increase. As currently there is no efficient way to deliver large genes to photoreceptors, chapter two explores the use of alternate viral vectors that might be used to deliver a large therapeutic transgene. Lastly, whilst our understanding of cone loss in RP remains incomplete, it is necessary to develop a broadly applicable therapy to slow or attenuate further cone loss in RP patients regardless of the underlying cause. In chapters three and four we examine the use of low molecular weight "growth factors‟, such as ciliary neurotrophic factor (CNTF), to preserve cone photoreceptors long-term using a rhodopsin knockout model of RP.

One of the most important roles for growth factors such as insulin and IGF-1 in certain cells is the promotion of cell survival and protection against apoptosis. Growth factors achieve this antiapoptotic signalling via complex intracellular signalling pathways mediated by protein kinases and phosphatases. A recently cloned protein kinase, protein kinase B (PKB) has been shown to be a key component of growth factor signalling pathways and in particular has been implicated as a crucial mediator in antiapoptoic signalling. In this study I report the cloning of PKBy, the third and least studied of the PKB isoforms, from rat and mouse brain tissue and production of two different constitutively active mutants of PKBγ, the first a membrane targeted mutant and the second a phosphorylation site active mutant. These mutants were then used to stabily transfect Madin-Darby Canine embryonic Kidney (MDCK) cells in order to investigate the role of PKBγ in antiapoptotic signalling. Using these stable cell lines it was established that GSK-3 is a downstream target of PKBγ and expression of the constitutively active PKBγ mutants was found to protect the stable cells against apoptosis induced by growth factor withdrawal. In addition, a dominant negative rat PKBγ was cloned and found to induce apoptosis when transfected into wild-type MDCK cells, even in the presence of serum. Taken together these results suggest that PKBγ has a crucial role to play in growth factor stimulated cell survival signalling. This research may have important implications for research into cancer, diabetes and neurodegenerative disease since PKBγ has been found to be upregulated in breast cancer and prostate cancer cell lines and downregulation of PKB may be a central cause of insulin resistance and Alzheimers disease.

FGF2 is expressed in renal podocytes as they differentiate in-vivo, as demonstrated by specific antibody staining within developing glomeruli of chicken metanephros. Mice lacking FGF2 have been generated in the laboratory by targeted deletion and kidneys of Fgf2 deficient mice appear to develop normally. Detailed histological analysis of adult kidneys from these mice however has revealed low frequency glomerular abnormalities. Additionally, a few obvious cases of glomerulosclerosis with severe podocyte damage were observed specifically in mutant mice. Taken together these observations indicate FGF2 plays a role in podocyte development and/or function. A novel culture system that allows the induction of podocyte cell differentiation in-vivo has recently been developed. The system is based on isolation of conditionally immortalised podocyte cells derived from H-2K^btsA58 transgenic (immorto) mice. Isolation of podocyte cells from renal glomeruli of wild-type and FGF2 deficient immorto mice was performed to address the functional relevance of FGF2 in podocyte development. Conditionally immortalised wild-type podocyte cells (wild-type MPCs) display characteristic features of podocytes in-vivo, however Fgf2 null MPCs show striking morphological and molecular abnormalities. Mutant podocyte cells do not undergo the epithelial-to-mesenchymal transformation (EMT) associated with normal podocyte maturation and, correspondingly, fail to differentiate. The EMT mediator Slug is up-regulated as wild-type cells differentiate but is missing in mutant cells, suggesting this transcription factor acts downstream of FGF signalling to mediate EMT associate maturation of podocytes. Fgf7 and Fgf10 expressions are lost in Fgf2 deficient MPC cells, but not in Fgf2 deficient kidney cortex, affording an explanation to the emergence of a stronger defect in-vitro.

The Masquelet technique is a two-staged procedure that uses an induced biological membrane and bone graft to reconstruct critical-sized bone defects. However, unpredictable clinical outcomes result due to the variable durability and the transient vascular network of the induced membrane, as well as high incidences of osteomyelitis. To this end, we have engineered a resorbable multifunctional electrospun wrap that guides formation of the induced membrane with improved durability and enhanced angiogenesis while simultaneously preventing infection. We achieve this by developing and combining an antimicrobial poly(lactic-co-glycolic) acid (PLGA) mesh and an angiogenic crosslinked gelatin mesh. We first confirmed the ability of electrospun PLGA to provide sustained release of gentamicin sulfate or gallium maltolate above its minimum inhibitory concentration (MIC). Studies that evaluated antimicrobial activity indicated that osteomyelitis-derived bacteria was not susceptible to released gallium maltolate at the hypothesized MIC and further established the accurate gallium maltolate MIC. The inhibitory concentration of each antimicrobial on osteoblasts was compared to the respective MIC to determine if they were safe and effective at released concentrations. Results concluded that the gentamicin sulfate-loaded PLGA mesh is safer and more effective mesh. Next, the bioactivity retention of vascular endothelial growth factor (VEGF) released from electrospun photo-crosslinked gelatin-methacrylate was confirmed. Subcutaneous implantation of the VEGF-loaded mesh in a rat corroborated resorption and the capacity for sustained release. A multifunctional electrospun wrap was then engineered to prevent osteomyelitis and guide formation of the induced membrane by combining the antimicrobial and angiogenic platforms with co-electrospinning. The combination of the two fiber populations was confirmed microscopically and offered independently tuned bimodal release of gentamicin sulfate and VEGF. Overall, this work provides the fundamentals to advance the development of a multifunctional electrospun wrap that can guide formation of the induced membrane and prevent osteomyelitis for improved clinical outcomes with the Masquelet technique. This work offers a substrate that can recruit and support cellular adhesion, provide a template for matrix deposition and tissue remodeling, and enable bimodal release of bioactive agents. These studies also enhance the capacity of electrospun platforms to serve as stand-alone therapies or combinatorial therapies in various bone regeneration applications.

Abstract: Osteoarthritis (OA) is a disease affecting >10% of Canadians and is associated with a huge economic burden on the Canadian economy and healthcare system. The disease is characterized by a variety of symptoms including chronic pain, inflammation, and decreased joint mobility. Therefore, the development of effective treatments is being investigated. Within the knee joint, the state of meniscus health is a direct correlate of healthy joint physiology. Meniscal trauma is frequently corrected through surgical debridement in the form of partial meniscectomies; however this treatment predisposes the patient for OA development due to increased stress on surrounding articular cartilage.Meniscus tissue engineering aims to develop new repair or replacement strategies to maintain proper joint physiology in cases of meniscal injury. Because the menisci’s functional properties are derived from its extracellular matrix (ECM), tissue engineering focuses on creating biomimetic constructs that maintain biomechanical functionality by promoting meniscus-like ECM formation. This may be achieved by combining candidate cell types, experimental models, and growth factor supplementation. Fibroblast growth factors have demonstrated potent anabolic effects in a number of cell types, however they are relatively understudied in cells isolated from healthy, adult meniscal tissue.Here we examined the effects of supplementing fibroblast growth factor 18 on differentiated, primary MFCs and expanded MFCs to assess the capacity for FGF-18 to drive the redifferentiation of MFCs to produce meniscus-like ECM formation. Our findings suggest that FGF-18 is insufficient to restore a differentiated ECM-forming phenotype in expanded MFCs.

Tendon injuries significantly impact quality of life and are often career ending. Mesenchymal stem cell (MSC) therapy is known to augment intrinsic tendon healing, however, little is known of the stem cells endogenous to tendon, the microenvironmental cues that induce tendon differentiation, and whether individual cells in an inflammatory milieu respond differently to these cues. To address these questions, a three-dimensional tenogenesis assay was developed as an efficient and reproducible metric of cellular capacity to differentiate toward tendon. In contrast to more complex assays of tenogenesis, this design incorporates a simple apparatus using commercially available plasticware for the application of uniaxial static strain in in a type I collagen cell-seeded hydrogel construct. Tendon-related gene expression, glycosaminoglycan levels, elongated cell morphologies and parallel cell alignments are enhanced with BMP-12 induction over ten days of culture. This dissertation provides novel insight to the roles of growth factors in MSC tenogenesis. Tendon healing in vivo is dependent on endogenous tendon stem cells (TSC) that mediate the inflammatory response to injury and promote synthesis of collagen and matrix remodeling, among other extracellular processes. Recent evidence suggests that these cells exist on a spectrum of differentiation potencies, and may be differently committed to the tendon fate. Individual stem cells were isolated from the tendon, and their capacities for proliferation, tri-lineage differentiation and tenogenesis were evaluated. Three distinct TSC phenotypes were revealed, and significant, positive correlations were found in quadra-differentiation potency (toward four lineages) and the expression of a strong, composite tendon phenotype. These studies have important implications in the current standard-of-care in regenerative therapies for tendon. Our benchtop tenogenesis assay can be used to determine the therapeutic potential of allogeneic MSC lines and MSCs from novel sources for 'off-the-shelf' treatments. Our study of TSCs lends valuable insight to the diversity of cell phenotypes found in tendon, and the potential contributions of each phenotype to tendon healing and homeostasis. These results further strengthen the status of tendon as a superior source of stem cells for tendon repair.

Human acidic fibroblast growth factor (hFGF1) is a protein well known for its role in cell growth and differentiation. To elicit these cell-signaling processes, hFGF1 non-selectively binds to any one of the seven cell surface hFGF receptor isoforms. Due to its significant involvement in tissue repair activity, hFGF1 is a prime candidate for novel wound healing therapeutics. However, one drawback toward its use as a novel wound healing therapeutic is the poor inherent thermal stability of hFGF1, as it has been found to unfold near physiological temperature. The cause of this instability is strong electrostatic repulsion created by a dense cluster of positively charged amino acids near the c-terminus. This instability leads to proteolytic degradation of the unfolded protein, which severely limits the bioavailability of hFGF1. To counteract this instability, hFGF1 binds with high affinity to the heavily sulfated glycosaminoglycan, heparin, which eliminates the charge-charge repulsion via electrostatic interactions with the positively charged residues near the c-terminus in the region known as the heparin-binding pocket. However, recently several disadvantages have been acknowledged with the use of heparin in hFGF1 wound-healing therapeutics. Thus, to address these issues, we have genetically engineered several rationally designed point mutations within and near by the heparin-binding region of hFGF1 to modulate the heparin-binding affinity and to increase the thermal stability and cell proliferation activity of the protein. Study of each mutation is performed with biophysical experiments as well as molecular dynamics simulations (which are found as supplementary files).

The morphology and biochemical phenotype of cells are closely linked. This relationship is important in progenitor cell bioengineering, which generates functional, tissue-specific cells from uncommitted precursors. Advances in biofabrication have demonstrated that cell shape can regulate cell behavior and alter phenotype-specific functions. Establishing accessible and rigorous techniques for quantifying cell shape will therefore facilitate assessment of cellular responses to environmental stimuli, and will enable more comprehensive understanding of developmental, pathological, and regenerative processes. For progenitor cells being induced into specific lineages, this ability becomes a pertinent means for validating their degree of differentiation and may lead to novel strategies for controlling cell phenotype. In our approach, we used the differentiation of adult human mesenchymal stem cells (MSCs) into smooth muscle cells (SMCs) as a model system to investigate the relationship between cell shape and phenotype. These cell types are responsive to mechanical and biochemical stimuli and the shape of SMCs is a recognized marker of differentiated state, providing a system in which morphological and biochemical phenotype are both understood and inducible. By applying exogenous stimuli, we changed cell shape and examined the corresponding cellular phenotype. In the first Aim, we applied stretch to MSCs on 2D collagen sheets to promote differentiation. Using mathematical shape factors, we quantified the morphological changes in response to defined stretch parameters. In the second Aim, we investigated the use of input energy as a means of controlling cell shape and corresponding differentiation. We examined how combinations of stretch parameters that produce equal energy input impacted morphology, and postulated that cell shape is a function of energy input. In the third Aim, we translated our method of quantifying shape factors into 3D culture, and validated the method by investigating the differentiation of MSCs into SMCs by mechanical and growth factor stimulation. We used the shape factors to quantify morphological differences and compared these changes to biochemical markers. Our results demonstrate that mechanical stretch influences multiple aspects of MSC phenotype, including cell morphology. Shape factors described these changes objectively and quantitatively, and enabled the identification of relationships between SMC shape and differentiated state. Similar morphological responses could be induced using different combinations of stretch parameters that resulted in equal energy input. Cell shape followed a linear relationship with energy input despite the variance introduced by using MSCs from different patients. Only one SMC gene marker directly exhibited this relationship; however, partial least squares regression analysis revealed that other genes were also associated with shape factors. Translation of the shape quantification method into 3D systems revealed that while the additional dimensionality hindered comparison of morphology between 2D and 3D samples, these shape factors were still applicable within 3D systems. Differences in cell morphology caused by growth factors and mechanical stretch in 3D constructs were elucidated by shape analysis, and these phenotypic changes were corroborated through biochemical assays. Taken together, these results validate the use of cell shape as means of characterizing phenotype and the process of progenitor cell differentiation. The automated method we developed generates a robust set of morphological parameters that provide a way to characterize the differentiation of MSCs into SMCs. This work has implications in our understanding of the relationship between cell morphology and phenotype, and may lead to new ways to control and improve differentiation efficiency in a variety of cell and tissue systems.

Growth factors are extracellular signals that regulate cell proliferation and total body mass. Some animal growth factors can work on plant tissues and vice versa. Here we show that the mammalian growth factor adrenomedullin (AM) induces growth in tobacco plants. Addition of synthetic AM resulted in a dose-dependent growth of tobacco calluses. Furthermore, AM transgenic plants showed enhanced survival and significant increases in stem diameter, plant height, leaf length, weight of all organs, and a reduction in the time to flowering when compared to plants transformed with the control vector. These differences were maintained when organs were dried, resulting in a mean total biomass increase of 21.3%. The levels of soluble sugars and proteins in the leaves were unchanged between genotypes. AM transgenic plants had a significantly higher expression of cyclin D3 and the transcription factor E2FB than controls, suggesting that cell cycle regulation may be part of the intracellular signaling of AM in plants. In summary, mammalian AM increases vascular plants' survival and biomass with no apparent detriment of plant's morphological and/or biochemical properties, thus this strategy could be useful for crop productivity improvement.

Peripheral arterial disease affects more than 27 million patients in the United States. PAD can lead to peripheral limb ischemia and result in non-healing foot ulcers. Currently, surgical therapies such a stenting, grafting, and bypass, exist for treatment of ischemia, but these treatments are prone to long-term failure. These treatments also only successfully yield beneficial results about 25% of the time. Alternatively, regenerative therapies that stimulate the growth of new vasculature have great potential for treating peripheral and myocardial ischemia. Growth factor based therapies that induce neovascularization have shown promising results in animal models, but have limited success in clinical trials. This discrepancy is most likely a result of growth factor resistance brought on by diseases that lead to peripheral vascular disease. Here, we have developed a new method for enhancing the activity of growth factors in growth factor resistant disease states such as diabetes and hyperlipidemia. Our novel method delivers the growth factor co-receptor glypican-1 embedded in a liposomal carrier to create a glypican-1 proteoliposome (a “glypisome”). By co-delivering the co-receptor glypican-1 along with the growth factor, we hope to overcome growth factor resistance associated with long-term disease. Here we optimize glypisome composition to maximize angiogenic response when co-delivered with growth factors, through the use of in vitro endothelial assays and explore what mechanisms bring about this change in activity. Then we determine therapeutic potential of co-delivering glypisomes with growth factors in vivo by assessing neovascularization in healthy and disease mouse models of ischemia. We also explore overcoming disease mediated growth factor resistance by delivering glioblastoma-derived exosomes as a naturally occurring alternative to the glypisomes that we have developed. We test these exosomes in both in vitro endothelial assays and in vivo with a mouse hind limb ischemia model. We demonstrate that delivering these co-receptors in conjunction with the growth factor will allow us to overcome the disease phenotype and lead to a viable growth factor therapy for peripheral arterial disease.

This work utilises models of normal and abnormal human hematopoiesis to improve the ex vivo expansion of cord blood hematopoietic stem and progenitor cells (HSPC) for therapeutic use and to increase our understanding of ERG in human leukemogenesis. The majority of experimental work in this study has been published in peer reviewed journals Cytotherapy and Leukemia. HSPC transplantation remains the most effective curative therapy for leukemia, and cord blood HSPC have considerable advantages compared to other sources, however their use is limited by the low number of cells harvested. Therefore, improvements to the ex vivo expansion of cord blood HSPC have high clinical relevance. This study addresses both the limited investigation of the combinatorial effects of growth factors and oxygen level, and the difficulty in translating laboratory research into clinical practice. Systematic investigation using phenotypic markers, colony forming assays and gene expression identified enhanced HPC ex vivo expansion with growth factor combination TSFI in 10% oxygen. Additionally, through use of clinically approvable methods and reagents, this study established a baseline methodology for the assessment of additional factors to increase cord blood transplantable cell dose. Ets transcription factor ERG is a member of self-sustaining transcriptional networks that maintain human and murine HSPC, and ERG is a prime human leukemogenic candidate. High ERG expression is prognostic of poor patient outcome in acute myeloid leukemia (AML) and Tcell lymphoblastic leukemia (T-ALL), and high ERG expression induces both AML and T-ALL in murine models. However, no human model of high ERG expression in normal HSPC exists. This study established a human model of high ERG expression in cord blood CD34+ HSPC, which allowed investigation of the effect of high ERG expression in primitive HSPC and lineage committed progenitors. High ERG expression induced a gene expression signature that recapitulated hematopoietic stem cells and signatures of both AML and T-ALL. This signature was associated with enhanced expansion of functional progenitors, which potentially provide the opportunity for the acquisition and propagation of mutations that may lead to the development of leukemia. As such, high ERG may mark a pre-disposition to development of high risk leukemia.

Several uterine-derived paracrine factors have been implicated as critical regulators of conceptus development in cattle, but it remains unclear how these factors work together to establish and maintain pregnancies. The primary objectives of this work were to establish if cooperative interactions between epidermal growth factor (EGF), fibroblast growth factor-2 (FGF2) and insulin-like growth factor-1 (IGF1) promote bovine trophoblast cell proliferation, and to decipher the intracellular signaling mechanisms employed by these growth factors to regulate cell proliferation. Pilot studies established effective concentrations for each growth factor on a bovine trophoblast cell line (CT1). The first set of studies examined how each factor worked individually or in conjunction with each other to impact CT1 proliferation. Mitotic index (percentage of EdU-positive nuclei after a 45 min challenge) was increased (P

The field of orthopaedic tissue engineering includes a broad range of treatment options that seek to regenerate or accelerate the repair of bone tissue. Formation of new bone by the activity of connective tissue progenitor cells (CTPs) is a central feature of each of these treatment options. CTPs are defined as the heterogeneous population of stem and progenitor cells that are resident in native tissues and are capable of proliferation to give rise to progeny that will differentiate to express one or more connective tissue phenotypes. The strategy of using a cell-based therapy to achieve bone regeneration may include: targeting CTPs, and guiding their downstream progression towards the osteogenic pathway using a combination of scaffolds, bioactive factors, and mechanical and biophysical methods. Recent advancements in biomaterials science include promising technologies for modifying biomaterial surfaces with pro-survival or pro-osteogenic bioactive ligands. Before testing the pre-clinical efficacy of these surface modified biomaterials in animal models, it is important to test and optimize the influence of these materials on cells in an in vitro setting. The purpose of this dissertation is to introduce and demonstrate a new method to assess the response of CTPs to surface-modified biomaterials using a standardized automated colony forming unit (CFU) assay. Individual components of the method are verified for their compliance with the technical specifications. The method is validated using ß-TriCalcium Phosphate (TCP), a bone scaffold material that was modified with Epidermal Growth Factor (EGF), a pro-survival and proliferation bioactive ligand, using a novel TCP-binding peptide. The amount of tethered EGF on the TCP surface was quantified, and its stability was demonstrated. Surfaces modified with tethered EGF were shown to enhance the colony forming efficiency of CTPs obtained from human bone marrow and the trabecular surface of bone. These data support the potential of tethered EGF presentation on the surface of implantable TCP biomaterials as a means of enhancing the performance of local and transplanted CTPs in a setting of bone repair or other tissue engineering applications.

Grafts currently used for the repair of anterior cruciate ligament (ACL) ruptures integrate poorly with bone due to a significant mismatch in properties between graft and bone. Specifically, conventional grafts (e.g., hamstring tendon) are unable to recapitulate intricate gradients in mechano-chemical properties and extracellular matrix (ECM) architecture found at natural bone-ligament (B-L) transitions, and thus result in stress-concentrations at the graft-bone interface leading to graft failure. In contrast, tissue-engineered scaffolds possessing gradients in properties can potentially guide the establishment of phenotypic gradients in bone marrow stromal cells (BMSCs), and thus aid the regeneration of B-L transitions in the long-term. Towards the eventual goal of regenerating complex tissue transitions, this project employs three complementary strategies to fabricate graded scaffolds. The three strategies involve the presentation of gradients in 1) mineral content, 2) scaffold architecture and 3) growth factor (GF) concentration within scaffolds to control BMSC morphology and phenotype. The first strategy involved co-electrospinning two polymers (one doped with hydroxyapatite) from offset spinnerets onto a rotating drum to produce scaffolds possessing a gradient in mineral content. Post-electrospinning, these graded scaffolds were treated with a simulated body fluid to further enhance the gradient. Analysis of mRNA expression of osteoblastic makers by BMSCs and the deposition of bone-specific ECM proteins indicated that the scaffolds could guide the formation of an osteoblastic phenotypic gradient. The second strategy involved electrospinning two polymer solutions onto a custom-designed dual-drum collector to fabricate scaffolds possessing region-wise differences in fiber alignment, diameter and chemistry. Specifically, electrospinning onto the dual-drum collector resulted in the deposition of aligned fibers from one polymer solution in the gap region between the drums, randomly oriented fibers from the other polymer solution on one of the drums and a mixture of fibers from both polymer solutions in the overlap region in between. The topographical cues within these scaffolds were shown to result in region-dependent BMSC morphology and orientation. Although the long-term goal of the third strategy was to create a co-electrospun scaffold possessing a gradient in GF concentration, a new technique to protect GF activity within electrospun scaffolds via the use of gelatin microspheres was first validated. Preliminary results from these studies indicate that microspheres can protect and deliver a model protein (lysozyme) in active conformation from electrospun scaffolds. These results further suggest that gradients of GF concentration can be achieved in the long-term by protecting GFs within microspheres and co-electrospinning as described in the first strategy. In conclusion, the results from this project suggest that graded scaffolds can help guide the formation of gradients in cell morphology, orientation and phenotype, and thus potentially promote the regeneration of B-L transitions in the long-term. The three strategies described in this project can be employed in concert to create scaffolds intended for the regeneration of complex tissue transitions.

Introduction: The management of bone defects continues to present surgical challenges, and alternatives to autograft remain sought after. Upon activation, platelets release an array of growth factors which regulate and contribute to healing. Therefore, combining platelet rich plasma (PRP) with synthetic bone graft substitutes has the potential to reduce the reliance on autograft. Currently, the use of PRP is controversial. The progression of PRP is hindered by the absence of robust pre-clinical investigations, which has led to conflicting outcomes on its efficacy within the literature. This thesis investigates the potential of PRP for bone augmentation in an older age sheep model. Specifically, PRP dose is controlled to provide clearer indications for its clinical use. Methods: Clinical application of bovine thrombin is avoided, yet, many animal studies report outcomes with its use as a PRP activator. The first study investigates ovine PRP activation methods in vitro, and confirms a relevant alternative to bovine thrombin for use in the animal study. Such pre-clinical animal studies require appropriate characterisation of the model to ensure treatment outcomes are judged appropriately. Therefore, the second study develops and characterises an aged ovine multi-site bone defect model suitable for the aims of this thesis. The final study assembles the knowledge gained from the preceding chapters, and investigates the use of PRP at varying doses when combined with a biphasic calcium phosphate to treat critical size bone defects. Results: Autologous thrombin-activated PRP stimulated new bone formation within the bone defect with a dose dependant response. When the PRP was combined with a biphasic calcium phosphate, new bone formation within the defect was significantly greater than if the calcium phosphate was used on its own, demonstrating the benefit of PRP in a pre-clinical setting. Additionally, the role of other PRP constituents is revealed. In particular, the potential benefit of fibrinogen in the PRP. Overall, the PRP appeared to share chemotactic and mitogenic cell signalling functions similar to that of autograft at four weeks. However, the autograft displayed a more immediate healing response compared to PRP. Conclusion: PRP induces new bone formation at four weeks in aged sheep with a dose dependant response. Relevance: Clinical investigations of PRP are impeded by conflicting indications for its use. If a biphasic calcium phosphate is used to treat bone defects, the addition of an autologous thrombin-activated 3-fold PRP, mixed at a 1cc:1mL ratio, can improve the early bone defect healing response.

Cardiovascular diseases remain the first cause of morbidity and mortality in the developed countries and are a major problem not only in the western nations but also in developing countries. Current standard approaches for treating patients with ischemic heart disease include angioplasty or bypass surgery. However, a large number of patients cannot be treated using these procedures. Novel curative approaches under investigation include gene, cell, and protein therapy. This review focuses on potential growth factors for cardiac repair. The role of these growth factors in the angiogenic process and the therapeutic implications are reviewed. Issues including aspects of growth factor delivery are presented in relation to protein stability, dosage, routes, and safety matters. Finally, different approaches for controlled growth factor delivery are discussed as novel protein delivery platforms for cardiac regeneration.

Breast cancer is one of the prevailing cancers diagnosed among women today and the second leading cause of cancer death in women. Modeling breast cancer cell growth would be a useful tool in identifying therapeutically relevant targets while reducing the amount of spent resources. We have compiled a detailed signal transduction network incorporating epidermal growth factor receptor (EGFR) signaling and downstream components, such as PLC-[gamma], MAPK, PI3K/Akt, cell cycle signaling, transcription, and translation. Using mass-action kinetics, the model was formulated as a set of ordinary differential equations (ODEs). This resulted in more than 8,000 unknown parameters and more than 3,000 ODEs. Partitioning the original model into smaller sub-models and solving them individually may reduce run-time, while maintaining qualitatively similar results as the unpartitioned model. Experiments were performed on the MDA-MB-231 cell line to observe the effects of growth factor treatment on targets such as transcription factors and post-translationally modified proteins. Combination treatments of different growth factors resulted in negative synergy with respect to the chosen targets, which suggests interference between the different pathways involved in growth. This experimental data serves as a starting point to estimate an initial parameter set that can be used to obtain ensembles of parameters that emulate experimental results. In conclusion we have identified an approach to solving large-scale systems that can be used in conjunction with experimental data to predict novel therapeutic targets.

External stresses surrounding bone can stimulate heat shock proteins (HSPs), which are involved in anti-apoptosis, cell proliferation, and differentiation. In vitro stress modulation and HSP induction may be critical factors for enhancing bone regeneration. We investigated whether applying individual or combinatorial stress conditioning (thermal, tensile, and biochemical) and effective HSP modulation could induce in vitro responses in preosteoblasts indicating mitogenic/osteogenic/angiogenic/anti-osteoclastic effects. A preosteoblast cell line (MC3T3-E1) was exposed to conditioning protocols utilizing thermal stress applied with a water bath, tensile stress using a Flexcellâ„¢ bioreactor, and biochemical stress with the addition of growth factors (GFs) (i.e. transforming growth factor-beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2)). Furthermore, the role of HSP70 in osteogenesis under normal conditions and in response to heat was investigated by transfecting preosteoblasts with HSP70 small interfering RNA alone or in combination with thermal stress and measuring cellular response. Heating at 44°C (for 8 minutes) rapidly induced osteocalcin (OCN), osteopontin (OPN), osteoprotegerin (OPG), vascular endothelial growth factors (VEGF), and cyclooxygenase 2 (COX-2) mRNA at 8 hour post-heating (PH). The addition of GFs with heating induced OPG and VEGF genes more than heating or GF addition alone. OPN, OCN, and OPG secretions increased with the addition of GFs. However, matrix metalloproteinase-9 (MMP-9) secretion was inhibited by heating, with more significant declines associates with GF inclusion. Equibiaxial tension (5%, 0.2 Hz, 10 seconds tension/10 seconds rest, 6 days) with GFs enhanced proliferation than tension or GF addition alone. MMP-9 secretion decreased in response to tension alone or more with GFs. Tension (1-5%, 24 hours) with GFs induced prostaglandin E synthase 2 (PGES-2), OPG, and VEGF genes more than tension or GFs alone. Combinatorial conditioning with thermal stress (44°C, 8 minutes) and tension (3%, 0.2 Hz, 10 seconds tension/10 seconds rest, 4 hours for HSP gene and 24 hours for VEGF secretion and MMP-9 gene) induced HSP27 and HSP70, secretion of VEGF (protein), and suppression of MMP-9 (gene) more than heating or tension alone. HSP70 silencing followed by heating (44°C, 8 minutes) enhanced expression of HSP27. Mitogenic activity was inhibited by heating with more significant decrease occurring by heating and HSP70 silencing. At 10 hours PH, TGF-β1, MMP-9, and ALP mRNA decreased in response to heating and HSP70 silencing. At 48 hours PH, heating following HSP70-silencing induced VEGF secretion significantly. In conclusion, effective application of individual or combinatorial conditioning utilizing heating, tension, and GFs could be beneficial as a bone healing-strategy by rapidly inducing stress proteins (HSPs), angiogenic factor (e.g. VEGF), anti-osteoclastogenic cytokines (e.g. OPG), and bone matrix proteins (e.g. OPN and OCN) with anti-resorptive activity by inhibiting MMP-9.

The design of scaffold materials that could be used to assist tissue regeneration has been based on the extracellular matrix (ECM), in structure and in the presentation of molecular cues. Synthetic hydrogels resemble the ECM with their structure, high water content, and elasticity, but they lack bioactivity and recognition. Therefore, the incorporation of biomolecules into synthetic hydrogels is a growing current area of research. Heparan sulfate (HS), a glycosaminoglycan present in basement membrane, has been known to bind and signal various ECM proteins. Sustained presentation of biomolecules like HS could be achieved by covalent linking to scaffolds; however it poses a challenge on the preservation and expression of the biomolecules’ activity after incorporation. Biosynthetic hydrogels derived from heparin (a model for HS) and poly(vinyl alcohol) (PVA) were formed by photopolymerisation. PVA and heparin were both functionalised with photopolymerisable methacrylate groups prior to crosslinking, and the effect of this modification on heparin was examined. PVA/heparin co-hydrogels were made with varying compositions and assessed in terms of their structure, strength and growth factor presentation. The activity of the co-hydrogels following incubation with platelet extract (PE) was also studied, to simulate responses that might occur when the hydrogels, as tissue engineered scaffolds, come in contact with blood products. Heparin remained structurally intact and biologically active following methacrylate modification and UV exposure. The addition of up to 2.5 wt% of heparin increased the hydrogel swelling capacity without compromising the strength of the resulting hydrogel network. The specific FGF-2-signalling activity of heparin in the PVA/heparin co-hydrogels was demonstrated, with results indicating that co-hydrogels may be formulated with a minimal amount of heparin (≥0.05 wt%), thus limiting any effects on structural integrity. PE treatment of the hydrogel-bound heparin diminished its anticoagulation properties but increased the FGF-2 signalling, suggesting the heparanase activity in PE cleave at the antithrombin binding site to yield fragments that can signal cell receptors. This work has demonstrated the formation of biosynthetic co-hydrogels, capable of presenting growth factors to cells, and provides a novel insight on the molecular activation of heparin-based hydrogels upon enzymatic degradation.

Given the pivotal role G protein-coupled receptors (GPCRs) play in physiological responses, various mechanisms have evolved to ensure the tight regulation of GPCR signaling. Classically, agonist exposure leads to an attenuation of receptor responsiveness (i.e. desensitization) through multiple mechanisms including receptor phosphorylation by second messenger-activated protein kinases (e.g. PKA and PKC) and/or GPCR kinases (i.e. GRKs), receptor internalization, and receptor down-regulation. Our laboratory recently discovered that p90 ribosomal S6 kinase 2 (RSK2), a downstream effector of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade, interacts with the 5-hydroxytryptamine 2A (serotonin 2A, 5-HT2A) receptor and exerts a “tonic brake” on agonist-mediated receptor signaling. Importantly, genetic deletion of RSK2 potentiates 5-HT2A signaling in fibroblasts without affecting its subcellular distribution, global G protein function, and expression of serotonergic pathway genes. Furthermore, re-introduction of wild-type RSK2—but not ‘kinase-dead’ RSK2 (K451A)—into fibroblasts restores normal 5-HT2A signaling. Therefore it was hypothesized that RSK2 regulates 5-HT2A receptor signaling through direct receptor phosphorylation. In this thesis I significantly advanced our understanding of 5-HT2A receptor regulation by showing that RSK2 directly phosphorylates the 5-HT2A receptor third intracellular (i3) loop at the conserved residue Ser314, thereby modulating 5-HT2A receptor signaling. I arrived at these conclusions via multiple lines of evidence including in vitro kinase experiments, tandem mass spectrometry, and site-directed mutagenesis. These findings were further validated using phospho-specific Western blot analysis, metabolic labeling studies and whole-cell signaling experiments. These results support a novel regulatory mechanism whereby a downstream effector of the ERK/MAPK pathway directly interacts with, phosphorylates, and modulates signaling of the 5-HT2A serotonin receptor. Furthermore I discovered that RSK2 differentially regulates 5-HT2A agonist signaling, thereby leading to altered patterns of functional selectivity. These studies ultimately led to the larger discovery that RSK2 is required for receptor tyrosine kinase (RTK)-mediated desensitization of 5-HT2A receptors. It is attractive to speculate that RTK signaling may be relevant for regulating 5-HT2A receptor signaling in vivo given the important role of growth factor signaling in normal physiology. Moreover, this novel regulatory mechanism may have far-reaching implications for human disease in which RTK activity is altered (e.g. neuropsychiatric disorders and cancer).

The main purpose of dental implants is to restore the function of the dentition. Despite utilizing structural analyses to investigate treatment outcomes, the therapeutic effect is sometimes unclear due to a lack of direct relevance to the biomechanical function of the peri-implant tissue. While the effective function of tissue depends on the growth pattern, maturation, and load bearing situation of the apparatus, in this dissertation I homogenized the peri-implant tissue parameters under simulated loading situations to generate functional bone apparent modulus (FBAM) and functional composite tissue apparent modulus (FCAM) through the finite element (FE) optimization process. Both FBAM and FCAM were correlated to the structural parameters, and FCAM was determined to be more relevant to interfacial biomechanical characteristics with a pre-existing extraction defect, whereas FBAM within a 200 m peri-implant concentric layer was more relevant to the implant osteotomy defect. Platelet-derived growth factor (PDGF) has been utilized for periodontal tissue regeneration based on its effects on chemotaxis and mitogenesis, which are also key events which occur during early osseointegration. However, the bioactivity of recombinant growth factor application may be significantly reduced due to its rapid degradation and diffusion in vivo. To ensure the efficiency of PDGF expression, we delivered PDGF to peri-implant osseous defects using adenovirus gene therapy vectors (AdPDGF-B) and evaluated the treatment outcome histologically, radiographically, and functionally. The results demonstrate that AdPDGF-B significantly accelerates defect fill and promotes early bone-implant contact (BIC) in a dose-dependent manner. AdPDGF-B also facilitated favorable functional implant support in the early stages of osseointegration. There also exist some considerations regarding the potential of adenovirus-mediated gene therapy to induce virus-related pathologic changes. Thus, the local and systemic safety profile of AdPDGF-B was thoroughly examined in this dissertation in order to alleviate concerns about future gene therapy applications for clinical use. AdPDGF-B was eliminated within two weeks without significant dissemination in vivo, and no histopathologic changes or alterations of systemic parameters were noted. Taken together, this dissertation contributes a novel methodology to functionally evaluate the dynamics of osseointegration and demonstrates the feasibility of AdPDGF-B for accelerating osseointegration while maintaining an acceptable safety profile.

Many attempts have been made to improve the in vitro production of cattle embryos by refining in vitro maturation (IVM) and culture systems. Cysteine supplementation to IVM media of bovine oocytes increases cellular glutathione production, which reduces reactive oxygen species (ROS). Similarly, beneficial effects of growth factors for improving the rate of blastocyst development have been reported, but combined effects are unknown. This study was conducted to determine the additive effect of the antioxidant cysteine with epidermal growth factor (EGF) and/or insulin-like growth factor-I (IGF-I) on subsequent embryo development. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199 (control), with or without the addition of 0.6 mM cysteine (C) at 0 or 12 h of maturation. After in vitro fertilization, embryos were allocated to culture treatments containing synthetic oviductal fluid medium. Culture treatments included fetal calf serum (FCS, 4%) alone; IGF-I (100 ng/mL); EGF (10 ng/mL); and IGF-I+EGF (100 ng/mL+10 ng/mL) for all IVM treatments. Although rates for blastocysts development were not different among treatments, an increased proportion of embryos attaining morula formation was achieved when cysteine was added to the IVM media (12 h C IGF-I+EGF, 41.4%; 0 h C EGF, 40.0%) as compared to control (FCS: 34.6%). When cysteine treatments were combined, percent cleavage was greater for IGF-I+EGF (70.8%) compared to FCS (61.2%). The abundance of mRNA from the apoptotic genes, Bax and Bcl-2, and the oxidative stress genes, copper (Cu)-zinc (Zn) superoxide dismutase (SOD; SOD1) and manganese (Mn) SOD (SOD2) in embryos was assessed. No significant treatment effect was observed on the expression of apoptotic and oxidative stress genes. Bax was expressed strongly (4-fold) in morulae with the addition of IGF-I, but was less prevalent in all other morula and blastocyst groups relative to FCS. There was slightly less expression of both SOD1 and SOD2 with treatments compared to FCS in morulae and blastocysts, indicative of low mitochondrial activity and/or a low level of oxidative stress in treatments. There was no significant treatment effect on total cell number, apoptotic nuclei, or apoptotic index. In conclusion, supplementation of cysteine during IVM of oocytes, in conjunction with growth factors could effectively be used as a replacement for FCS.

A mixed self assembling monolayer (mSAM) chip with attached heparin was developed to analyze heparin-protein interactions using a Reichert Inc, SR7000, surface plasmon resonance (SPR) instrument. The heparin was attached via streptavidin-biotin linkage where the streptavidin was covalently coupled to the mSAM and biotinylated heparin bound to it. These chips were then used to quantify the interactions of fibroblast growth factor-2 (FGF-2) with the surface bound heparin. Kinetic rate constants of association and disassociation were calculated. The association data of FGF-2 with heparin was fit to a single compartment, well-mixed model as the data did not exhibit mass transfer limitations. The results suggested that rebinding was prevalent and observed disassociation rates differed significantly in the presence of competing soluble heparin during disassociation. Our results indicate that the Reichert instrument and mSAM chips can be used to analyze heparin-protein interactions but that a careful protocol, outlined in this thesis, should be followed to obtain optimal data.

The efficiency of skeletal muscle regeneration following injury is decreased with age, and a deficient macrophage response may be part of the cause. Since several important growth factors and cytokines are produced by macrophages, a deficient macrophage response may alter these important cellular signals, further inhibiting the regenerative process. PURPOSE: The primary aim of the study was to observe the response of three macrophage subpopulations (ED1+, ED2+, and IA+) and to quantify the appearance of IL-1β, FGF-2, TGF-β1 and IGF-I following exercise induced muscle damage in both young (11 months) and old (22-23 months) rats. These subpopulations are thought to be involved in different phases of muscle regeneration. METHODS: Male Fischer 344 rats (n=35 old, n=42 young) were sacrificed as controls, or at 2 hours, 1, 2, 4, 8 or 12 days after 90 minutes of running on a -16° slope at 11 m/min, and their soleus muscles removed. After sectioning (8μm), macrophage supopulations were visualized via light microscopy using immunohistochemical staining and quantified at each time point. Muscle damage was assessed by observing H&E treated sections. RESULTS: Following exercise, there were significant increases (p≤.05) in ED1+ macrophages at day 2 in young rats, in IA+ macrophages at day 1 in old rats, and in ED2+ and IA+ macrophages at days 8 and 12 in both age groups. Despite sustaining significantly more muscle damage than young rats, increases in soleus macrophage subpopulations in old rats seemed to be generally less robust than in young. This was indicated by 1) the absence of a significant increase in ED1 + macrophages in old animals, and 2) a somewhat slower increase in both IA+ and ED2+ macrophage populations in the elderly age group. Among growth factors/cytokines, only IL-1β (increased at 2 hours in elderlyover control and young) and FGF-2 (decreased at 1 and 2 days in old rats) were changed with the exercise stimulus. CONCLUSION: These results suggest that a deficient macrophage response may play a role in the delayed regeneration of damaged skeletal muscles in elderly animals, but this deficiency does not seem to directly affect growth factor concentrations.

Insulin-like growth factor-I is a small growth factor known to signal in a variety of mammalian cells through the IGF-I cell surface receptor (IGF-IR). A unique feature of the IGF-I system is the regulation of this binding by soluble IGF binding proteins. Recent studies from our laboratory show that there is a pH dependence in the association of IGF-I with the cell surface in the presence of IGFBP-3 which suggested increased association of IGF-I with IGFBP-3 at low pH. We studied cell free interaction of IGF-I and IGFBP-3 as a function of pH using surface plasmon resonance (SPR) in order to understand the mechanism that causes the increased association. In our studies three different SPR instruments with different surfaces for immobilization of one of the binding partners were used: a Leica Bio-SPR 9000 with a low molecular weight carboxymethylated dextran (CMD) surface, a BIAcore 2000 with a high molecular weight CMD surface and a Leica SPR 2001 Alpha with a planar mixed self-assembled monolayer (mSAM) surface. Since the experimental system we used was transport sensitive, only the mSAM surface, under optimized conditions, produced results that fit to a single site model. Results suggest that use of CMD layers for immobilization of one partner of a high-affinity binding complex can result in transport limited binding for which simple analysis is inappropriate. Future studies are planned to expand the work with the mSAM surface to elucidate whether a significant difference between the binding parameters as a function of pH exists.

Prolactin (PRL) is a 23 kDa hormone that targets the breast, but its role in breast cancer is controversial. An N-terminal 16K PRL fragment suppresses endothelial cell proliferation, but its ability to inhibit tumor growth had not been tested. Our hypotheses were: 1) locally-produced PRL promotes breast cancer growth and 2) 16K PRL inhibits tumor vascularization and growth by suppressing angiogenesis. Using MDA-MB-435 (MDA) breast cancer cells as our model, the specific aims were to: 1) determine the effects of 23K human PRL (hPRL) on cell proliferation and PRL receptor (PRL-R) expression in vitro , 2) test whether 23K hPRL accelerates tumor growth in nude mice, 3) confirm the angiostatic activity of recombinant 16K hPRL in vitro and 4) compare the inhibitory properties of 16K hPRL and endostatin on angiogenesis and tumor growth in nude mice. Exogenous hPRL increased MDA cell proliferation and PRL-R mRNA expression. MDA clones overexpressing 23K hPRL were generated, and PRL production/secretion were confirmed by RT-PCR, Western blotting and the Nb2 assay. When incubated in charcoal-stripped serum, the PRL clones proliferated faster and expressed higher levels of the PRL-R than controls; PRL-overexpressing tumors grew 2-4 times faster than vector or wild type MDA tumors when injected into nude mice. Western analysis demonstrated increased hPRL, PRL-R, and bcl-2 proteins in the PRL-overexpressing tumors compared to controls. These data support a role for local PRL as a mitogenic/anti-apoptotic factor in breast cancer and suggest it may serve as a new therapeutic target for treating breast cancer.Recombinant 16K and 23K hPRL (rhPRL) were produced by baculovirus expression. 16K rhPRL failed to suppress the proliferation of endothelial cells unless contaminated with endotoxins. MDA clones overexpressing 16K hPRL or endostatin were generated. MDA clones overexpressing 16K hPRL did not inhibit angiogenesis or tumor growth in nude mice, but tumors from the endostatin clones were 4-5 fold smaller than vector tumors. The apoptotic index was significantly higher while the blood vessel density was lower in tumors expressing endostatin than in vector controls. These data confirm the potent anti-angiogenic activity of endostatin but do not support a similar function for 16K hPRL.

Adrenomedullin (AM) is a pluripotent regulatory peptide initially isolated from a human pheochromocytoma (adrenal tumor) and subsequently shown to play a critical role in cancer cell division, tumor neovascularization, and circumvention of programmed cell death, thus it is an important tumor cell survival factor underlying human carcinogenesis. A variety of neural and epithelial cancers have been shown to produce abundant amounts of AM. Recent findings have implicated elevation of serum AM with the onset of malignant expression. In addition, patients with tumors producing high levels of this peptide have a poor prognostic clinical outcome. Given that most human epithelial cancers display a microenvironment of reduced oxygen tension, it is interesting to note that AM and several of its receptors are upregulated during hypoxic insult. The existence of such a regulatory pathway has been implicated as the basis for the overexpression of AM/AM-R in human malignancies, thereby generating a subsequent autocrine/paracrine growth advantage for the tumor cell. Furthermore, AM has been implicated as a potential immune suppressor substance, inhibiting macrophage function and acting as a newly identified negative regulator of the complement cascade, protective properties which may help cancer cells to circumvent immune surveillance. Hence, AM's traditional participation in normal physiology (cited elsewhere in this issue) can be extended to a primary player in human carcinogenesis and may have clinical relevance as a biological target for the intervention of tumor progression.