Plasma Lipoproteins and Preeclampsia in Women with Type 1 Diabetes: A Prospective Study

Context: In nondiabetic pregnancy, cross-sectional studies have shown associations between maternal dyslipidemia and preeclampsia (PE). In type 1 diabetes mellitus (T1DM), the prevalence of PE is increased 4-fold, but prospective associations with plasma lipoproteins are unknown.

Objectives: The aim of this study was to define lipoprotein-related markers and potential mechanisms for PE in T1DM.

Design and Settings: We conducted a multicenter prospective study in T1DM pregnancy.

Patients: We studied 118 T1DM women (26 developed PE, 92 remained normotensive). Subjects were studied at three visits before PE onset [12.2 1.9, 21.6 1.5, and 31.5 1.7 wk gestation (means SD)] and at term (37.6 2.0 wk). Nondiabetic normotensive pregnant women (n 21) were included for reference.

Main Outcome Measures: Conventional lipid profiles, lipoprotein subclasses [defined by size (nuclear magnetic resonance) and by apolipoprotein content], serum apolipoproteins (ApoAI, ApoB, and ApoCIII), and lipolysis (ApoCIII ratio) were measured in T1DM women with and without subsequent PE.

Results: In women with vs. without subsequent PE, at the first and/or second study visits: lowdensity lipoprotein (LDL)-cholesterol, particle concentrations of total LDL and large (but not small) LDL, serum ApoB, and ApoB:ApoAI ratio were all increased (P 0.05); peripheral lipoprotein lipolysis was decreased (P0.01). These early differences remained significant in covariate analysis (glycated hemoglobin, actual prandial status, gravidity, body mass index, and diabetes duration) but were not present at the third study visit. High-density lipoprotein and very low-density lipoprotein subclasses did not differ between groups before PE onset.

Conclusions: Early in pregnancy, increased cholesterol-rich lipoproteins and an index suggesting decreased peripheral lipolysis were associated with subsequent PE in T1DM women. Background maternal lipoprotein characteristics, perhaps masked by effects of late pregnancy, may influence PE risk.

Intraretinal Leakage and Oxidation of LDL in Diabetic Retinopathy

Purpose: The pathogenesis of diabetic retinopathy (DR) is not fully understood. Clinical studies suggest that dyslipidemia is associated with the initiation and progression of DR. However, no direct evidence supports this theory.

Methods: Immunostaining of apolipoprotein B100 (ApoB100, a marker of low-density lipoprotein [LDL]), macrophages, and oxidized LDL was performed in retinal sections from four different groups of subjects: nondiabetic, type 2 diabetic without clinical retinopathy, diabetic with moderate nonproliferative diabetic retinopathy (NPDR), and diabetic with proliferative diabetic retinopathy (PDR). Apoptosis was characterized using the TUNEL assay. In addition, in cell culture studies using in vitro-modi?ed LDL, the induction of apoptosis by heavily oxidized-glycated LDL (HOG-LDL) in human retinal capillary
pericytes (HRCPs) was assessed.

Results: Intraretinal immuno?uorescence of ApoB100 increased with the severity of DR. Macrophages were prominent only in sections from diabetic patients with PDR. Merged images revealed that ApoB100 partially colocalized with macrophages. Intraretinal oxidized LDL was absent in nondiabetic subjects but present in all three diabetic groups, increasing with the severity of DR. TUNEL-positive cells were present in retinas from diabetic subjects but absent in those from nondiabetic subjects. In cell culture, HOG-LDL induced the activation of caspase, mitochondrial dysfunction, and apoptosis in
HRCPs.

Conclusions: These ?ndings suggest a potentially important role for extravasated, modi?ed LDL in promoting DR by promoting apoptotic pericyte loss.

Glycation does not alter LDL-induced secretion of tissue plasminogen activator and plasminogen activator inhibitor-1 from human aortic endothelial cells

Diabetes may induce both quantitative and qualitative changes in lipoproteins, especially low-density lipoprotein (LDL). Effects of LDL glycation on endothelial cell secretion of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) have not been fully elucidated. Human aortic endothelial cell (HAEC) tPA and PAI-1 production were determined after incubation with LDL (50 to 500 microg/mL protein, 24 h) from three sources: (1) nondiabetic LDL (N-LDL) modified in vitro to form six preparations: native, nonmodified (N); glycated (G); minimally oxidized (MO); minimally oxidized and glycated (MOG); heavily oxidized (HO); and heavily oxidized and glycated (HOG); (2) in vivo glycated and relatively nonglycated LDL subfractions from type 1 diabetic patients; (3) LDL from type 1 diabetic patients and matched controls, which was subfractionated using density gradient ultracentrifugation. In experiments using LDL modified in vitro, the rate of tPA release by HAECs incubated with N-LDL (83 +/- 4 ng/mg cell protein/24 h) did not differ significantly from those incubated with G-LDL (73 +/- 7), MO-LDL (74 +/- 13), or MOG-LDL (66 +/- 15) and was not influenced by LDL concentration. The rate of PAI-1 release was similar in HAECs incubated with N-LDL (5.7 +/- 0.6 mug/mg cell protein/24 h), G-LDL (5.7 +/- 0.7), MO-LDL (5.5 +/- 0.8), or MOG-LDL (5.7 +/- 0.9) and was not influenced by LDL concentration. In contrast, tPA release was significantly decreased in cells incubated with LDL (10 microg/mL) modified extensively by oxidation, and averaged 45.2 +/- 5.0 and 43.7 +/- 9.9 ng/mg/24 h for HO-LDL and HOG-LDL, respectively, and was further decreased with increasing concentrations of the heavily oxidized LDL preparations. PAI-1 release was not significantly decreased relative to N-LDL in cells incubated with low concentrations (5 to 50 microg/mL) of HO-LDL and HOG-LDL, but was decreased to 3.2 +/- 0.5 and 3.1 +/- 0.7 microg/mg/24 h for HO-LDL and HOG-LDL at 200 microg/mL, respectively. Results using in vivo glycated versus nonglycated LDL showed that tPA and PAI-1 release did not differ between subfractions. Release of tPA averaged 5.11 +/- 0.6 and 5.12 +/- 0.7 ng/mg/24 h, whereas release of PAI-1 averaged 666 +/- 27 ng/mg/24 h and 705 +/- 30 ng/mg/24 h for nonglycated and glycated LDL subfractions, respectively. Using LDL of different density subclasses, tPA and PAI-1 release in response to LDL from diabetic patients compared with control subjects did not differ when HAECs were incubated with LDLs of increasing density isolated from each subject pair. We conclude that oxidation of LDL, but not glycation, may contribute to the altered fibrinolysis observed in diabetes.

Lipoproteins in the DCCT/EDIC cohort:associations with diabetic nephropathy

Lipoproteins may contribute to diabetic nephropathy. Nuclear magnetic resonance (NMR) can quantify subclasses and mean particle size of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL), and LDL particle concentration. The relationship between detailed lipoprotein analyses and diabetic nephropathy is of interest.

Glycation and oxidation:a role in the pathogenesis of atherosclerosis

Reactions involving glycation and oxidation of proteins and lipids are believed to contribute to atherogenesis. Glycation, the nonenzymatic binding of glucose to protein molecules, can increase the atherogenic potential of certain plasma constituents, including low-density lipoprotein (LDL). Glycation of LDL is significantly increased in diabetic patients compared with normal subjects, even in the presence of good glycemic control. Metabolic abnormalities associated with glycation of LDL include diminished recognition of LDL by the classic LDL receptor; increased covalent binding of LDL in vessel walls; enhanced uptake of LDL by macrophages, thus stimulating foam cell formation; increased platelet aggregation; formation of LDL-immune complexes; and generation of oxygen free radicals, resulting in oxidative damage to both the lipid and protein components of LDL and to any nearby macromolecules. Oxidized lipoproteins are characterized by cytotoxicity, potent stimulation of foam cell formation by macrophages, and procoagulant effects. Combined glycation and oxidation, "glycoxidation," occurs when oxidative reactions affect the initial products of glycation, and results in irreversible structural alterations of proteins. Glycoxidation is of greatest significance in long-lived proteins such as collagen. In these proteins, glycoxidation products, believed to be atherogenic, accumulate with advancing age: in diabetes, their rate of accumulation is accelerated. Inhibition of glycation, oxidation, and glycoxidation may form the basis of future antiatherogenic strategies in both diabetic and nondiabetic individuals.

High-density lipoprotein subfractions display proatherogenic properties in overweight and obese children

BACKGROUND: In adults, obesity-driven inflammation can lead to increased cardiovascular disease (CVD). However, information regarding childhood obesity and its inflammatory sequelae is less well defined. Serum amyloid-A (SAA) is an inflammatory molecule that rapidly associates with high-density lipoproteins (HDLs) and renders them dysfunctional. Therefore, SAA may be a useful biomarker to identify increased CVD potential in overweight and obese children.

METHODS: Young Hearts 2000 is a cross-sectional cohort study in which 92 children who were obese were matched for age and sex with 92 overweight and 92 lean children. HDL2 and HDL3 (HDL2&3) were isolated from plasma by a three-step rapid-ultracentrifugation procedure. SAA was measured in serum and HDL2&3 by an enzyme-linked immunosorbent assay procedure, and the activities of cholesterol ester transfer protein (CETP) and lecithin cholesteryl acyltransferase (LCAT) were measured by fluorimetric assays.

RESULTS: Trends across the groups indicated that SAA increased in serum and HDL2&3 as BMI increased, as did HDL2-CETP and HDL2-LCAT activities.

CONCLUSION: These results have provided evidence that overweight and obese children are exposed to an inflammatory milieu that impacts the antiatherogenic properties of HDL and that could increase CVD risk. This supports the concept that it is important to target childhood obesity to help minimize future cardiovascular events.

Extravascular modified lipoproteins: a role in the propagation of diabetic retinopathy in a mouse model of type 1 diabetes

Aims/hypothesis: We aimed to determine whether plasma lipoproteins, after leakage into the retina and modification by glycation and oxidation, contribute to the development of diabetic retinopathy in a mouse model of type 1 diabetes.

Methods: To simulate permeation of plasma lipoproteins intoretinal tissues, streptozotocin-induced mouse models of diabetes and non-diabetic mice were challenged with intravitreal injection of human ‘highly-oxidised glycated’ low-density lipoprotein (HOG-LDL), native- (N-) LDL, or the vehicle PBS.Retinal histology, electroretinography (ERG) and biochemical markers were assessed over the subsequent 14 days.

Results: Intravitreal administration of N-LDL and PBS had noeffect on retinal structure or function in either diabetic or non-diabetic animals. In non-diabetic mice, HOG-LDL elicited a transient inflammatory response without altering retinal function,but in diabetic mice it caused severe, progressive retinal injury, with abnormal morphology, ERG changes, vascular leakage, vascular endothelial growth factor overexpression, gliosis, endoplasmic reticulum stress, and propensity to apoptosis.

Conclusions/interpretation: Diabetes confers susceptibility to retinal injury imposed by intravitreal injection of modified LDL. The data add to the existing evidence that extravasated, modified plasma lipoproteins contribute to the propagation of diabetic retinopathy. Intravitreal delivery of HOG-LDL simulates a stress known to be present, in addition to hyperglycaemia, in human diabetic retinopathy once blood retinal barriers are compromised.

BELFAST centenarians: A Case of Optimal Cardiovascular Risk?

Introduction: Centenarians are reservoirs of genetic and environmental information to successful ageing and local centenarian groups may help us to understand some of the factors that contribute to longevity. The current centenarian cohort in Belfast survived the 1970s epidemic of death from coronary heart disease in Northern Ireland, where cardiovascular mortality was almost highest in the world. These centenarians provided an opportunity to assess biological and genetic factors important in cardiovascular risk and ageing. Methods: Thirty-five (27 female, 8 male) centenarians, participants of the Belfast Elderly Longitudinal Free-living Ageing STudy (BELFAST), were community-living and of good cognition at enrolment. Results: Centenarians showed median Body Mass Index (BMI) at 25.7, systolic blood pressure 140mmHg and diastolic blood pressure 90mmHg, and fasting glucose of 5.54 mmol/l with no sex-related difference. Lipoproteins showed median cholesterol 5.3, High Density Lipoprotein (HDL) 1.10 and Low Density Lipoprotein (LDL) 3.47umol/l respectively. Centenarian smokers showed no different blood pressure or lipid measurements compared with non-smokers. Malondialdehyde, a measure of lipid peroxidation, was low at 1.19 umol/l, and measures of antioxidant status were varied. Male centenarians did not carry any of the vascular risk genotypes studied-ApoE4 for Apolipoprotein E (ApoE), DD for Angiotensinogen Converting Enzyme (ACE) and tt for 5,10-methylenetetrahydrofolate reductase (MTFHR), though this was not true for female centenarians.. Conclusions: This small local study shows that Belfast centenarians carry a reasonably favourable risk profile, except for age, with respect to cardiovascular disease. There is also some evidence that vascular risk factors and genotypes may be tolerated differently between the male and female centenarians. Maintaining a favourable cardiovascular risk profile seems likely to improve the chance of becoming a centenarian, especially for males.

Saturated and Trans Fatty Acids and Coronary Heart Disease

Dietary intake of both saturated and trans fatty acids has been associated with an increase in the risk of coronary heart disease (CHD). Evidence comes mainly from controlled dietary experiments with intermediate end points, such as blood lipoproteins, and from observational studies. A few small, randomized controlled trials with clinical end points have been carried out in which saturated fat was replaced with polyunsaturated fat, leading to a reduction in low-density lipoprotein cholesterol and a reduction in CHD risk. However, no such studies exist for trans fatty acids. More high-quality, randomized controlled trials on fatty acids and CHD are required, but public health recommendations to reduce intake of both saturated and trans fatty acids are appropriate based on the current evidence.

Low cytosine triphosphate synthase 2 expression renders resistance to 5-fluorouracil in colorectal cancer

Understanding the determinants of resistance of 5-fluorouracil (5FU) is of significant value to optimizing administration of the drug, and introducing novel agents and treatment strategies. Here, the expression of 92 genes involved in 5FU transport, metabolism, co-factor (folate) metabolism and downstream effects was measured by real-time PCR low density arrays in 14 patient-derived colorectal cancer xenografts characterized for 5FU resistance. Candidate gene function was tested by siRNA and uridine modulation, and immunoblotting, apoptosis and cell cycle analysis. Predictive significance was tested by immunohistochemistry of tumors from 125 stage III colorectal cancer patients treated with and without 5FU. Of 8 genes significantly differentially expressed between 5FU sensitive and resistant xenograft tumors, CTPS2 was the gene with the highest probability of differential expression (p = 0.008). Reduction of CTPS2 expression by siRNA increased the resistance of colorectal cancer cell lines DLD1 and LS174T to 5FU and its analog, FUDR. CTPS2 siRNA significantly reduced cell S-phase accumulation and apoptosis following 5FU treatment. Exposure of cells to uridine, a precursor to the CTPS2 substrate uridine triphosphate, also increased 5FU resistance. Patients with low CTPS2 did not gain a survival benefit from 5FU treatment (p = 0.072), while those with high expression did (p = 0.003). Low CTPS2 expression may be a rationally-based determinant of 5FU resistance.

Structural and electronic properties of Ce overlayers and low-dimensional Pt-Ce alloys on Pt{111}

The structural, thermal, chemisorptive, and electronic properties of Ce on Pt{111} are studied by photoemission, Auger spectroscopy, scanning tunnel microscope (STM), and low-energy electron diffraction (LEED). Stranski-Krastanov-like growth of low-density Ce layers is accompanied by substantial valence charge transfer from Ce to Pt: in line with this, the measured dipole moment and polarizability of adsorbed Ce at low coverages are 7.2 x 10(-30) C m and similar to 1.3x10(-29) m(3), respectively. Pt-Ce intermixing commences at similar to 400 K and with increasing temperature a sequence of five different ordered surface alloys evolves. The symmetry, periodicities, and rotational epitaxy observed by LEED are in good accord with the STM data which reveal the true complexity of the system. The Various bimetallic surface phases are based on growth of crystalline Pt5Ce, a hexagonal layer structure consisting of alternating layers of Pt2Ce and Kagome nets of Pt atoms. This characteristic ABAB layered arrangement of the surface alloys is clearly imaged, and chemisorption data permit a distinction to be made between the more reactive Pt2Ce layer and the less reactive Pt Kagome net. Either type of layer can appear at the surface as the terminating structure, thicker films exhibiting unit mesh parameters characteristic of the bulk alloy.

Apolipoprotein C-III protein concentrations and gene polymorphisms in type 1 diabetes:associations with lipoprotein subclasses

Serum apolipoprotein C-III (apoCIII) concentration and apoCIII gene polymorphisms have been shown to be a risk factor for cardiovascular disease; however, the underlying mechanisms remain unclear. In addition, no studies have been performed that address these issues in type 1 diabetes. The current study investigated apoCIII protein and apoCIII gene variation in a normotriglyceridemic (82 +/- 57 mg/dL) population of patients with type 1 diabetes, the Diabetes Control and Complications Trial/Epidemiology of Diabetes Intervention and Complications (DCCT/EDIC) cohort. Blood samples were obtained in 409 patients after an overnight fast. Serum apoCIII concentration was highly correlated with multiple changes in lipids and lipoproteins that resulted in an adverse cardiovascular disease risk profile. Higher apoCIII concentrations were associated (P <.0001) with increased triglycerides (r = 0.78), total (r = 0.61) and low-density lipoprotein (LDL) (r = 0.40) cholesterol, apoA-I (r = 0.26), and apoB (r = 0.50), and these relationships persisted after controlling for age, gender, body mass index (BMI), and hemoglobin A1c (HbA1c). Nuclear magnetic resonance (NMR) lipoprotein subclass analyses demonstrated that apoCIII was correlated with an increase in very-low-density lipoprotein (VLDL) subclasses (P = .0001). There also was a highly significant positive relationship between serum apoCIII concentration and the LDL particle concentration in both men (r = 0.49, P = .001) and women (r = 0.40, P = .001), and a highly significant negative relationship between serum apoCIII levels and average LDL particle size in both men (r = -0.37, P = .001) and women (r = -0.22, P = .001) due primarily to an augmentation in the small L1 subclass (r = 0.42, P = .0001). Neither the T(-455) --> C polymorphism affecting an insulin response element in the apoCIII gene promoter nor a SacI polymorphism in the 3'UTR were associated with any alterations in circulating apoCIII concentrations, serum lipids, apolipoprotein concentrations, lipoprotein composition, or parameters measured by NMR lipoprotein subclass analyses. In summary, elevated apoCIII concentration was associated with risk factors for cardiovascular disease in normolipidemic type 1 diabetic patients through associated changes in lipoprotein subfraction distributions, which were independent of apoCIII genotype.

Lipoprotein subclass profiles of hyperlipidemic diabetic mice measured by nuclear magnetic resonance spectroscopy

Dyslipidemia accelerates vascular complications of diabetes. Nuclear magnetic resonance (NMR) analysis of lipoprotein subclasses is used to evaluate a mouse model of human familial hypercholesterolemia +/- streptozotocin (STZ)-induced diabetes. A double knockout (DKO) mouse (low-density lipoprotein receptor [LDLr] -/-; apolipoprotein B [apoB] mRNA editing catalytic polypeptide-1 [Apobec1] -/-) was studied. Wild-type (WT) and DKO mice received sham or STZ injections at age 7 weeks, yielding control (WT-C, DKO-C) and diabetic (WT-D, DKO-D) groups. Fasting serum was collected when the mice were killed (age 40 weeks) for Cholestech analysis (Cholestech Corp, Hayward, CA) and NMR lipoprotein subclass profile. By Cholestech, fasting triglyceride and total cholesterol increased in DKO-C versus WT-C. Diabetes further increased total cholesterol in DKO. High-density lipoprotein cholesterol (HDL-C) was similar among all groups. NMR revealed that LDL in all groups was present in a subclass the size of large human LDL and was increased 48-fold in DKO-C versus WT-C animals, but was unaffected by diabetes. HDL was found in a subclass equivalent to large human HDL, and was similar among groups. In conclusion, NMR analysis reveals lipoprotein subclass distributions and the effects of genetic modification and diabetes in mice, but lack of particles the size of human small LDL and small HDL may limit the relevance of the present animal model to human disease.

Aminoguanidine and the effects of modified LDL on cultured retinal capillary cells

Compared with normal low density lipoprotein (N-LDL), LDL minimally modified in vitro by glycation, minimal oxidation, or glycoxidation (G-, MO-, GO-LDL) decreases survival of cultured retinal capillary endothelial cells and pericytes. Similar modifications occurring in vivo in diabetes may contribute to retinopathy. The goal of this study was to determine whether low concentrations of aminoguanidine might prevent cytotoxic modification of LDL and/or protect retinal capillary cells from previously modified LDL.

The advanced glycation end product, Nepsilon-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions

Nepsilon-(Carboxymethyl)lysine (CML) is an advanced glycation end product formed on protein by combined nonenzymatic glycation and oxidation (glycoxidation) reactions. We now report that CML is also formed during metal-catalyzed oxidation of polyunsaturated fatty acids in the presence of protein. During copper-catalyzed oxidation in vitro, the CML content of low density lipoprotein increased in concert with conjugated dienes but was independent of the presence of the Amadori compound, fructoselysine, on the protein. CML was also formed in a time-dependent manner in RNase incubated under aerobic conditions in phosphate buffer containing arachidonate or linoleate; only trace amounts of CML were formed from oleate. After 6 days of incubation the yield of CML in RNase from arachidonate was approximately 0.7 mmol/mol lysine compared with only 0.03 mmol/mol lysine for protein incubated under the same conditions with glucose. Glyoxal, a known precursor of CML, was also formed during incubation of RNase with arachidonate. These results suggest that lipid peroxidation, as well as glycoxidation, may be an important source of CML in tissue proteins in vivo and that CML may be a general marker of oxidative stress and long term damage to protein in aging, atherosclerosis, and diabetes.