Kevin J Williams, Ph.D. - US grants

The overall objective of this research is to determine the mechanism by which phospholipid infusions produce rapid, substantial regression of experimental atherosclerosis. This knowledge will allow rational modifications in the treatment and may eventually allow its application to the human disease. The present proposal will focus on the metabolism of infused phospholipid vesicles. Under the proper circumstances, these particles persist in the circulation for hours, acquire large amounts of endogenous cholesterol and apolipoprotein-E, and appear to be gradually taken up by the liver. Initial studies will be in three areas. First, to explore the mechanism by which vesicles may transport cholesterol from the periphery to the liver, the role of hepatic LDL receptors in hepatic uptake of vesicular particles will be determined. Hepatic clearance will be measured in rabbits with stimulated, suppressed, saturated, or absent hepatic LDL receptors. Second, because vesicles block the ability of B-VLDL, and atherogenic lipoprotein, to cholesterol-load cultured macrophages, blockage of B-VLDL uptake by aortic macrophages will be measured in vivo. Aortic uptake of 125 I-dilactitol tyramine-labeled B-VLDL will be compared in phospholipid-infused and saline-infused animals. Third, the abilities of infused vesicles of different size and composition to acquire endogenous cholesterol, to undergo hepatic clearance, and to block macrophage uptake of B-VLDL will be compared. Subsequent studies will depend on the results of these initial studies. If hepatic LDL receptors are found to be involved in hepatic uptake of vesicular particles, agents that stimulate hepatic LDL receptors will be examined for their ability to accelerate phospholipid-induced regression of atherosclerosis. Conversely, if substantial hepatic uptake of vesicles occurs in the absence of hepatic LDL receptors, the alternate pathways for uptake will be investigated in vitro. Also, the ability of phospholipid infusions to prevent or reverse atherosclerosis in receptor-deficient rabbits will be determined. This last study will be of particular interest, because atherosclerosis in receptor- deficient rabbits closely resembles the human disease in histology and anatomic distribution. The ultimate goal is a rational, non-invasive treatment that produces regression of atherosclerosis in humans.

We have discovered a novel pathway for cellular catabolism of apoB-rich atherogenic lipoproteins: a non-enzymatic action of lipoprotein lipase (LpL) enhances their binding to cell-surface heparin sulfate proteoglycans (HSPGs), and thereby mediates substantial retention, internalization, and lysosomal degradation of the lipoproteins. The pathway is independent of the LDL and scavenger receptors and is not regulated by cellular sterol content. Aim I is to identify and characterize the relevant HSPGs from hepatocytes, the major cell to clear LpL from plasma. We will focus on the HSPG core proteins, which form distinct genetic and metabolic families. Based on our initial success with 21-mer anti-sense thioated oligonucleotides against the syndecan family of HSPG core proteins, we propose a series of molecular manipulations of intact cells. We shall prepare full- or long-length sense and anti-sense cDNA constructs driven by a strong promoter, for use in transient transfections to determine the roles of specific core proteins in the uptake of LpL-apoB complexes. Some HSPG core patients are phosphatidylinositol (PI) anchored, and so we shall also determine the effects of digestion with PI-specific phospholipase C. Because these manipulations are limited to known species or classes of core proteins, we also propose a standard protein purification. We will rely on unusual features of the HSPGs, namely, rapid metabolic incorporation of 35SO4, high negative charge, and binding to LpL-lipoprotein complexes. Previously cloned hepatic HSPGs will be identified immunologically. Core proteins from previously uncharacterized HSPGs will be microsequenced by Edman degradation, and degenerate oligonucleotides based on the microsequences will be used to screen cDNA libraries, to obtain probes for molecular studies. Aim II is to test directly the atherogenicity of the LpL-HSPG pathway in vivo. We have hypothesized that this pathway is anti-atherogenic in liver because it promotes hepatic re-uptake of nascent apoB-rich atherogenic lipoproteins, thereby reducing their net secretory output, yet it would be atherogenic in the arterial wall because it promotes cellular and interstitial accumulation of cholesteryl esters in peripheral cells. As part of a separately funded pilot program, we are making transgenic mice that express normal and mutated human Lpl in their hepatocytes. In this application, we propose to examine these liver- specific transgenics for their resistance to atherosclerosis from diet or from a genetic absence of apoE. We also propose to make transgenics that express LpL in vascular smooth muscle cells. Smooth muscle-specific transgenics will be examined for their susceptibility to atherosclerosis. Overall, these studies will establish the molecular basis and the physiologic important of this novel, non-enzymatic action of LpL in atherogenesis.

The key instigating event that provokes a normal artery to become atherosclerotic appears to be the sub-endothelial retention of apoB-rich lipoproteins by arterial matrix. In this proposal, we will examine the role known to avidly bind apoB-rich lipoproteins. To allow a molecular approach, we have cloned, sequenced, and expressed the human cDNA for chondroitin-6-sulfotransferase (C6ST), the key enzyme in C6S biosynthesis. We have documented expression in human endothelial cells and obtained and characterized genomic clones. Aim I: Regulation of endothelial proteoglycan assembly and function in vitro. We will focus on four regulatory stimuli, each of which changes proteoglycan structure and has been linked to atherogenesis: shear stress, hypoxia, oxidized lipoproteins, and cytokines. Proteoglycan assembly by cultured endothelial cells will be assessed by the C6:C4 sulfate ration, expression of C6ST mRNA & protein, C6ST mRNA transcription & stability, C6ST phosphorylation & subcellular distribution, and expression of C6ST promoter constructs. Proteoglycan function will be assessed by affinity co-electrophoresis (ACE) gels to establish binding constructs to human LDL, and by our cell-culture model of lipoprotein retention. Aim II: Atherogenesis in endothelial-specific C6ST transgenics. To directly examine the effects of endothelial matrix variations on atherogenesis in vivo, we propose to create C6ST transgenics with expression limited to large-vessel endothelium. Distribution of C6ST message and protein will be examined microscopically, and aortic proteoglycans will be assessed as described in Aim I. Retention of LDL in aortae ex vivo and in vivo, endothelial function, and atherosclerotic lesion development after crossing to hyperlipidemic apoE knock-out mice will then be determined. Aim III: The role of C6ST in human disease. We will determine the distribution of C6ST message and protein in normal and atherosclerotic human arteries: screen for C6ST polymorphisms in patients proven by cardiac catheterization to be with or without disease; & test linkage of the C6ST locus with a disease of low C6S. Overall, these proposed studied will substantially enhanced our understanding of endothelial matrix assembly, which is likely to contribute to the large variation in atherosclerotic lesion development between arterial sites and amongst individuals with similar plasma lipid profiles.

[unreadable] DESCRIPTION (provided by applicant): Human immunodeficiency virus type 1 (HIV-1) enters the brain soon after serconversion. Consequently, the central nervous system (CNS) becomes a sanctuary for virus, and it can be damaged by virus or virally encoded proteins. A prominent model for viral entry into the CNS is the "Trojan Horse" hypothesis, in which HTV- 1-infected CD4+ T-lymphocytes and monocytes transmigrate across the blood-brain barrier (BBB). Consistent with prior work, we observed that HIV-1 infection enhances T-cell and monocyte transmigration in vitro across a simple acellular barrier of Matrigel, which mimics basement membrane. More importantly, we made the novel discovery that clinically available inhibitors of cholesterol biosynthesis (statins) potently inhibit HTV-1 -induced transmigration. This finding arose from an essential synergy between two laboratories in different fields, lipid metabolism (Dr. Williams) and neurovirology (Drs. Mukhtar and Pomerantz). In addition, as noted in the Introduction and revised Preliminary Studies, we recently found a neuroprotective effect of statins as well. Our central hypothesis is that HIV-1 infection alters the expression of specific genes that play a central role in transmigration and cytotoxicity, and that statins restore the expression of these genes towards normal levels. Experimental systems available to us include primary human T-cells and monocytes, our acellular model barrier of Matrigel, and a sophisticated cellular BBB model that we created using well-characterized human BMVECs and astrocytes grown in transwell inserts over human neurons. There are two Specific Aims: Aim I; Molecular mechanisms by which statins reduce transmigration of HIV-1-infected CD4+ T-cells and monocytes through an acellular model basement membrane. Three sub-Aims will study whether this effect results from (i) cholesterol-dependent or -independent (pleiotropic) effects of statins, (ii) involvement of specific genes identified on our focused gene array as altered by infection but restored by statins, e.g., MMPs, TIMPs, paxillin, and rho-related proteins, and (iii) alterations in specific inflammatory or cytotoxic cytokines. Aim II: Effects of statins on transmigration of HIV-1-infected T-cells and monocytes through a cellular blood brain barrier model in vitro. Sub-aims will somewhat parallel Aim I, focusing on the effects of infection and statin treatment on specific gene expression, cytokine release, endothelial integrity, and transmigration. Overall, our proposed studies will provide new insights into HIV-1 neuropathogenesis. In addition, our work may provide a new use for statins to prevent or treat AIDS-related dementia and to deplete or eradicate the CNS as an HIV-1 sanctuary. [unreadable] [unreadable]

Two classes of molecules - the LDL receptor (LDLr) family and cell-surface heparan sulfate proteoglycans (HSPGs) - are key participants in the transport of hydrophobic nutrients by plasma lipoproteins. We have discovered a novel pathway, in which endocytosis of lipoproteins and other ligands is mediated directly by syndecan HSPGs. Using a chimera, FcR-Synd, that consists of an IgG Fc receptor ectodomain linked to the transmembrane (TM) and cytoplasmic regions of syndecan-1, we found that efficient endocytosis is triggered by clustering of syndecan or the chimera. Clustering causes rapid movement into cholesterol-rich, detergent-insoluble, membrane rafts, and then the actual uptake into the cell requires recruitment of tyrosine kinases and the actin cytoskeleton. Surprisingly, we found that constructs containing either the LDLr TM or the syndecan-1 TM domain localized equally well to rafts upon clustering. Sequence comparisons revealed an unexpected 15- residue consensus between the inner (C-terminal) portions of the syndecan and LDLr TM domains, which was not shared by a protein excluded from rafts. Importantly, this consensus may explain unusual features of the way these two molecules have been shown to process multivalent ligands, such as large apoE-rich remnant lipoproteins. Thus, the central hypothesis of this proposal is that specific motifs of syndecan, including the raft-localizing segment shared with the LDL receptor, direct the sub-cellular trafficking of nutrient-bearing ligands, with specific functional consequences. There are two Aims. Aim 1: Detailed definition of novel trafficking motifs in the LDLr gene family and in syndecan. In Aim 1a, we will use CHO cells andMcArdle hepatocytes to map determinants of raft localization within the TM domain of the LDLr, other members of the LDLr gene family, and syndecan. In Aim 1b, we will map trafficking determinants in the syndecan cytoplasmic tail. In Aim Ic, we will test these determinants in another key cell type, the macrophage, which is of particular interest becauseofits variant endocytic pathway through the LDLr. Aim 2: Functional roles for the novel raft-localizing motif in the LDLr transmembrane domain. In Aim 2a, we will determine the role of TM raft-localizing motifs from Aim 1 in the marked stimulation of ACAT that occurs in macrophages when the LDLr binds multivalent lipoproteins. In Aim 2b, the role of these TM motifs in LDLr-mediated regulation of apoB secretion via re-uptake will be investigated in hepatocytes. These proposed studies will clarify basic mechanisms and functional consequences of these novel endocytic determinants within the LDLr and syndecans, including the role of raft localization during nutrient delivery.

DESCRIPTION (provided by applicant): Vascular disease is a major cause of morbidity and mortality in patients with Type 1 diabetes. Hyperglycemia causes endothelial dysfunction, a pathological state consisting of reduced nitric oxide (NO) bioavailability, increased cellular superoxide production and activation of an inflammatory cascade with increased expression of adhesion molecules at the endothelial cell surface, that leads to the initiation and progression of vascular disease in diabetes. In preliminary studies, we have found that adiponectin, an abundant circulating plasma protein, suppresses high glucose-induced endothelial superoxide generation and enhances NO production in vascular endothelial cells. We and others have also found that adiponectin activates AMP kinase in various cell types including endothelial cells. In this project, we will characterize the signaling mechanism(s) used by adiponectin to improve endothelial function under high glucose conditions. By studying endothelial cells in culture in vitro and mesenteric microvascular endothelial cells in situ by intravital microscopy, we will test the hypotheses that the globular domain of adiponectin (gAd) expressed in a bacterial system and the full-length adiponectin protein (fAd) expressed in a recombinant eukaryotic system: (1) suppress superoxide production by endothelial cells treated with high glucose, possibly via an NAD(P)H oxidase-linked pathway regulated by protein kinase C; (2) enhance NO production by endothelial cells treated with high glucose, possibly via an AMP kinase-linked pathway; and (3) ameliorate endothelial dysfunction in vivo in rodent models of hyperglycemia as evidenced by salutary effects on leukocyte-endothelial interactions, expression of cell adhesion molecules and NO production. The in vivo studies will also be facilitated by using adiponectin knock-out mice, available to us by a research collaboration, which should demonstrate augmented vascular effects of adiponectin when the various forms are administered on a background of no endogenous circulating adiponectin. This powerful combination of in vitro and in vivo techniques will provide insight into adiponectin signal transduction in endothelial cells and may lead to new targets to reduce the heightened vascular risk associated with type 1 diabetes.

DESCRIPTION (provided by applicant): Cardiovascular disease accounts for an overwhelming proportion of the morbidity and mortality suffered by patients with diabetes mellitus. Insulin resistance in obesity and type 2 diabetes, characterized by excess circulating non-esterified fatty acids (NEFA) and cytokines such as TNFalpha, and the hyperglycemia of overt diabetes are associated with endothelial dysfunction that contributes to the atherosclerotic process. Adiponectin is an abundant plasma protein secreted from adipose tissue that exhibits potent anti-inflammatory effects in the vasculature as well as insulin-sensitizing properties in metabolically-active tissues. In extensive preliminary studies, we have made the novel observations that the recombinant globular domain of adiponectin (gAd) exhibits a number of salutary effects in cultured endothelial cells, including enhanced nitric oxide (NO) production associated with activation of AMP kinase, and reduced superoxide generation induced by oxidized LDL (oxLDL) which is associated with cellular NAD(P)H oxidase activity. In addition, gAd inhibits superoxide production in endothelial cells exposed to high glucose, blocks the oxidation of native LDL by endothelial cells, and suppresses cell proliferation and MAP kinase activation stimulated by oxLDL. By quantitative intravital microscopy in the db/db mouse, we have also found in our initial studies that overexpression of gAd by adenoviral gene delivery in vivo ameliorates the increased leukocyte/endothelial interactions characteristic of the endothelial dysfunction in this model of insulin resistant type 2 diabetes. We propose here to combine studies of endothelial cells in vitro with intravital microscopy in situ to examine the cellular responses and signaling mechanisms of the two major forms of adiponectin (full-length and gAd) and test the hypotheses that adiponectin: (1) enhances NO production in states of endothelial dysfunction via an AMP kinase-linked pathway; (2) suppresses superoxide production by endothelial cells treated with oxLDL or high glucose, possibly via an NAD(P)H oxidase-linked pathway; and (3) ameliorates endothelial dysfunction in vivo in rodent models of obesity with insulin resistance and/or diabetes as evidenced by salutary effects on leukocyte/endothelial interactions, expression of cell adhesion molecules and NO production. These studies will provide insight into the cellular mechanisms employed by adiponectin to ameliorate endothelial dysfunction in states of insulin resistance and type 2 diabetes, and may lead to improved strategies to reduce the excessive cardiovascular risk associated with these disorders.

DESCRIPTION (provided by applicant): Reversible tyrosine phosphorylation of the insulin receptor and its cellular substrate proteins is a fundamental regulatory process for insulin signal transduction. The long-term goals of this project are to elucidate the signaling roles and cellular regulation of specific protein-tyrosine phosphatases (PTPs) in insulin action and in pathological states of insulin resistance. During the last funding cycle, we have identified a novel regulatory pathway whereby H2O2 generated by cellular insulin stimulation leads to the reversible inhibition of specific PTPs, via oxidation of the catalytic thiol moiety, which enhances receptor substrate tyrosine phosphorylation and facilitates insulin action. In particular, this process involves PTP1B, an intracellular PTP strongly implicated as a negative regulator of insulin signaling; additional oxidation-sensitive signaling proteins are also likely be regulated by this process. We have provided compelling evidence that the NADPH oxidase homolog Nox4 is the source of insulin-stimulated H2O2 and the redox regulation of PTP catalytic activity in insulin-sensitive cells. The current revised proposal will test four major hypotheses: (1) Nox4 plays an integral role in insulin-stimulated H2O2 generation and is coupled to specific molecular components shown to regulate other NADPH oxidases, including p22phox, Rac, Galphai2, NOXO1, NOXA1 or related proteins; (2) Insulin stimulation of NADPH oxidase in isolated plasma membranes is functionally linked to specific Nox regulatory subunits; that high glucose enhances insulin-stimulated cellular H2O2 by PKC stimulation of Nox4; (3) Global loss of Nox4 expression in knock-out mice, or tissue-specific loss of Nox4 in transgenic models leads to impaired insulin signaling due to excess PTP activity in vivo; (4) Insulin-stimulated H2O2 leads to the oxidation and reversible inactivation of a limited set of susceptible cellular proteins involved in insulin signal transduction (including PTPs) that can be identified using novel biochemical reagents. Each of these aims will provide insight into novel regulatory mechanisms that may help identify new targets to enhance insulin sensitivity in common insulin-resistant states such as obesity and type 2 diabetes.

DESCRIPTION (provided by applicant): Atherosclerotic cardiovascular disease remains the major cause of death in patients with type 1 and type 2 diabetes mellitus (T1DM, T2DM). Atherosclerosis arises from the retention of cholesterol-rich, apolipoprotein-B (apoB)-lipoproteins within the vessel wall. Importantly, diabetic patients suffer from a unique and typically neglected aspect of cardiovascular risk, namely, the striking persistence of intestinally derived apoB-lipoproteins, called `remnants,'in their plasma after each meal. The cause is a defect in hepatic clearance of these harmful particles. A major impediment in this area has been our ignorance regarding pathways for remnant uptake into the liver. A quarter century ago, hepatic uptake of remnants was shown to be independent of LDL receptors. This realization launched a long, difficult search for the responsible molecules. In 1991-1992, seminal work from our laboratory implicated heparan sulfate proteoglycans (HSPGs) in remnant lipoprotein uptake. Each HSPG molecule consists of a protein strand onto which the cell assembles sugar polymers, called heparan sulfate, that we showed could capture lipoproteins. In a major, recent breakthrough, we found that T2DM induces HSPG degradative enzymes in liver. Our central hypothesis is that the identification of compounds that accelerate uptake of atherogenic remnant lipoproteins into liver cells will substantially advance our physiologic understanding while also opening exciting, new avenues for potentially life-saving therapeutics in diabetes. In Aim 1, we propose to develop screens for inducers of hepatocyte uptake of model remnant lipoproteins. Compound screens using whole-cell read-outs are the ideal approach to manage the biologic complexity of HSPG assembly. Aim 1a will automate and optimize our new fluorescent assay for HSPG-mediated uptake of model remnant lipoproteins, in preparation for moderate-throughput screening. Aim 1b will perform titration-based screening of three libraries of diverse, active compounds. In Aim 2, we propose to evaluate hit compounds. Aim 2a will rule out artifacts, establish selectivity for HSPG-mediated uptake, and prioritize compounds. Aim 2b will determine the molecular effects of prioritized compounds. Based on our new data, our favored mechanism is inhibition of HSPG degradative enzymes. Aim 2c will define metabolic responses to enhanced uptake of remnants, to choose potential therapeutic leads. Overall, these proposed Aims will substantially advance our molecular knowledge of remnant lipoprotein clearance, as well as our ability to correct diabetic postprandial dyslipidemia. Our ultimate goal will be to avert the tremendous excess burden of cardiovascular disease in diabetes, to which postprandial dyslipidemia makes a substantial, and potentially avoidable, contribution. PUBLIC HEALTH RELEVANCE: Project relevance to public health Patients with diabetes mellitus suffer from fatal and disabling atherosclerotic cardiovascular disease that results in part from the striking persistence of harmful intestinally derived lipoproteins, called `remnants,'in their plasma after each meal. Based on our seminal work implicating a crucial role for heparan sulfate proteoglycans (HSPGs) in the rapid, healthy disposal of remnant lipoproteins by the liver, we now seek novel compounds to enhance HSPG display by hepatocytes and thereby accelerate the uptake of these harmful lipoproteins. The ultimate goal will be to avert the tremendous excess burden of cardiovascular disease in diabetes.

DESCRIPTION (provided by applicant): Atherosclerotic cardiovascular disease remains the major cause of death in patients with type 1 and type 2 diabetes mellitus (T1DM, T2DM). Atherosclerosis arises from the retention of cholesterol-rich, apolipoprotein-B (apoB)-containing lipoproteins within the vessel wall. Importantly, diabetic patients suffer from a unique and typically neglected aspect of cardiovascular risk, namely, the striking persistence of intestinally derived apoB-lipoproteins, called 'remnants,'in their plasma after each meal. The cause is a defect in hepatic clearance of these harmful particles. A major impediment in this area has been our ignorance regarding pathways for remnant uptake into liver. Over a quarter century ago, hepatic uptake of remnants was shown to be independent of LDL receptors. This realization launched a long, difficult search for the responsible molecules. In 1991-1992, seminal work from our laboratory implicated heparan sulfate proteoglycans (HSPGs) in remnant lipoprotein uptake. Each HSPG molecule consists of a protein strand onto which the cell assembles sugar polymers, called heparan sulfate, that we showed could capture lipoproteins. Despite the existence of roughly 50 genes that are directly involved in hepatic HSPG assembly and disassembly, our results so far indicate dysregulation of only two of them in diabetes. Moreover, T1DM and T2DM induce distinct molecular derangements. First, we identified Ndst1, a key enzyme in heparan sulfate assembly, as specifically suppressed in T1DM liver in vivo. Second, in a major, recent breakthrough, we found that T2DM induces a novel HSPG degradative enzyme in liver. Thus, our central hypothesis is that the atherogenic, postprandial dyslipidemias of T1DM and T2DM each arise from dysregulation of a surprisingly small number of key molecules that directly affect hepatic HSPG structure. Aim 1 will use specific gene transfer to test the hypothesis that Ndst1 suppression is responsible for impaired remnant clearance in T1DM. Because Ndst1 deficiency can mask defects in other HSPG assembly enzymes, we will compre- hensively characterize hepatic HSPG structure, molecular biology, and function as remnant recep- tors in vivo in T1DM, without and with Ndst1 gene transfer. Aim 2 will use a specific knock-down in vivo to test the hypothesis that the overexpressed degradative enzyme impairs remnant clearance in T2DM. To ensure a comprehensive survey, we will characterize hepatic HSPG fine structure, molecular biology, and postprandial dyslipidemia in T2DM, without and with the knock-down. Overall, these proposed Aims will define the structural and molecular derangements in HSPG assembly that are responsible for diabetic postprandial dyslipidemias. The work will expand our understanding of excess cardiovascular disease in diabetes and provide novel therapeutic targets. PUBLIC HEALTH RELEVANCE: Project relevance to public health Patients with type 1 and type 2 diabetes mellitus suffer from fatal and disabling atherosclerotic cardiovascular disease that results in part from the striking persistence of harmful intestinally derived lipoproteins, called 'remnants,'in their plasma after each meal. Based on our seminal work implicating a crucial role for heparan sulfate proteoglycans (HSPGs) in the rapid, healthy disposal of remnant lipoproteins by the liver, we now seek to characterize the structural and molecular derangements responsible for impaired hepatic HSPG function in T1DM and T2DM. By expanding our understanding of the pathophysiology of diabetic postprandial dyslipidemias, we may be able to avert the tremendous excess burden of cardiovascular disease in diabetes.

DESCRIPTION (provided by applicant): The overwhelming majority of patients with type 2 diabetes mellitus (T2DM) and related syndromes die from accelerated atherosclerosis. These patients exhibit a striking persistence of postprandial TG-rich lipoproteins, called 'remnants,' in their plasma after each meal. Importantly, remnants have been linked to human cardiovascular events. Because the basis for delayed remnant clearance in T2DM patients has been poorly understood, no therapeutic strategies are available to target these harmful particles. Our laboratory has made a series of fundamental advances in this area. First, we identified the syndecan-1 heparan sulfate proteoglycan (HSPG) as a remnant receptor. Second, using an array, we found dysregulation of exactly one gene that would impair syndecan-1 function in remnant clearance - namely, sulfatase-2 (Sulf2), which is 10-fold overexpressed in T2DM liver. SULF2 impedes syndecan-1-mediated catabolism of remnants by liver cells. Third, we just published that inhibition of hepatic Sulf2 in vivo flattens plasma TG excursions after corn-oil gavage in T2DM mice. Fourth, we discovered that insulin suppresses SULF2 protein posttranscriptionally, and that this effect becomes insulin-resistant in T2DM liver owing to a defect in NOX4 function that impairs AKT activation. By focusing on SULF2, we will improve our under- standing of postprandial dyslipidemia and facilitate the translation of our work into clinicl utility. Aim 1: Role of sulfatase-2 in atherosclerosis: lipoprotein and non-lipoprotein effects. Hypothesis 1: Inhibition of SULF2 will slow atherosclerosis through two effects: in the liver by improving the plasma lipoprotein profile and in the arterial wall by impeding local pro-atherogenic signaling pathways, particularly Wnt. We will examine atherosclerotic lesion development in SULF2-deficient mice, bred into major atherosclerosis models. Beneficial effects of SULF2 deficiency will further bolster our therapeutic focus. Aim 2: Molecular mechanisms for overexpression of sulfatase-2 in T2DM liver. Hypothesis 2: Hepatic SULF2 overexpression in T2DM occurs through key nodes that are potential therapeutic targets. We will define the crucial signaling intermediates downstream of the insulin receptor that normally suppress SULF2, as well as novel molecular mediators that affect SULF2 protein synthesis and degradation. Aim 3: Novel strategies to correct hepatic SULF2 overexpression in T2DM liver, and hence attenuate postprandial dyslipoproteinemia. Hypothesis 3: Inhibition of SULF2 is a viable therapeutic strategy, and we will take this concept beyond our previous ASO method. In Aim 3a, we will correct hepatic insulin signaling defects in T2DM liver in vivo, focusing on chaperones of NOX4 that are dysregulated in T2DM. Aim 3b will manipulate in vivo the novel participants in SULF2 regulation that we identify in Aim 2. Overall, these proposed Aims will substantially advance our molecular understanding and our abilities to correct the devastating burden of accelerated atherosclerosis in T2DM.