Central Role of ß-1,4-GalT-V in Cancer Signaling, Inflammation, and Other Disease-Centric Pathways.

UDP-Galactose: Glucosylceramide, ß-1,4-Galactose transferase-V (ß-1,4-GalT-V), is a member of a large glycosyltransferase family, primarily involved in the transfer of sugar residues from nucleotide sugars, such as galactose, glucose mannose, etc., to sugar constituents of glycosphingolipids and glycoproteins. For example, UDP-Galactose: Glucosylceramide, ß-1,4-galactosyltransferase (ß-1,4-GalT-V), transfers galactose to glucosylceramide to generate Lactosylceramide (LacCer), a bioactive "lipid second messenger" that can activate nicotinamide adenine dinucleotide phosphate(NADPH) oxidase (NOX-1) to produce superoxide's (O2-) to activate several signaling pathways critical in regulating multiple phenotypes implicated in health and diseases. LacCer can also activate cytosolic phospholipase A-2 to produce eicosanoids and prostaglandins to induce inflammatory pathways. However, the lack of regulation of ß-1,4-GalT-V contributes to critical phenotypes central to cancer and cardiovascular diseases, e.g., cell proliferation, migration, angiogenesis, phagocytosis, and apoptosis. Additionally, inflammation that accompanies ß-1,4-GalT-V dysregulation accelerates the initiation and progression of cancer, cardiovascular diseases, as well as inflammation-centric diseases, like lupus erythematosus, chronic obstructive pulmonary disease (COPD), and inflammatory bowel diseases. An exciting development in this field of research arrived due to the recognition that the activation of ß-1,4-GalT-V is a "pivotal" point of convergence for multiple signaling pathways initiated by physiologically relevant molecules, e.g., growth factors, oxidized-low density lipoprotein(ox- LDL), pro-inflammatory molecules, oxidative and sheer stress, diet, and cigarette smoking. Thus, dysregulation of these pathways may well contribute to cancer, heart disease, skin diseases, and several inflammation-centric diseases in experimental animal models of human diseases and in humans. These observations have been described under post-transcriptional modifications of ß-1,4- GalT-V. On the other hand, we also point to the important role of ß-1-4 GalT-V-mediated glycosylation in altering the formation of glycosylated precursor forms of proteins and their activation, e.g., ß-1 integrin, wingless-related integration site (Wnt)/-ß catenin, Frizzled-1, and Notch1. Such alterations in glycosylation may influence cell differentiation, angiogenesis, diminished basement membrane architecture, tissue remodeling, infiltrative growth, and metastasis in human colorectal cancers and breast cancer stem cells. We also discuss Online Mendelian Inheritance in Man (OMIM), which is a comprehensive database of human genes and genetic disorders used to provide information on the genetic basis of inherited diseases and traits and information about the molecular pathways and biological processes that underlie human physiology. We describe cancer genes interacting with the ß-1,4-GalT-V gene and homologs generated by OMIM. In sum, we propose that ß-1,4-GalT-V gene/protein serves as a "gateway" regulating several signal transduction pathways in oxidative stress and inflammation leading to cancer and other diseases, thus rationalizing further studies to better understand the genetic regulation and interaction of ß-1,4-GalT-V with other genes. Novel therapies will hinge on biochemical analysis and characterization of ß-1,4-GalT-V in patient-derived materials and animal models. And using ß-1,4-GalT-V as a "bonafide drug target" to mitigate these diseases.

Glycosphingolipids in Diabetes, Oxidative Stress, and Cardiovascular Disease: Prevention in Experimental Animal Models.

Diabetes contributes to about 30% morbidity and mortality world-wide and has tidal wave increases in several countries in Asia. Diabetes is a multi-factorial disease compounded by inflammation, dyslipidemia, atherosclerosis, and is sometimes accompanied with gains in body weight. Sphingolipid pathways that interplay in the enhancement of the pathology of this disease may be potential therapeutic targets. Thus, the application of advanced sphingolipidomics may help predict the progression of this disease and therapeutic outcomes in man. Pre-clinical studies using various experimental animal models of diabetes provide valuable information on the role of sphingolipid signaling networks in diabetes and the efficacy of drugs to determine the translatability of innovative discoveries to man. In this review, we discuss three major concepts regarding sphingolipids and diabetes. First, we discuss a possible involvement of a monosialodihexosylceramide (GM3) in insulin-insulin receptor interactions. Second, a potential role for ceramide (Cer) and lactosylceramide (LacCer) in apoptosis and mitochondrial dysfunction is proposed. Third, a larger role of LacCer in antioxidant status and inflammation is discussed. We also discuss how inhibitors of glycosphingolipid synthesis can ameliorate diabetes in experimental animal models.

Convergence: Lactosylceramide-Centric Signaling Pathways Induce Inflammation, Oxidative Stress, and Other Phenotypic Outcomes.

Lactosylceramide (LacCer), also known as CD17/CDw17, is a member of a large family of small molecular weight compounds known as glycosphingolipids. It plays a pivotal role in the biosynthesis of glycosphingolipids, primarily by way of serving as a precursor to the majority of its higher homolog sub-families such as gangliosides, sulfatides, fucosylated-glycosphingolipids and complex neutral glycosphingolipids-some of which confer "second-messenger" and receptor functions. LacCer is an integral component of the "lipid rafts," serving as a conduit to transduce external stimuli into multiple phenotypes, which may contribute to mortality and morbidity in man and in mouse models of human disease. LacCer is synthesized by the action of LacCer synthase (ß-1,4 galactosyltransferase), which transfers galactose from uridine diphosphate galactose (UDP-galactose) to glucosylceramide (GlcCer). The convergence of multiple physiologically relevant external stimuli/agonists-platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), stress, cigarette smoke/nicotine, tumor necrosis factor-α (TNF-α), and in particular, oxidized low-density lipoprotein (ox-LDL)-on ß-1,4 galactosyltransferase results in its phosphorylation or activation, via a "turn-key" reaction, generating LacCer. This newly synthesized LacCer activates NADPH (nicotinamide adenine dihydrogen phosphate) oxidase to generate reactive oxygen species (ROS) and a highly "oxidative stress" environment, which trigger a cascade of signaling molecules and pathways and initiate diverse phenotypes like inflammation and atherosclerosis. For instance, LacCer activates an enzyme, cytosolic phospholipase A2 (cPLA2), which cleaves arachidonic acid from phosphatidylcholine. In turn, arachidonic acid serves as a precursor to eicosanoids and prostaglandin, which transduce a cascade of reactions leading to inflammation-a major phenotype underscoring the initiation and progression of several debilitating diseases such as atherosclerosis and cancer. Our aim here is to present an updated account of studies made in the field of LacCer metabolism and signaling using multiple animal models of human disease, human tissue, and cell-based studies. These advancements have led us to propose that previously unrelated phenotypes converge in a LacCer-centric manner. This LacCer synthase/LacCer-induced "oxidative stress" environment contributes to inflammation, atherosclerosis, skin conditions, hair greying, cardiovascular disease, and diabetes due to mitochondrial dysfunction. Thus, targeting LacCer synthase may well be the answer to remedy these pathologies.

Management of metabolic syndrome and reduction in body weight in type II diabetic mice by inhibiting glycosphingolipid synthesis.

Metabolic syndrome is defined by hyperlipidemia and cardiovascular complications. We have examined whether inhibition of glycosphingolipid synthesis can interfere with metabolic syndrome in a male mouse model of type II diabetes (db/db). The db/db and control mice (C57/BL6) (n = 6) fed chow for 30 weeks received vehicle (5% Tween-80 in PBS; 100 µl), or a biopolymer-encapsulated D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (BPD) glycosphingolipid synthesis inhibitor daily via oral gavage for 6 weeks. Echocardiography revealed increased Ao-IMT in db/db mice compared to control. However, BPD decreased Ao-IMT, monohexosylceramide and dihexosylceramide, LDL, triglycerides, glucose, and raised HDL levels in db/db mice. This was due to increased gene expression of HMG-CoA reductase, LDLr, SREBP2, and bile acids: Cy7-a hydroxylase, LXR and FXR, lipoprotein lipase, VLDL receptor and PPAR. Treatment also increased the expression of superoxide dismutase-II to reduce the pro-oxidant status in these mice. We observed that decreased cholesterol levels correlated with decreased cholesterol sensing proteins e.g. NPC1 gene/protein expression and mammalian target of rapamycin (mTORC-1) and reduced body weight. Thus, glycosphingolipid synthesis inhibition is a novel approach to manage metabolic syndrome and reduce body weight in diabetic mice and with potential applications in humans.

Lactosylceramide synthase ß-1,4-GalT-V: A novel target for the diagnosis and therapy of human colorectal cancer.

Little is known about an oncogenic signal transducer ß-1,4-galactosyltransferase-V (ß-1,4-GalT-V), in human colorectal cancer. Using quantitative RT-PCR, immunohistochemical staining and ELISA assays, we determined that ß-1,4-GalT-V gene/protein expression is specifically increased in human colorectal cancer (CRC) tumors, compared to visibly normal tissue. Furthermore, we observed a marked increase in its enzymatic activity, and its product lactosylceramide. Moreover, we found increased dihydrosphingolipid metabolites, in particular dihydrosphingomyelin in cancer tissue compared to normal. Further, inhibition of glycosphingolipid synthesis by the synthetic ceramide analog, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), concurrently inhibited colorectal cancer cell (HCT-116) proliferation, as well as ß-1,4-GalT-V mass and several glycosphingolipid levels. We conclude that ß-1,4-GalT-V may serve as a diagnostic and therapeutic biomarker for the progression of human colorectal cancer, and consequently, inhibition of GSL synthesis may be a novel approach for the treatment of this life-threatening disease.

Inhibition of glycosphingolipid synthesis ameliorates atherosclerosis and arterial stiffness in apolipoprotein E-/- mice and rabbits fed a high-fat and -cholesterol diet.

BACKGROUND: Glycosphingolipids, integral components of the cell membrane, have been shown to serve as messengers, transducing growth factor-initiated phenotypes. Here, we have examined whether inhibition of glycosphingolipid synthesis could ameliorate atherosclerosis and arterial stiffness in transgenic mice and rabbits. METHODS AND RESULTS: Apolipoprotein E(-/-) mice (12 weeks of age; n=6) were fed regular chow or a Western diet (1.25% cholesterol, 2% fat). Mice were fed 5 or 10 mg/kg of an inhibitor of glycosphingolipid synthesis, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), solubilized in vehicle (5% Tween-80 in PBS); the placebo group received vehicle only. At 20 and 36 weeks of age, serial echocardiography was performed to measure aortic intima-media thickening. Aortic pulse-wave velocity measured vascular stiffness. Feeding mice a Western diet markedly increased aortic pulse-wave velocity, intima-media thickening, oxidized low-density lipoprotein, Ca(2+) deposits, and glucosylceramide and lactosylceramide synthase activity. These were dose-dependently decreased by feeding D-PDMP. In liver, D-PDMP decreased cholesterol and triglyceride levels by raising the expression of SREBP2, low-density lipoprotein receptor, HMGCo-A reductase, and the cholesterol efflux genes (eg, ABCG5, ABCG8). D-PDMP affected very-low-density lipoprotein catabolism by increasing the gene expression for lipoprotein lipase and very-low-density lipoprotein receptor. Rabbits fed a Western diet for 90 days had extensive atherosclerosis accompanied by a 17.5-fold increase in total cholesterol levels and a 3-fold increase in lactosylceramide levels. This was completely prevented by feeding D-PDMP. CONCLUSIONS: Inhibition of glycosphingolipid synthesis ameliorates atherosclerosis and arterial stiffness in apolipoprotein E(-/-) mice and rabbits. Thus, inhibition of glycosphingolipid synthesis may be a novel approach to ameliorate atherosclerosis and arterial stiffness.

Integrated glycoprotein immobilization method for glycopeptide and glycan analysis of cardiac hypertrophy.

Post-translational modifications of proteins can have a major role in disease initiation and progression. Incredible efforts have recently been made to study the regulation of glycoproteins for disease prognosis and diagnosis. It is essential to elucidate glycans and intact glycoproteins to understand the role of glycosylation in diseases. Sialylated N-glycans play crucial roles in physiological and pathological processes; however, it is laborious to study sialylated glycoproteins due to the labile nature of sialic acid residues. In this study, an integrated platform is developed for the analysis of intact glycoproteins and glycans using a chemoenzymatic approach for immobilization and derivatization of sialic acids. N-Glycans, deglycosylated proteins, and intact glycoproteins from heart tissues of wild type (WT) and transverse aortic constriction (TAC) mouse models were analyzed. We identified 291 unique glycopeptides from 195 glycoproteins; the comparative studies between WT and TAC mice indicate the overexpression of extracellular proteins for heart matrix remodeling and the down-regulation of proteins associated with energy metabolism in cardiac hypertrophy. The integrated platform is a powerful tool for the analysis of glycans and glycoproteins in the discovery of potential cardiac hypertrophy biomarkers.

Prevention of cardiac hypertrophy by the use of a glycosphingolipid synthesis inhibitor in ApoE-/- mice.

ApoE-/- mice fed a high fat and high cholesterol (HFHC) diet (20% fat and 1.25% cholesterol) from 12 weeks of age to 36 weeks revealed an age-dependent increase in the left ventricular mass (LV mass) and decline in fractional shortening (FS%), which worsened with HFHC diet. These traits are indicative of maladaptive pathological cardiac hypertrophy and dysfunction. This was accompanied by loading of glycosphingolipids and increased gene expression of ANP, BNP in myocardial tissue. Masson's trichrome staining revealed a significant increase in cardiomyocyte size and fibrosis. In contrast, treatment with 5 and 10 µM D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of glucosylceramide synthase and lactosylceramide synthase, dose-dependently decreased the load of glycosphingolipids and preserved fractional shortening and maintained left ventricular mass to normal 12-week-old control levels over a 6 month treatment period. Our mechanistic studies showed that D-PDMP inhibited cardiac hypertrophy by inhibiting the phosphorylation of mitogen-activated protein kinase (MAPK). We propose that associating increased glycosphingolipid synthesis with cardiac hypertrophy could serve as a novel approach to prevent this phenotype in experimental animal models of diet -induced atherosclerotic heart disease.

Molecular imaging of inflammation in the ApoE -/- mouse model of atherosclerosis with IodoDPA.

BACKGROUND: Atherosclerosis is a common and serious vascular disease predisposing individuals to myocardial infarction and stroke. Intravascular plaques, the pathologic lesions of atherosclerosis, are largely composed of cholesterol-laden luminal macrophage-rich infiltrates within a fibrous cap. The ability to detect those macrophages non-invasively within the aorta, carotid artery and other vessels would allow physicians to determine plaque burden, aiding management of patients with atherosclerosis. METHODS AND RESULTS: We previously developed a low-molecular-weight imaging agent, [(125)I]iodo-DPA-713 (iodoDPA), which selectively targets macrophages. Here we use it to detect both intravascular macrophages and macrophage infiltrates within the myocardium in the ApoE -/- mouse model of atherosclerosis using single photon emission computed tomography (SPECT). SPECT data were confirmed by echocardiography, near-infrared fluorescence imaging and histology. SPECT images showed focal uptake of radiotracer at the aortic root in all ApoE -/- mice, while the age-matched controls were nearly devoid of radiotracer uptake. Focal radiotracer uptake along the descending aorta and within the myocardium was also observed in affected animals. CONCLUSIONS: IodoDPA is a promising new imaging agent for atherosclerosis, with specificity for the macrophage component of the lesions involved.

Lactosylceramide promotes hypertrophy through ROS generation and activation of ERK1/2 in cardiomyocytes.

Hypertrophy is central to several heart diseases; however, not much is known about the role of glycosphingolipids (GSLs) in this phenotype. Since GSLs have been accorded several physiological functions, we sought to determine whether these compounds affect cardiac hypertrophy. By using a rat cardiomyoblast cell line, H9c2 cells and cultured primary neonatal rat cardiomyocytes, we have determined the effects of GSLs on hypertrophy. Our study comprises (a) measurement of [(3)H]-leucine incorporation into protein, (b) measurement of cell size and morphology by immunofluorescence microscopy and (c) real-time quantitative mRNA expression assay for atrial natriuretic peptide and brain natriuretic peptide. Phenylephrine (PE), a well-established agonist of cardiac hypertrophy, served as a positive control in these studies. Subsequently, mechanistic studies were performed to explore the involvement of various signaling transduction pathways that may contribute to hypertrophy in these cardiomyocytes. We observed that lactosylceramide specifically exerted a concentration- (50-100 µM) and time (48 h)-dependent increase in hypertrophy in cardiomyocytes but not a library of other structurally related GSLs. Further, in cardiomyocytes, LacCer generated reactive oxygen species, stimulated the phosphorylation of p44 mitogen activated protein kinase and protein kinase-C, and enhanced c-jun and c-fos expression, ultimately leading to hypertrophy. In summary, we report here that LacCer specifically induces hypertrophy in cardiomyocytes via an "oxygen-sensitive signal transduction pathway."

Glycosphingolipid-associated ß-1,4 galactosyltransferase is elevated in patients with systemic lupus erythematosus.

OBJECTIVE: ß-1,4 galactosyltransferase-V (ß-1,4 GalT-V) is an enzyme that synthesises a glycosphingolipid known as lactosylceramide, which has been implicated in general inflammation and atherosclerosis. We asked if ß-1,4 GalT-V was present at elevated levels in patients with SLE, a disease which is associated with increased risk of atherosclerosis. METHODS: In this case-control observational study, serum samples were obtained from patients with SLE who are part of the Johns Hopkins Lupus Cohort. Control serum samples were obtained from healthy adult community members recruited from the Baltimore area. All serum samples (n=50 in the SLE group and n=50 in the healthy control group) were analysed with enzyme-linked immunoassays. These assays used antibodies raised against antigens that enabled us to measure the absorbance of oxidised phosphocholines per apolipoprotein B-100 (ox-PC/apoB) and the concentration of lipoprotein(a) (Lp(a)) and ß-1,4 GalT-V. RESULTS: Absorbance of ox-PC/apoB and concentrations of Lp(a) and ß-1,4 GalT-V were significantly higher in the SLE serum samples as compared with the control serum (p<0.0001). CONCLUSIONS: We conclude that patients with SLE have elevated levels of ß-1,4 GalT-V and ox-PC, which have previously been recognised as risk factors for atherosclerosis.

The Yin and Yang of lactosylceramide metabolism: implications in cell function.

Although lactosylceramide (LacCer) plays a pivotal role in the biosynthesis of nearly all the major glycosphingolipids, its function in regulating cellular function has begun to emerge only recently. Our current opinion is that several physiologically critical molecules such as modified/oxidized LDL, growth factors, pro-inflammatory cytokines and fluid shear stress converge upon and activate lactosylceramide synthase to generate LacCer. In turn, LacCer activates an "oxygen-sensitive" signaling pathway involving superoxides, nitric oxide, p21 Ras GTP loading, kinase cascade, PI3kinase/Akt activation, nuclear factor up-regulation ultimately contributing to phenotypic changes such as cell proliferation, adhesion, migration and angiogenesis. Since dys-regulation of such phenotypic changes constitute a hallmark in several diseases of the cardiovascular system, proliferative disorders such as cancer, polycystic kidney disease and inflammatory diseases, LacCer synthase and LacCer provide novel targets for the development of therapeutics aimed at these health conditions.

VEGF recruits lactosylceramide to induce endothelial cell adhesion molecule expression and angiogenesis in vitro and in vivo.

Angiogenesis is largely driven by vascular endothelial growth factor (VEGF). However, the role of lipid second messengers such as lactosylceramide (LacCer) and LacCer synthase in angiogenesis is not well understood. We have determined the distribution of various LacCer synthase mRNA transcripts using sequential analysis of gene expression (SAGE). Endothelial cells from colon cancer tissues had a 4.5-fold increase in a LacCer synthase transcript (beta1,4GalT-V) as compared to normal colon tissue endothelial cells. Consequently, our focus turned to understanding the role of this enzyme in regulating VEGF-induced angiogenesis in vitro and in vivo. Herein, we show that in human endothelial cells, VEGF-induced angiogenesis is mitigated by dimethylsphingosine and suramin; inhibitors of sphingosine kinase 1(SphK-1) and sphingosine1-phosphate receptor 1(S1P (1)), respectively, and this were bypassed by LacCer but not by S1P. VEGF and basic fibroblast growth factor-induced angiogenesis was mitigated by PDMP; an inhibitor of glucosylceramide synthase and LacCer synthase in human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC). Likewise, GalT-V gene ablation using corresponding siRNA also mitigated VEGF-induced angiogenesis. In Matrigel plug angiogenesis assay in nude mice, angiogenesis was markedly inhibited by D-PDMP with concordantly diminished LacCer synthase activity. Mechanistic studies revealed that the use of LY294002, a PI3 kinase inhibitor, mitigated VEGF-induced expression of platelet-endothelial cell adhesion molecule (PECAM-1/CD31); the trans-endothelial migration of a monocyte cell line (U-937) and angiogenesis in HAEC cells. Since this enzyme is a target for VEGF action and LacCer serves as a lipid second messenger in inducing angiogenesis in vitro and in vivo, novel therapeutic approaches may be developed using our findings to mitigate colon cancer.

Use of a novel anti-proliferative compound coated on a biopolymer to mitigate platelet-derived growth factor-induced proliferation in human aortic smooth muscle cells: comparison with sirolimus.

Drug eluting stents (DES) have become a common mode of treatment for stenosis in coronary arteries. However, currently, the use of sirolimus/paclitaxel-coated DES has come under scrutiny, because of their pro-thrombotic effects leading to potential adverse outcomes in the long run. We have previously documented that D: -threo-1-phenyl-2-decanoylamino-3-morholino propanol (D-PDMP); an inhibitor of glucosylceramide synthase and lactosylceramide (LacCer) synthase markedly inhibited platelet-derived growth factor (PDGF)-induced cell proliferation. We have fabricated DES wherein, D-PDMP or sirolimus was coated on to a double layer of poly (lactic-co-glycolic acid) on a bare metal stent. The in vitro release of D-PDMP from biopolymer and its consequent effect on PDGF induced proliferation and apoptosis was assessed in human aortic smooth muscle cells (ASMC). D-PDMP was released from biopolymers in a dose-dependent fashion and was accompanied with a decrease in PDGF-induced cell proliferation, but not apoptosis. In contrast, sirolimus markedly increased apoptosis in these cells in addition to inhibiting proliferation. Our mechanistic studies revealed that D-PDMP, but not sirolimus decreased the cellular level of glucosyl and lactosylceramide that accompanied inhibition of PDGF-induced cell proliferation. Our short-term (14 days) in vivo studies in rabbits also attested to the safety and biocompatibility of the D-PDMP coated stents. Our data reveal the superiority of D-PDMP coated biopolymers over sirolimus coated biopolymers in mitigating ASMC proliferation. Such D-PDMP coated stents may be useful for localized delivery of drug to mitigate neo-vascular hyperplasia and other proliferative disorders.

Drug eluting stents: friend or foe? A review of cellular mechanisms behind the effects of Paclitaxel and sirolimus eluting stents.

Coronary artery disease continues to be an important cause of mortality and morbidity. Sirolimus and paclitaxel eluting stents have become an important treatment for patients undergoing revascularization from coronary blockages. These drug eluting stents have enjoyed great success initially in preventing recurrences of adverse cardiac events and decreasing the incidences of repeat revascularizations. However, adverse effects, such as thrombosis, emanating from the use of these drug eluting stents has recently come to focus. Hence a better understanding of the mechanism of action of these drugs in preventing restenosis is important for the long term success and potential betterment of drug eluting stent technology. Herein we review and discuss the pathophysiology of restenosis, the basic mechanism of action of sirolimus and paclitaxel eluting stents and their limitations so as to create a scope for more efficient and novel drug eluting stents in the future.

The Glycoproteomics-MS for Studying Glycosylation in Cardiac Hypertrophy and Heart Failure.

With recent advancements of analytical techniques and mass spectrometric instrumentations, proteomics has been widely exploited to study the regulation of protein expression associated with disease states. Many proteins may undergo abnormal change in response to the stimulants, leading to regulation of posttranslationally modified proteins. In this review, the physiological and pathological roles of protein glycosylation in cardiac hypertrophy is discussed, and how the signal pathways regulate heart function and leading to heart failure. The analytical methods for analysis of protein glycosylation, including glycans, glycosite, occupancy, and heterogeneity is emphasized. The rationale on glycoproteins as disease biomarkers is also discussed. The authors also propose potential research in this field and challenges in the diagnosis and treatment of this disease.

Inhibition of glycosphingolipid synthesis reverses skin inflammation and hair loss in ApoE-/- mice fed western diet.

Sphingolipids have been accorded numerous biological functions however, the effects of feeding a western diet (diet rich in cholesterol and fat) on skin phenotypes, and color is not known. Here, we observed that chronic high-fat and high-cholesterol diet intake in a mouse model of atherosclerosis (ApoE-/-) decreases the level of ceramides and glucosylceramide. At the expense of increased levels of lactosylceramide due to an increase in the expression of lactosylceramide synthase (GalT-V). This is accompanied with neutrophil infiltration into dermis, and enrichment of tumor necrosis factor-stimulated gene-6 (TSG-6) protein. This causes skin inflammation, hair discoloration and loss, in ApoE-/- mice. Conversely, inhibition of glycosphingolipid synthesis, by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), unbound or encapsulated in a biodegradable polymer (BPD) reversed these phenotypes. Thus, inhibition of glycosphingolipid synthesis represents a unique therapeutic approach relevant to human skin and hair Biology.

MRI visualized neo-intimal dissection and co-localization of novel apoptotic markers apolipoprotein C-1, ceramide and caspase-3 in a Watanabe hyperlipidemic rabbit model.

PURPOSE: Apoptotic arterial wall vascular smooth muscle cell death is known to contribute to plaque vulnerability and rupture. Novel apoptotic markers like apolipoprotein C-I have been implicated in apoptotic human vascular smooth muscle cell death via recruiting a neutral sphingomyelinase (N-SMase)-ceramide pathway. In vivo relevance of these observations in an animal model of plaque rupture has not been shown. METHODS AND RESULTS: Using Watanabe rabbits, we investigated three different groups (group 1, three normal Watanabe rabbits; group 2, six Watanabe rabbits fed with high cholesterol diet for 3 months; group 3, five Watanabe rabbits with similar diet but additional endothelial denudation). We followed progression of atherosclerosis to pharmacologically induced plaque rupture non-invasively using novel 3D magnetic resonance Fast-Field-Echo angiography (TR=7.2, TE=3.6 ms, matrix=512 x 512) and Fast-Spin-Echo vessel wall imaging methods (TR=3 heart beats, TE=10.5 ms, matrix=304 x 304) on 1.5 T MRI. MRI provided excellent image quality with good MRI versus histology vessel wall thickness correlation (r=0.8). In six animals of group 2/3 MRI detected neo-intimal dissection in the abdominal aorta which was accompanied by immuno-histochemical demonstration of concomitant aforementioned novel apoptotic markers, previously implicated in the apoptotic smooth muscle cell death in vitro. CONCLUSIONS: Our studies suggest a potential role for the signal transduction pathway involving apolipoprotein C-I for in vivo apoptosis and atherosclerotic plaque rupture visualized by MRI.

High-resolution three-dimensional aortic magnetic resonance angiography and quantitative vessel wall characterization of different atherosclerotic stages in a rabbit model.

PURPOSE: Atherosclerosis results in a considerable medical and socioeconomic impact on society. We sought to evaluate novel magnetic resonance imaging (MRI) angiography and vessel wall sequences to visualize and quantify different morphologic stages of atherosclerosis in a Watanabe hereditary hyperlipidemic (WHHL) rabbit model. MATERIAL AND METHODS: Aortic 3D steady-state free precession angiography and subrenal aortic 3D black-blood fast spin-echo vessel wall imaging pre- and post-Gadolinium (Gd) was performed in 14 WHHL rabbits (3 normal, 6 high-cholesterol diet, and 5 high-cholesterol diet plus endothelial denudation) on a commercial 1.5 T MR system. Angiographic lumen diameter, vessel wall thickness, signal-/contrast-to-noise analysis, total vessel area, lumen area, and vessel wall area were analyzed semiautomatically. RESULTS: Pre-Gd, both lumen and wall dimensions (total vessel area, lumen area, vessel wall area) of group 2 + 3 were significantly increased when compared with those of group 1 (all P < 0.01). Group 3 animals had significantly thicker vessel walls than groups 1 and 2 (P < 0.01), whereas angiographic lumen diameter was comparable among all groups. Post-Gd, only diseased animals of groups 2 + 3 showed a significant (>100%) signal-to-noise ratio and contrast-to-noise increase. CONCLUSIONS: A combination of novel 3D magnetic resonance angiography and high-resolution 3D vessel wall MRI enabled quantitative characterization of various atherosclerotic stages including positive arterial remodeling and Gd uptake in a WHHL rabbit model using a commercially available 1.5 T MRI system.