The development of atherosclerosis can be influenced by genetic or pharmacologic disruptions of cellular cholesterol homeostasis. Cholesterol homeostasis in macrophages is of critical importance because these cells have a pivotal function in the vessel wall and in the development of atherosclerotic lesions. Macrophages scavenge modified lipoproteins in the walls of blood vessels and transform into foam cells as cholesterol accumulates intracellularly. Research on apolipoprotein (apo) E, apoAI, and the ATP-Binding Cassette (ABC) A1 transporter suggests that cholesterol efflux has a role in macrophage cholesterol homeostasis that directly affects atherosclerosis risk. Few studies have been done to characterize the interactions between efflux and the other processes of cholesterol balance in the macrophages of atherosclerosis models. The objective of this thesis was to identify the connections between cholesterol efflux and cellular cholesterol homeostasis in macrophages with genotypes that are known to affect the progression of atherosclerosis. To fulfill this objective, we measured the efflux, storage, uptake, and synthesis of cholesterol in peritoneal macrophages from different genetically engineered mice. Our results show that cholesterol storage deficits resulting from the genetic deletion of the cholesterol esterifying enzyme, acyl-coenzyme A: cholesterol acyltransferase (ACAT), disrupted cholesterol efflux, increased lipoprotein uptake, increased cholesterol synthesis, and altered cellular morphology. Our studies also show that macrophages that endogenously synthesize apoE or transgenic apoAI have increased cholesterol efflux due to the concentration of these cholesterol acceptors in the extracellular space and to the stimulation of cholesterol efflux pathways. The current in vitro studies support the idea that cholesterol efflux helps to maintain cholesterol homeostasis in macrophages. Together, these studies suggest that cholesterol efflux is a mechanism that protects against foam cell formation and atherosclerosis.

Bifunctional alkylating agents that cause DNA cross-linking are implicated in the pathogenesis of myelodysplastic syndrome (MDS) and MDS related acute myeloid leukemia (MDR-AML). Therefore, characterizing hematopoietic cell responses to DNA cross-link damage may be relevant to elucidating the molecular pathogenesis of MDS and MDR-AML. To search for genes selectively involved in the cellular response to DNA cross-linking agents, HL-60 cells were treated with bifunctional cross-linking agents. In cDNA microarray gene expression profiles, S100P mRNA was selectively upregulated in bifunctional alkylating agent treated HL-60 cells but not in cells treated by monofunctional agents. This upregulation was confirmed by virtual Northern blot and real time PCR analysis and was both drug dose and time dependent. S100P protein was induced with kinetics similar to that of S100P mRNA. The upregulation of S100P by HN2 suggests that it may have a role in the cellular response to cross-link DNA damage and pathogenesis of MDS and AML.

This project is focused on understanding the molecular mechanisms of prostate tumor initiation due the loss of the tumor suppressor gene Nkx3.1. Here, we examined the dosage-sensitive and stochastic target gene regulation by Nkx3.1 as a mechanism of haploinsufficient prostate tumor suppression. Our results showed that the dosage-sensitive and insensitive Nkx3.1 target genes are regulated by differential H3/H4 acetylation and Nkx3.1 occupancy in vivo. Our findings underscore the importance of chromatin accessibility in dosage-sensitive target gene regulation by Nkx3.1. We next established the functional significance of dosage-sensitive Nkx3.1 target gene intelectin / omentin in prostate tumorigenesis. We found that intelectin suppresses prostate cell growth in vitro and in vivo suggesting its tumor suppressor-like activity in prostate cancer. In summary, our results provide an example of how a genetic lesion such as haploid loss of the Nkx3.1 tumor suppressor can engender epigenetic changes such as alterations in histone H3/H4 acetylation that selectively inactivate a dosage-sensitive target gene important for suppressing tumorigenicity.

This project is concerned with the lysosomal accumulation of lipid in the developing macrophage foam cell as occurs in the atherosclerotic lesion. Lysosomal lipid accumulation in atherosclerosis is a feature of advanced atherosclerotic lesions, but an under explored area of research. In this dissertation I explored the potential mechanisms behind the lysosomal accumulation of lipid. We found that, upon incubation of macrophages with physiological lipid particles, there was an increase in lysosome pH over time such that the number of lysosomes having an active pH decreased. This could be duplicated by pharmacologically sequestering free cholesterol within the lysosome. To analyze for the potential mechanism behind lysosomal inactivation a procedure for isolating lysosomes and monitoring their activation in vitro was established. The studies presented here are the first to monitoring how cholesterol affects the activation of the human macrophage vacuolar-ATPase, which is responsible for maintaining the acidic nature of the lysosome. These experiments determined that exogenously increasing the lysosomal membrane cholesterol inhibited the vacuolar-ATPase. Furthermore, data indicate that the entire vacuolar-ATPase complex is inhibited and not just the pumping of hydrogen ions into the lysosomal lumen. This inhibition of the vacuolar-ATPase does appear to be a reversible process if the membrane cholesterol levels are returned to normal. These data suggest that, if the cholesterol could be removed from the lesion, the lysosomes could potentially recover their v-ATPase activity and become reactivated. These studies should aid in the understanding of lysosome biology and provide a more comprehensive understanding of macrophage foam cell metabolism in the atherosclerotic lesion.

The focus of this project is determining if an ankyrin-G-dependent pathway controls the membrnae expression of the voltage-gated sodium channel 1.5 in cardiomyocytes. Disruption of the normal localization of Nav1.5 can result in Brugada syndrome and has been linked to myopathic disease. This project defines that an ankyrin-G-based pathway is required for the expression, localization and function of Nav1.5 at the cariomyocyte plasma membrane.

Bone marrow-derived mesenchymal stem cells (MSCs) are an attractive candidate for cell-mediated wound repair. Due to their plasticity, MSCs have been utilized in several preclinical and clinical trials of tissue regeneration. MSCs have been able to repair infarcted myocardium, bone, and soft tissue, albeit with varying degrees of success. These promising results are inconsistent partly due to the low levels of engraftment of MSCs within the injured tissues. Hence, strategies to increase survival and engraftment within the wound may enhance MSC therapy. We compared MSCs isolated from MRL/MpJ mice, known to demonstrate enhanced regenerative capacity, to those from C57BL/6 wild-type (WT) mice. Genomic and functional analysis indicated a down regulation of the canonical Wnt pathway in MRL-MSCs characterized by significant up-regulation of secreted frizzled-related protein 2 (sFRP2). These results led us to generate WT-MSCs overexpressing sFRP2 (sFRP2-MSCs) by retroviral transduction. sFRP2-MSCs, when implanted in vivo, resulted in enhanced engraftment, vascular density, reduced infarct size, and increased cardiac function after myocardial injury in mice. Besides increasing the proliferative index of MSCs, sFRP2 also decreased MSC apoptosis and inhibited both osteogenic and chondrogenic lineage commitment. sFRP2 activity occurred through the inhibition of both Wnt and BMP signaling pathways. We found that sFRP2-MSC-treated hearts and wound tissue had less ectopic calcification. We hypothesized that sFRP2 also increased MSC-directed wound repair by regulating their secretome and proteomic analysis of the conditioned media identified Connective Tissue Growth Factor (CTGF) to play physiological role in early wound repair. This work provides important new insight into the mechanisms by which sFRP2 increases MSC self-renewal leading to superior tissue engraftment and enhanced wound healing. These findings implicate sFRP2 as a key molecule for the biogenesis of a superior regenerative phenotype in MSCs.

Atherosclerosis is a chronic vascular disease which is the underlying cause of over half the deaths in the United States each year. Variations in local vascular hemodynamics predispose select sites in the vasculature to atherosclerosis and the atherosclerotic lesions, in turn, alter the biomechanical functioning of the local microenvironment, the consequences of which are not well understood on a molecular level. Work from our lab demonstrated that the small proline rich repeat protein 3 (SPRR3), which is known to be biomechanically responsive in stratified epithelia, is selectively expressed in vascular smooth muscle cells (VSMCs) in the atherosclerotic microenvironment. SPRR3 has stable head and tail domains that contain amino acid domains which are substrates for transglutaminase. Additionally, SPRR3 has a highly flexible, proline-rich central domain that is believed to confer elasticity. We have shown that in VSMCs, SPRR3 transcripts are upregulated by prolonged cyclic strain, as sensed through integrin α1β1 integrin binding to type I collagen. Furthermore, we have shown that SPRR3 overexpression in VSMCs promotes migration and inhibits contraction, though this effect is independent of transglutamination of the protein. Ongoing and future work will demonstrate a role for SPRR3 in signaling pathways, such as in the Akt pathway. Ultimately, the study of SPRR3 will provide insight into the molecular pathogenesis of atherosclerosis.

PATHOLOGY <p>LOCAL SIGNALING MICRODOMAINS IN EXCITABLE CELLS: DEFINING NOVEL ROLES FOR ANKYRIN-B IN ION CHANNEL TARGETING AND REGULATION <p>CRYSTAL FAITH KLINE <p>Dissertation under the direction of Professor Peter J. Mohler <p> The focus of this project was to evaluate the role of ankyrin-B in the targeting and regulation of ion channels to membrane microdomains in excitable cells. Specifically, the project evaluates the role of ankyrin-B for the targeting and metabolic regulation of the KATP channel and defines the minimal binding region of the InsP3 receptor for ankyrin-B binding. Alterations in KATP channel localization and regulation are associated with severe abnormalities in glucose homeostasis; namely, neonatal diabetes and congenital hyperinsulinemia of infancy. Disruption in the proper localization of the InsP3 receptor to the ER/SR membrane has been linked to abnormal intracellular Ca2+ dynamics in cardiomyocytes, resulting in arrhythmia. <p> Using an array of in vivo and in vitro techniques, this project presents evidence supporting a role for ankyrin-B in the targeting and metabolic regulation of Kir6.2, the pore-forming subunit of the KATP channel. Specifically, in vitro binding analysis demonstrates that an eight amino acid motif in the C-terminal domain of Kir6.2 is both necessary and sufficient for interaction with ankyrin-B. Furthermore, when three key acidic residues in this motif are mutated to an opposite charge, association with ankyrin-B is lost. Most exciting is the discovery that a known human mutation, E322K, resides in this motif and is associated with permanent neonatal diabetes mellitus. In vivo work demonstrates that the E322K mutation results in two phenotypes (disruption of Kir6.2 targeting to the plasma membrane and a decrease in ATP sensitivity) that can be resolved using a mathematical model of beta cell electrical activity. The observation of an ankyrin-B/Kir6.2 interaction represents a novel discovery, but more importantly, provides a potential target for therapeutic modulation of KATP channel activity. <p> Similar in vitro and in vivo techniques were used to determine a minimal binding region on the InsP3 receptor necessary for association with ankyrin-B. This determination resolves a long-standing debate regarding the validity of in vivo ankyrin-B/InsP3 receptor interactions. Furthermore, identification of a plausible ankyrin-B-binding domain on the InsP3 receptor has far-reaching implications with regard to the formation of macromolecular ER/SR signaling complexes that are involved in the targeting and regulation of InsP3 receptor activity in vivo. Approved_______________________________________ Date_____________

Activation of coagulation factor IX is a pivotal reaction in the formation and maintenance of a fibrin clot. Factor IX is activated by two structurally unrelated enzymes; factor XIa of the intrinsic coagulation cascade and the extrinsic cascade complex of factor VIIa and tissue factor. Factor IX is activated by cleavage of two internal peptide bonds, resulting in the release of an activation peptide. Both factor VIIa/tissue factor and factor XIa make the same cleavages in factor IX, but appear to do so by different mechanisms that involve different rate limiting steps. While the factor VIIa/tissue factor complex has one catalytic domain per complex, factor XIa is a unique homodimer with up to two active serine protease domains per molecule. Factor XIa appears to cleave both scissile bonds in factor IX without releasing an intermediate, while factor VIIa/tissue factor releases a singly cleaved intermediate. This observation has led many investigators to postulate that factor XIa, acting via a processive mechanism, can make both cleavages in factor IX simultaneously without generating any singly cleaved intermediate forms of factor IX. Based on observations suggesting that factor XIa contributes to obstructive (thrombotic) clot formation to a greater degree than to normal coagulation at a wound site, there is increased interest in the mechanism by which factor XIa is formed, and by which factor XIa cleaves factor IX. Regulation of clotting factors follows their form, and most pro-coagulant molecules are directed to assemble on injured surfaces by common accessory domains. Factor XIa is unique in this regard in that it does not appear to require either co-factors or damaged surfaces for its activation or for cleavage of factor IX once activated. The relationship between the unique structural features of factor XIa and its unusual mode of action is poorly understood. Here we present evidence that factor XIa with only one active subunit per molecule can be formed, is produced in plasma, and can activate factor IX without intermediate accumulation. Our data, along with the recently solved structure of the zymogen precursor of factor XIa, factor XI, suggests that the structure of factor XIa allows its subunits to act independently of each other, a behavior that has not been described previously for a serine protease.

Macrophage foam cells are prominent in atherosclerotic lesions. In late stage disease, much of the cholesterol accumulation within foam cells is found in large, swollen, lysosomes. Tissue culture models using human macrophages incubated with various modified LDLs indicate that cholesterol accumulation within lysosomes can disrupt lysosome function leading to foam cells with significant lysosomal free and esterified cholesterol, similar to cells found in atherosclerotic lesions. The cholesterol is trapped and not accessible for efflux, even in the presence of strong efflux promoters. In the artery wall, however, the foam cells are bathed not only in modified LDLs but other lipid particles as well, including triglyceride-rich particles (TRPs), such as VLDL. Little is known about how metabolism of TRPs might affect cholesterol metabolism and, specifically, the formation of cholesterol-rich macrophage foam cells. The studies presented in this dissertation explore the effect of TRPs on intracellular cholesterol metabolism. Results show that cellular and, specifically, lysosomal triglyceride (TG) enrichment reduces cholesteryl ester (CE) accumulation by over 50% in THP-1 macrophage foam cells. TG, delivered to the cell as a component of TRP, decreases the volume of lysosomes providing further evidence of increased lysosomal cholesterol clearance. Cholesterol accumulation in lysosomes inhibits acidification of lysosomes. In contrast, lysosomal TG enrichment reduced this inhibition and allowed lysosomes to remain active. Maintained lysosomal activity results in enhanced degradation and clearance of internalized CE and facilitates the movement of cholesterol out of the lysosome, to sites where it can be utilized for cholesterol efflux. In sum, our results show that introduction of TG into CE-laden foam cells influences CE metabolism and, potentially, atherogenesis.