The development and maintenance of a vascular network is critical to the growth and survival of a tissue and ultimately an organism. An understanding of the mechanisms which regulate angiogenesis within and surrounding currently used polymeric devices would contribute to the success of these implants by establishing methods to enhance tissue in-growth and new vessel development. Furthermore, tissue-engineering currently used polymers such as expanded polytetrafluoroethylene (ePTFE) may create an angiogenic material that can be used to induce new microvessel formation in infarcted myocardium. Myocardial infarcts represent a pathology that affects a large percentage of the patient population who suffer from coronary heart disease. Disease of the coronary vasculature can lead to narrowing of the coronary vasculature and result in regions of ischemia which can progress to infarction. Studies in this dissertation evaluate two different tissue-engineered polymer constructs for their ability to stimulate a new collateral network in infarcted myocardium. The results from these studies indicate that the tissue site of implantation is an important factor in influencing the healing response. Therefore, it is important to evaluate future polymer devices in tissues where the device will ultimately reside. Additionally, the physical and chemical characteristics of polymers were found to have a significant influence on the healing response. Furthermore, tissue-engineered polymer constructs stimulated a significant angiogenic response within infarcted myocardium. Tissue-engineered constructs that secreted soluble angiogenic agents were found to have the greatest depth of angiogenic effect into infarcted myocardium leading to the formation of arterioles, capillaries, and venules. Additionally, hearts treated with these devices demonstrated significantly greater left ventricular function in comparison to infarct-only hearts. Based on this work, it is apparent that tissue-engineered polymer constructs may have a future role as cardiac patches and thus provide the patient population with an additional therapy to revascularize infarcted cardiac tissues.

Inorganic phosphate (Pi) homeostasis is mainly regulated by absorption of dietary Pi in the small intestine and reabsorption of filtered Pi in the kidney. I have characterized the 5 '-flanking region and overall gene structure of the murine type IIb sodium-phosphate cotransporter (Na/Pi-IIb), a major apical Pi transporter. The Na/Pi-IIb cotransporter gene spans more than 18 kb and consists of 12 introns and 13 exons. Three promoter/reporter gene constructs, -159/+73, -429/+73 and -954/+73, showed significant luciferase activity when transfected into in rat intestinal epithelial (RIE-1) cells. Pi requirement during development is much higher than in adult life. In the next set of experiments, I sought to characterize expression of the intestinal Na/Pi-IIb cotransporter during mouse ontogeny and to assess the effects of methylprednisolone (MP) treatment. In control mice, Na/Pi uptake by intestinal brush-border membrane vesicles was highest at 14-days-of-age, lower at 21 days and further reduced at 8 weeks and 8--9 months of age. Na/Pi-IIb mRNA and immunoreactive protein levels in 14-d animals were markedly higher than in older groups. MP treatment significantly decreased Na/Pi uptake, and Na/P i-IIb mRNA and protein expression in 14-d mice. Additionally, the size of the protein was smaller in 14-d mice. Deglycosylation of protein from 14-d and 8-wk old animals with PNGase F reduced the molecular weight to the predicted size. I conclude that intestinal Na/Pi uptake and Na/Pi-IIb expression are highest at 14-d and decrease with age. Furthermore, MP treatment reduced intestinal Na/Pi uptake ∼3-fold in 14-d mice and this reduction correlates with reduced Na/Pi-IIb mRNA and protein expression. I also demonstrate that Na/Pi-IIb is an N-linked glycoprotein and that glycosylation is age-dependent. In conclusion, the mouse intestinal Na/Pi-IIb cotransporter is developmentally regulated at mRNA and protein levels. MP-treatment also reduces mRNA and protein expression during development. The Na/Pi-IIb gene promoter constructs identified in the first study will be a useful tool to investigate the possible transcriptional regulations. Furthermore, studying post-translational regulation including glycosylation will reveal developmental effects on Na/Pi-IIb cotransporter protein. These studies will help to decipher molecular mechanisms of Pi absorption in mammalian small intestine.

Sensory cells located in the pancreas, the liver, the stomach and the intestines sense nutrient and peptide levels. A change in blood glucose concentration is one of the primary signals monitored by this cell type and alterations in the sensing capabilities of these glucose sensing cells may underlie the pathophysiology associated with obesity and diabetes. To understand how these glucose sensing cells function and what components are essential for maintaining glucose homeostatic mechanisms in the body, we characterized these cells by isolating and characterized the responses of these cells to glucose using functional assays. The principal glucose sensing cells of interest for our research were the neurons located within the hypothalamus, which are believed to integrate signals from sensory cells throughout the body to maintain energy homeostasis. We proposed that the mechanisms by which hypothalamic neurons sense glucose are similar to those used by pancreatic beta cells. By suggesting that hypothalamic neurons and pancreatic beta cells use similar mechanisms to sense and respond to changes in glucose levels, we hoped to identify the shared components to help us learn more about this unique and rare cell type. We found that the enzyme glucokinase (GK) was expressed throughout all stages of development along with the GLUT-1, GLUT-3 and GLUTX-1 glucose transporters, but that the liver form of GKRP was not found in the hypothalamus. The enzyme GK was also found in tissues of the distal stomach and proximal intestine of adult rats. Each of the 3 methods we employed, RT-PCR, in vitro functional assays and the development of a transgenic animal enabled us to initiate a line of research that may one day lead to a further understanding of how the body maintains energy homeostasis.

Across species, glial cells in both peripheral and central nervous systems cooperate extensively with neurons to shape multiple aspects of neural development. In vertebrate and invertebrate olfactory systems, neuron-glia interactions are thought to underlie critical developmental events, including glomerulus formation, and the growth, sorting, and targeting of olfactory receptor neuron (ORN) axons. The olfactory system of the moth Manduca sexta has many similarities to vertebrate olfactory systems, and has been used extensively to explore intercellular interactions involved in the formation of the olfactory pathway. In particular, glial reduction experiments have implicated two types of central olfactory glia, the sorting zone and neuropil-associated glia, in axon sorting and glomerulus stabilization. The developmental roles of a third glial cell type, the antennal nerve glia, remain elusive, yet their peripheral origin and association with ORN axons are similar to mammalian olfactory ensheathing cells. The present body of work uses a defined co-culture system to characterize interactions between ORN axons growing from explants of olfactory receptor epithelium and glial cells from the primary olfactory system of Manduca. We have monitored how particular types of glia, known to influence the behavior of ORN axons in vivo, directly affect the behavior and morphology of individual ORN growth cones in vitro. Time-lapse imaging of neuron-glia cultures revealed that olfactory receptor growth cones elaborate extensively and cease advancement following contact with sorting zone and neuropil-associated glial cells. In contrast, growth cones advance along the surfaces of antennal nerve glial cells without prolonged changes in morphology. Cytoskeletal staining of fixed preparations reinforced live-cell findings, as contact with sorting zone and neuropil-associated glial cells caused statistically significant changes in growth cone morphology. Finally, ORN axons induce antennal nerve glia, but not sorting zone or neuropil glia, to form multicellular arrays through proliferation and process extension. These findings have led to the formation of hypotheses concerning the nature of neuron-glia interactions in vivo.

T cell development is regulated by signals generated in the interactions between developing thymocytes and the thymic stroma. Using fetal thymus organ culture (FTOC) as a model of T cell development, we investigated the ability of two potent signal modulators to influence this process. These studies show that both nicotine and tumor necrosis factor-alpha have the ability to influence T cell receptor (TCR) signaling and the maturational capacity of treated cultures. FTOC treated with low concentrations of nicotine produced more immature T cells and fewer mature T cells. These expanded populations of cells also expressed CD69, CD95 (FAS) and elevated levels of recombinase activating genes (RAG). This phenotype reflects the fact that these cells have received a positive selection signal, are for apoptosis and are likely attempting secondary TCR rearrangements. Nicotine effects were partially blocked by the nicotinic antagonist, d-tubocurarine. Furthermore, d-tubocurarine alone blocked the development of T cells entirely, suggesting the presence of an endogenous ligand that may engage nicotinic acetylcholine receptors and regulate normal thymopoeisis. These observations underscore the linkage between the nervous and the immune systems, not only in terms of shared resources, but also in terms of direct interactions between these two systems. In another study we used FTOC and an associated in vitro Type 1 diabetes mellitus model to reconcile the role of TNF-alpha in thymopoiesis with its role in diabetes. Our data indicate that thymocytes from NOD FTOC express lower levels of TNF receptors and produce more TNF-alpha compared to non-diabetic C57BL/6 (B6) FTOC. Neutralization of endogenous TNF-alpha in NOD FTOC with a soluble TNF receptor (sTNF R1) rescued insulin production in our in vitro diabetes model. NOD FTOC treated with TNF-alpha produced greater numbers of mature T cells and a higher percentage of cells expressing CD95L (Fas ligand). Treatment with sTNF R1 had the opposite effect. TNF-alpha's known ability to attenuate TCR signaling coupled with these observations suggest that its overproduction in these animals may be driving T cells to maturity, altering the process of negative selection and ultimately enhancing the survival of potentially diabetogenic T cells resulting in disease susceptibility in these animals.

The socioeconomic constraints generated by patients with cardiovascular disease necessitates the development of novel treatment strategies for the pathologies associated with disease progression. One promising field of active research and development is Cardiovascular Tissue Engineering. It is believed this discipline will ultimately provide alternative strategies for the development of vascular bypass conduit, bioprosthetic valves, functional microvascular networks, and solid organ replacement tissue. The primary goal of this project was to test the hypothesis that, following implantation, tissue engineered microvascular grafts are capable of inosculation with host coronary vasculature and attenuating the loss of ventricular function following acute myocardial infarction. To test this hypothesis, microvascular grafts were constructed of adipose-derived microvascular fragments suspended in a 3-dimensional matrix. These tissue engineered grafts were transplanted and evaluated in a number of in vivo research scenarios. Research protocols were designed to critically evaluate the potential of microvascular network grafting in multiple tissue sites, and in differing pathophysiologic conditions. Microvascular grafts were initially implanted and studied in a subcutaneous position in recipient animals. Following implantation, the microvessel network within the grafted construct established spontaneous anastomotic connections with the host. Inosculation of the grafted microvessles and host circulation occurred rapidly following surgical placement, with evidence of significant vascular remodeling within the graft. The experimental grafts were also evaluated in the cardiac position following acute cardiac injury. Perfusion was realized through the grafted microvascular tissue. The resulting microvasculature was complete with well-formed arterioles, venules, and capillaries. It was established that development of left ventricular dysfunction following experimental coronary artery occlusion was abated in animals treated with epicardial placement of microvascular grafts. Interestingly, while there was overwhelming evidence of microvascular remodeling in both the subcutaneous and cardiac position, there was a noted tissue-specific adaptation that occurred. Grafts in the cardiac position had a higher vascular density than those in the subcutaneous position, and developed a vessel-type distribution that was approximate to that observed in native epicardium. The results described in this dissertation project support the utility of tissue engineered microvascular grafts for the treatment of pathophysiologic tissue within the cardiovascular system proper, as well as in peripheral systems.

4-vinylcyclohexene diepoxide (VCD) is an environmental xenobiotic formed as a by-product in the manufacture of rubber and therefore potential human exposure is likely. VCD destroys half of the small pre-antral (25-100 μm) follicles in ovaries of rats following 15 days of dosing. The overall goal of this research, was to determine the mode and earliest time for identification of follicular destruction and examine the specificity of the response for 25-100 μm follicles. The particular involvement of protein synthesis and gene expression in this ovotoxic response was also examined. After daily dosing with VCD (80 mg/kg), the rate of protein synthesis in 25-100 μm follicles was inhibited following 3, 6, and 10 hr of in vitro incubation with VCD; whereas, the inhibition in the rate of protein synthesis at 3 hr in 25-100 μm follicles from untreated animals was reversed at 6 and 10 hr. Furthermore, follicular viability was compromised to a greater extent in 25-100 μm follicles from dosed versus untreated animals. Following 10 days of daily dosing with VCD, there was an increase in random DNA fragmentation in 25-100 μm follicles; however, there was not a reduction in the numbers of primordial and primary (25-100 μm) follicles. Morphological analysis showed changes characteristic of an apoptotic-like form of cell death in oocytes and granulosa cells of primordial and primary follicles 4 hr following 10 days of daily dosing. There was an increase in levels of mRNA for bax, manganese superoxide dismutase (MnSOD) and microsomal epoxide hydrolase (mEH) in 25-100 μm follicles following 10 days dosing with VCD, but the increase was not observed in large pre-antral (100-250 μm) follicles or liver. However, decreases in levels of mRNA for bax in liver and mEH in 100-250 μm follicles were observed. These results suggest that repeated dosing makes 25-100 μm follicles more susceptible to VCD-induced cellular changes and that VCD-induces an apoptotic-like form of cell death which is mediated through changes in levels of expression of genes associated with death of the follicular cells.

The overall objective of this dissertation was to estimate the in vivo hepatic clearance (CL(H)) of compounds using in vitro methods of drug metabolism. The isolated perfused rat liver, precision-cut liver slices and hepatocyte were used to estimate in vitro CL(H) and compared to in vivo CL(H) Two compounds, benzoic acid and tolbutamide were chosen as model compounds. An isolated perfused rat liver (IPRL) apparatus was developed to measure hepatic extraction ratio. Three compounds, antipyrine, ethanol and lidocaine were used to characterize the apparatus. The ability of the IPRL to utilize oxygen was also investigated. Antipyrine extraction ratio was independent of perfusate flow rate, while the extraction of ethanol and lidocaine were flow-dependent. The extraction ratios of benzoic acid and tolbutamide were determined. The CL(H) of benzoic acid and tolbutamide was 4.43 ± 0.84 mL/min and 1.52 ± 0.59 mL/min, respectively. The intrinsic clearance of benzoic acid and tolbutamide was determined in precision-cut liver slices and scaled to the whole liver using total protein. The CL(H) of benzoic acid in rat liver slices was 2.13 ± 0.71 mL/min, while the intrinsic clearance in humans was 270 mL/min. The CL(H) of tolbutamide in rat and human liver slices was 0.019 mL/min and 3.16 mL/min, respectively. The intrinsic clearance of benzoic acid and tolbutamide was determined in rat hepatocytes and scaled to the whole liver using total number of cells in the rat liver. The CL(H) of benzoic acid and tolbutamide in rat hepatocytes was 3.55 ± 1.29 mL/min and 0.57 ± 0.16 mL/min, respectively. The CL(H) of benzoic acid and tolbutamide on intravenous dosing in the rat was 8.02 ± 1.01mL/min and 0.49 ± 0.06 mL/min, respectively. Precision cut liver slices under-estimated the CL(H) of benzoic acid and tolbutamide. The under-estimation is probably due to the inability of the drug to permeate the liver slice over the short time course of the initial rate experiments. The CL(H) of benzoic acid and tolbutamide in rats was better predicted by the IPRL and hepatocytes.

MEQC (mvc embryonic quail cardiomyocytes) is a permanent cell line derived from cardiac tumors produced by infection of 3-day quail with the MC29 myelocytoma virus, which contains the v-myc proto-oncogene. This cell line can be induced to differentiate as evidenced by expression of muscle specific markers upon co-culture with NIH 3T3 fibroblasts. When MEQC are treated with low concentrations of BrdU before co-culture, they can no longer be induced to express phenotypic markers. In the current study, I have isolated BrdU-sensitive transcripts from MEQC cells by subtractive hybridization and investigated their distribution in developing chick embryos. In all, 29 transcripts were isolated, 14 of which could be identified by sequence comparison with the Genbank data base. Complete sequences were obtained for two of the remaining transcripts, pX19 and pX27. pX19 encodes a protein of 23 kDa that contains an amphipathic alpha-helix previously described only in plant seed embryos. By in situ hybridization pX19 was identified mainly in hemopoietic tissues; it was also found in cardiac cushion mesenchyme by PCR. The clone pX27 was identified as the avian homologue of mammalian glypican core proteins and was localized in early stages of development to the cephalic regions of the neural folds, rostral paraxial mesoderm, and newly formed somites. Later, glypican transcripts were found in the apical epidermal ridge of the limb buds, mantle zone of the telencephalon, and endocardial cushions of the atrioventricular canal and aortopulmonary outflow tract. An antibody raised against the glypican core protein was localized to the cell membrane in MEQC cells. Furthermore, upon withdraw of serum from cultures of MEQC expression of glypican transcripts was enhanced and the cells tended to clump. Administration of a glypican antisense oligonucleotide prevented cell clumping and blocked the migration of endocardial endothelial cells over collegen gel. Taken together, these results suggest that MEQC express transcripts unique to either myocardium or endocardium and provide a useful system from which transcripts expressed during development can be isolated.

The central hypothesis of this dissertation is that agonists and antagonists acting at the delta opioid receptor will have therapeutic applications in treating acute and chronic pain states and in the treatment of drug addiction. It is further hypothesized that delta compounds will have better therapeutic profiles than currently available opioids that act predominantly at the mu receptor. In advancing the central hypothesis, selective nonpeptidic delta compounds, that readily cross the blood brain barrier after systemic administration, were tested. BW373U86, a nonpeptidic ligand with moderate selectivity and activity at delta opioid receptors represented a lead compound. A structurally related molecule, SNC80, displayed an improved selectivity and activity profile compared to BW373U86. Importantly, SNC80 produced antinociception following systemic administration which was blocked by delta, but not mu, selective antagonists. The pharmacology of delta opioid receptors was further studied using antisense oligodeoxynucleotides that disrupted the synthesis of delta receptors in vivo and in vitro. The experiments provided further evidence for distinct delta receptor subtypes and demonstrated the utility of the antisense approache in studying neurochemical processes in vivo. Several studies addressed the phenomenon of opioid tolerance and physical dependence, two processes which compromise the clinical application of currently available opioid analgesics. The observation that NMDA receptor antagonists block the development of antinociceptive tolerance to repeated administrations of morphine was confirmed. The results were extended by demonstrating that NMDA antagonists did not block antinociceptive tolerance to more selective delta or mu agonists. These studies caution against the generalization that an effect seen with morphine is applicable to all opioid agonists. Further hypotheses regarding the mechanisms of opioid tolerance and physical dependence were tested using inhibitors of protein kinases and putative neutral and inverse opioid antagonists. These studies advanced the hypothesis that opioid receptor phosphorylation may play a critical role in the development of opioid antinociceptive tolerance and physical dependence. In summary, this dissertation has provided strong evidence that nonpeptidic delta selective opioid agonists and antagonists can be developed and that these compounds will have therapeutic applications in the treatment of pain and addictive disorders.