Forms Of Low-density Lipoprotein Research Articles

The in vitro effect of myeloperoxidase oxidized LDL on THP-1 derived macrophages.

Cardiovascular diseases (CVDs) linked to atherosclerosis remains the leading cause of death worldwide. Atherosclerosis is primarily caused by the accumulation of oxidized forms of low density lipoprotein (LDL) in macrophages (MΦs) in the subendothelial layer of arteries leading to foam cell and fatty streak formation. Many studies suggest that LDL that is modified by myeloperoxidase (MPO) is a key player in the development of atherosclerosis. MΦs can adopt a variety of functional phenotypes that include mainly the proinflammatory M1 and the anti-inflammatory M2 MΦ phenotypes which are both implicated in the process of atherogenesis. In fact, MΦs that reside in atherosclerostic lesions were shown to express a variety of phenotypes ranging between the M1- and M2 MΦ types. Recently, we pointed out the involvement of MPO oxidized-LDL (Mox-LDL) in increasing inflammation in MΦs by reducing their secretion of IL-10. Since little is known about Mox-LDL-mediated pro-atherosclerostic responses in MΦs, our study aimed at analyzing the in vitro effects of Mox-LDL at this level through making use of the well-established model of human THP-1-derived Mφs. Our results demonstrate that Mox-LDL has no effect on apoptosis, reactive oxygen species (ROS) generation and cell death in our cell model; yet, interestingly, our results show that Mox-LDL is significantly engulfed at a higher rate in the different MΦ subtypes supporting its key role in foam cell formation during the progression of the disease as well as previous data that were generated using another primary MΦ cell model of atherosclerosis.

Low-Density Lipoprotein Pathway Is a Ubiquitous Metabolic Vulnerability in High Grade Glioma Amenable for Nanotherapeutic Delivery

Metabolic reprogramming, through increased uptake of cholesterol in the form of low-density lipoproteins (LDL), is one way by which cancer cells, including high grade gliomas (HGG), maintain their rapid growth. In this study, we determined LDL receptor (LDLR) expression in HGGs using immunohistochemistry on tissue microarrays from intra- and inter tumour regions of 36 adult and 133 paediatric patients to confirm LDLR as a therapeutic target. Additionally, we analysed expression levels in three representative cell line models to confirm their future utility to test LDLR-targeted nanoparticle uptake, retention, and cytotoxicity. Our data show widespread LDLR expression in adult and paediatric cohorts, but with significant intra-tumour variation observed between the core and either rim or invasive regions of adult HGG. Expression was independent of paediatric tumour grade or identified clinicopathological factors. LDLR-expressing tumour cells localized preferentially within perivascular niches, also with significant adult intra-tumour variation. We demonstrated variable levels of LDLR expression in all cell lines, confirming their suitability as models to test LDLR-targeted nanotherapy delivery. Overall, our study reveals the LDLR pathway as a ubiquitous metabolic vulnerability in high grade gliomas across all ages, amenable to future consideration of LDL-mediated nanoparticle/drug delivery to potentially circumvent tumour heterogeneity.

Hypolipidemia activity testing protocol or Guizotia Abyssinica Cass. seed oil: An Experimental study report

Hyperlipidemia is characterized by increased level of cholesterol in the form of low-density lipoprotein (LDL), chylomicrons, VLDL. Guizotia abyssinica Cass (Ramtil) seeds are one among the sources for the edible oil, which are low in saturated fat, cultivated commonly in Northern part of Karnataka. Materials and Methods: Guizotia abyssinica Cass (Ramtil) seeds were collected authenticated seed oil extracted by Cold compression method. The experiment carried out in Wistar albino rats, inducing Hyperlipidemia by HFD, along with administration of test drugs (Guizotia abyssinica Cass. seed oil Single and double dose) and Standard Atorvastatin. Results & Discussion: G. abyssinica Cass seed oil found to decrease total cholesterol and LDL. Even not showed significant increase in HDL. Protective action over the Liver, Kidney and heart through histopathological study exhibit its safety aspect. Conclusion: G. abyssinica Cass seed oil in HFD induced Hyperlipidemia found effective and safe.

Comparative Analysis between Martin’s Formula and Friedewald’s Formula with Direct Homogenous Assay for Estimating Low Density Lipoprotein Cholesterol Level in Nepalese Population

Background: Low density lipoproteinforms a basis of decision making in treatment of hypercholesterolemic patients and primary target of intervention. Its cost effective and accurate measurementis a need for every clinical laboratories and different calculation methods has been adopted as a replacement to direct assays. This study aims to evaluate the Martin’s formula and Friedewald’s formula in a sample of Nepalese population compared against direct homogenous assay.
 Materials and Methods: This is a cross-sectional study conducted in Department of Biochemistry from Feb 2020 to January 2021. Serum samples of the participants were analysed for total cholesterol, triglyceride, high density lipoprotein and low density lipoprotein. Martin’s and Friedewald’s formula were applied to get calculated value of low density lipoprotein from both methods. Passing and Bablok regression analysis was used for methods comparison.
 Results: The mean age of participants was 54.2 ± 8.9 years. Passing-Bablok regression analysis showed Friedewald’s formula performed better than Martin’s formula as per systematic and proportional bias when compared with directassay. However at lowerserum low density lipoprotein level, underestimation of low density lipoprotein compared to direct assay was more common in Friedewald’s formula. At high triglyceride level more percentage error of difference of mean from direct assay was found for Friedewald’s formula.
 Conclusion: When compared to direct assay, Friedewald’s formula was found to be in better agreement than Martin’s formula. Martin’s formula had advantage over Friedewald’s formula at lower serum low density lipoprotein level and higher triglyceride level where Friedewald’s formula mostly underestimated low density lipoprotein.

Impact of Dyslipidemic Levels of Oxidized Cholesterol on Endothelial Membranes

Dyslipidemia, or abnormally high levels of blood cholesterol in the form of low-density lipoprotein (LDL), increases the chance of developing cardiovascular diseases, potentially leading to heart disease, diabetes, or stroke. Blood levels of oxidative modifications of low-density lipoprotein (oxLDL - a major proinflammatory and proatherogenic factor) are also increased under dyslipidemic conditions. In this study, we first performed mass spectrometric analyses of the sterol composition of human aortic endothelial cells (HAECs) exposed to the pathological levels of oxLDL observed in hypercholesterolemia in vivo. We found that exposure of HAECs to oxLDL does not lead to an increase in cellular cholesterol, but results instead in significant incorporation of oxysterols, particularly, 7-ketocholesterol. In this study, we computationally investigate the impact of the physiological increases of 7-ketocholesterol on the physical properties of endothelial membranes using coarse-grained molecular dynamics simulations to compare dyslipidemic lipid compositions to normal physiological profiles. A topology of 7-ketocholesterol based on the Martini coarse-graining scheme was created, validated, and subsequently incorporated into multi-component lipid bilayers representing endothelial membranes at both physiological and dyslipidemic concentrations, determined by mass spectrometric analyses. Molecular simulations show differential membrane perturbation effects of the various concentrations of oxidized sterol present, including increased fluidization and decreased packing of the membrane lipids accompanying increased 7-ketocholesterol levels, effects contrary to those expected to be caused by non-oxidized cholesterol. Differences in sterol diffusion rates, water, and lipid distribution within the model membranes were also observed. These changes in membrane structure and dynamics in response to varying levels of 7-ketocholesterol would suggest that, in addition to altering lipid composition, dyslipidemic conditions involving this oxidized sterol, along with several other bioactive molecules, disrupt the biomechanical integrity of the endothelial membrane.

A Fluorescence-Labeled Heptapeptide, (FITC)KP6, as an Efficient Probe for the Specific Detection of Oxidized and Minimally Modified Low-Density Lipoprotein.

Two oxidized forms of low-density lipoprotein (LDL), oxidized LDL (ox-LDL) and minimally modified LDL (MM-LDL), are believed to play a major role in the pathogenesis of atherosclerosis. Recently, we reported that a heptapeptide (Lys-Trp-Tyr-Lys-Asp-Gly-Asp, KP6) coupled through the ε-amino group of N-terminus Lys to fluorescein isothiocyanate, (FITC)KP6, bound to ox-LDL but not to LDL. In the present study, we investigated whether (FITC)KP6 could be used as a fluorescent probe for the specific detection of MM-LDL and ox-LDL. Results from polyacrylamide gel electrophoresis and surface plasmon resonance proved that (FITC)KP6 could efficiently bind to MM-LDL as well as ox-LDL in a dose-dependent manner and with high affinity (K D=3.16 and 3.54ng/mL protein for MM-LDL and ox-LDL, respectively). (FITC) KP6 bound to lysophosphatidylcholine and oxidized phosphatidylcholine, both present abundantly in ox-LDL and MM-LDL, respectively. In vitro, (FITC)KP6 was detected on the surface and/or in the cytosol of human THP-1-derived macrophages incubated with ox-LDL and MM-LDL, but not LDL. These results suggest that (FITC)KP6 could be an efficient fluorescent probe for the specific detection of ox-LDL and MM-LDL and can therefore contribute to the identification, diagnosis, prevention, and treatment of atherosclerosis.

Serum Lipoprotein(A) Concentration is a Better Predictor of Myocardial Infarction than Traditional Lipid Profile and Lipid Ratios

Background- Lipoprotein(a) is a variant form of Low Density Lipoprotein (LDL) which comprises of an LDL like particle in which apo B-100 is firmly linked to apo-a glycoprotein. A large number of genetic and epidemiologic studies have identified Lipoprotein(a) as a risk factor for atherosclerotic diseases such as coronary heart disease and stroke. Based on our knowledge, this is the first study comparing Lipoprotein(a) with lipid profile and lipid ratios in Nepal. Aim & Objective- To compare the serum Lipoprotein(a) and Lipid panel concentrations in myocardial infarction patients and normal healthy control subjects. Materials & Methods- Study was carried out in 45 myocardial infarction patients as cases and 45 healthy subjects as controls. Lipoprotein(a) and Lipid profile tests were measured in serum using HumaStar 600 fully automated analyzer, Human, Germany.Results- Mean serum Lipoprotein(a) concentration in myocardial infarction group was significantly higher as compared to healthy controls. Lipid panel assays were not statistically significant among the groups.Conclusion- Our study revealed that myocardial infarction patients have elevated levels of serum Lipoprotein(a) as compared to healthy subjects. High concentration of serum Lipoprotein(a) is strongly associated with the risk of coronary heart disease.DOI: http://dx.doi.org/10.3126/ijasbt.v1i3.8583 Int J Appl Sci Biotechnol, Vol. 1(3) 2013 : 106-109

Cloning and expression of an anti-LDL(-) single-chain variable fragment, and its inhibitory effect on experimental atherosclerosis

The in vivo modified forms of low-density lipoprotein (LDL) are important for the formation of foam cells and as mediators of the immuno-inflammatory process involved in the progression of atherosclerosis. Electronegative LDL, LDL(-), is a LDL subfraction with pro-inflammatory properties that is present in human blood. To investigate possible atheroprotective effects, an anti-LDL(-) single-chain variable fragment (scFv) was expressed in the methylotrophic yeast Pichia pastoris and its activity was evaluated in vitro against macrophages and in experimental atherosclerosis in Ldlr-/- mice. The recombinant 2C7 scFv was produced in a yield of 9.5 mg of protein/L. The specificity and affinity of purified 2C7 scFv against LDL(-) was confirmed by ELISA. To assess the activity of 2C7 scFv on foam cell formation, RAW 264.7 macrophages were exposed to LDL(-) in the presence or absence of 2C7 scFv. The 2C7 scFv inhibited the uptake of LDL(-) by macrophages in a dose-dependent manner, and internalization of LDL(-) by these cells was found to be mediated by the CD36 and CD14 receptor. In addition, compared with untreated cells, lipid accumulation in macrophages was decreased, and the expression of Cd36, Tlr-4 and Cox-2 was downregulated in macrophages treated with 2C7 scFv. Importantly, compared with untreated mice, the treatment of Ldlr-/- mice with 2C7 scFv decreased the atherosclerotic lesion area at the aortic sinus. In conclusion, our data show that 2C7 scFv inhibits foam cell formation and atherosclerotic plaque development by modulating the expression of genes relevant to atherogenesis. These results encourage further use of this antibody fragment in the development of new therapeutic strategies that neutralize the pro-atherogenic effects of LDL(-).

Immunity, atherosclerosis and cardiovascular disease

Atherosclerosis, the major cause of cardiovascular disease (CVD), is a chronic inflammatory condition with immune competent cells in lesions producing mainly pro-inflammatory cytokines. Dead cells and oxidized forms of low density lipoproteins (oxLDL) are abundant. The major direct cause of CVD appears to be rupture of atherosclerotic plaques. oxLDL has proinflammatory and immune-stimulatory properties, causes cell death at higher concentrations and contains inflammatory phospholipids with phosphorylcholine (PC) as an interesting epitope. Antibodies against PC (anti-PC) may be atheroprotective, one mechanism being anti-inflammatory. Bacteria and virus have been discussed, but it has been difficult to find direct evidence, and antibiotic trials have not been successful. Heat shock proteins could be one major target for atherogenic immune reactions. More direct causes of plaque rupture include pro-inflammatory cytokines, chemokines, and lipid mediators. To prove that inflammation is a cause of atherosclerosis and CVD, clinical studies with anti-inflammatory and/or immune-modulatory treatment are needed. The potential causes of immune reactions and inflammation in atherosclerosis and how inflammation can be targeted therapeutically to provide novel treatments for CVD are reviewed.

Model IgG Monoclonal Autoantibody–Anti-Idiotype Pair for Dissecting the Humoral Immune Response to Oxidized Low Density Lipoprotein

Increasing evidence implicates IgG autoantibodies against oxidized forms of low density lipoprotein (oxLDL) in the pathophysiology of atherosclerotic arterial disease. However, insufficient knowledge of their structure and function is a key gap. Using an elderly LDL receptor-deficient atherosclerotic mouse, we isolated a novel IgG3k against oxLDL (designated MAb LO1). LO1 reacts with copper-oxidized LDL, but minimally with native LDL. Further analysis showed that MAb LO1 also reacts in vitro with malondialdehyde-conjugated LDL (MDA-LDL), a known key epitope in copper-oxidized LDL preparations. By screening a phage library expressing single chain variable region antibodies (scFv), we selected an anti-idiotype scFv (designated H3) that neutralizes MAb LO1 binding to MDA-LDL. Amino acid substitutions between H3 and an irrelevant control scFv C12 showed that residues in the H3 CDRH2, CDRH3, and CDRL2 are all critical for MAb LO1 binding, consistent with a conformational epitope on H3 involving both heavy and light chains. Comparison of amino acids in H3 CDRH2 and CDRL2 with apoB, the major LDL protein, showed homologous sequences, suggesting H3 has structural similarities to the MAb LO1 binding site on MDA-LDL. Immunocytochemical staining showed that MAb LO1 binds epitopes in mouse and human atherosclerotic lesions. The MAb LO1-H3 combination therefore provides a very promising model for analyzing the structure and function of an individual IgG autoantibody in relation to atherosclerosis.

Phenotypic High-Throughput Screening in Atherosclerosis Research: Focus on Macrophages

Atherosclerosis is a complex disease characterized by arterial lesions consisting of macrophage foam cells, smooth muscle cells, lymphocytes and other cell types. As atherosclerotic lesions mature, they can rupture and thereby trigger thrombosis that can result in tissue infarction. Macrophage foam cells develop in the subendothelial space when cells take up cholesterol from modified forms of low-density lipoprotein (LDL) and other apolipoprotein B-containing lipoproteins. Current therapies to limit atherosclerosis focus on altering the plasma lipid composition, most commonly by reducing circulating LDL levels. No current therapy is specifically designed to alter the cellular composition of atherosclerotic lesions. To address this deficit, phenotypic high-throughput drug screens have been developed to identify compounds that reduce the uptake of oxidized LDL by macrophages or to identify compounds that increase the efflux of cholesterol from macrophages. Additional phenotypic screens can be envisaged that address cellular processes in active atherosclerotic lesions including macrophage apoptosis and efferocytosis.

Uptake of Oxidized Low Density Lipoprotein by CD36 Occurs by an Actin-dependent Pathway Distinct from Macropinocytosis

The class B scavenger receptor CD36 has numerous ligands that include modified forms of low density lipoprotein, fibrillar amyloid, apoptotic cells, and Plasmodium falciparum-infected red blood cells, linking this molecule to atherosclerosis, Alzheimer disease, malaria, and other diseases. We studied the signaling events that follow receptor engagement and lead to CD36 and ligand internalization. We show that oxidized low density lipoprotein or antibody-induced clustering of CD36 triggers macropinocytosis and internalization of the receptor-ligand complex. Remarkably, however, CD36 internalization is independent of macropinocytosis and occurs by a novel endocytic mechanism that depends on actin, but not dynamin. This actin-driven endocytosis requires the activation Src family kinases, JNK, and Rho family GTPases, but, unlike macropinocytosis, it is not affected by inhibitors of phosphatidylinositol 3-kinase or Na/H exchange. Manipulation of this unique mode of internalization may prove helpful in the prevention and management of the wide range of diseases in which CD36 is implicated.

Low level natural antibodies against phosphorylcholine: A novel risk marker and potential mechanism in atherosclerosis and cardiovascular disease

Atherosclerosis is an inflammatory condition characterised by an abundance of activated immunocompetent cells in plaques which cause cardiovascular disease (CVD) when they rupture. Oxidized forms of low density lipoprotein (OxLDL) are a major constituent of atherosclerotic plaques and have proinflammatory effects, making oxLDL a candidate factor promoting atherosclerosis. In previous studies we and others demonstrated that platelet-activating factor (PAF)-like lipids in oxLDL may cause oxLDL-induced immune-stimulatory effects. A common denominator is phosphorylcholine (PC), a hapten-like epitope which is exposed on OxLDL and some microorganisms. We recently demonstrated that anti-PC has anti-inflammatory properties and that low levels of anti-PC predict the development of stroke and myocardial infarction. We hypothesize that low anti-PC represents a novel paradigm as a cause of chronic inflammatory diseases such as atherosclerosis where oxidized and/or inflammatory phospholipids play a role. It is possible that anti-PC can be used as a novel diagnostic tool and therapy in atherosclerotic disease.

Hypercholesterolemic Mice Exhibit Lymphatic Vessel Dysfunction and Degeneration

Lymphatic vessels are essential for lipid absorption and transport. Despite increasing numbers of observations linking lymphatic vessels and lipids, little research has been devoted to address how dysregulation of lipid balance in the blood, ie, dyslipidemia, may affect the functional biology of lymphatic vessels. Here, we show that hypercholesterolemia occurring in apolipoprotein E-deficient (apoE(-/-)) mice is associated with tissue swelling, lymphatic leakiness, and decreased lymphatic transport of fluid and dendritic cells from tissue. Lymphatic dysfunction results in part from profound structural abnormalities in the lymphatic vasculature: namely, initial lymphatic vessels were greatly enlarged, and collecting vessels developed notably decreased smooth muscle cell coverage and changes in the distribution of lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1). Our results provide evidence that hypercholesterolemia in adult apoE(-/-) mice is associated with a degeneration of lymphatic vessels that leads to decreased lymphatic drainage and provides an explanation for why dendritic cell migration and, thus, immune priming, are compromised in hypercholesterolemic mice.

The macrophage scavenger receptor at 30 years of age: current knowledge and future challenges

It is now thirty years since the original observation that macrophages take up and degrade modified forms of low density lipoprotein (LDL). Molecular cloning has identified multiple scavenger receptors that can endocytose modified LDL and binding studies continue to identify a wide range of scavenger receptor ligands displayed by bacterial pathogens, modified self proteins and apoptotic cells. Scavenger receptors drive macrophage foam cell development in vitro but their exact role in the development of atherosclerosis remains difficult to assess critically. This could be due in part to functional redundancy or it could perhaps reflect our incomplete appreciation of all the potential roles of this family of receptors in host defense and tissue homeostasis. In this brief overview of the extensive literature in this area we emphasize the continuing duality of scavenger receptors as sensors of both nonself (pathogens) and modified self (modified forms of LDL and apoptotic cells). We highlight some under-appreciated roles of scavenger receptors in macrophage biology (e.g., regulation of macrophage cytokine responses) and we speculate on potential approaches to target the activity of this diverse family of receptors for therapeutic benefit in cardiovascular disease.

Cholesterol complacency in Australia: time to revisit the basics of cardiovascular disease prevention

Community awareness of the importance of hypercholesterolemia and the need for appropriate therapy is an important part of global efforts to reduce the population burden of cardiovascular disease. The aim of this study was to assess the knowledge and attitudes about cholesterol and to determine adherence to taking cholesterol-lowering medication among patients at high risk for cardiovascular events. In spite of the availability of lifestyle and medical treatments for lipid management, an estimated 50% of adult Australians (6.4 million) remain at risk for a cardiovascular-related event because they have total blood cholesterol levels which exceed recommended limits. It is within this context that a significant gap remains in meeting cholesterol goals, despite easy to meet targets with readily available therapeutic options. A two-page national self-report postal survey was conducted from August-October 2006. A total of 508 Australian adults previously treated for hypercholesterolemia were surveyed to determine their understanding about cholesterol and their adherence to treatment. The mean age of participants was 67 (SD 10) years (72% male). Many participants (72%) were at risk of a cardiovascular event based on a prior history and 18% had type II diabetes. Participants had been prescribed lipid-lowering therapy (94% statin therapy) for an average of 10 years and visited their general practitioner on average three times per year. For those who knew their most recent cholesterol reading (67%), the total cholesterol was on average 4.5 (SD 1.1) mmol/l. This level was above the recommended limits for 40% of participants. Overall, 85% of participants reported knowing that there was high- and low-density lipoprotein forms of cholesterol, but only 56% and 38%, respectively, said that they understood or showed signs of clearly understanding the different types of cholesterol when their knowledge was assessed further. On the whole, therefore, participants had a limited understanding about cholesterol and its potential impact on cardiovascular events. Moreover, 25% of participants admitted to being non-compliant in taking their medication and only 51% correctly identified modifiable risk factors as most important for heart disease. Encouragingly, despite 85% of participants finding lifestyle changes challenging, most still identified their potential benefits. This study highlights that there are many unresolved issues in relation to educating high-risk patients who regularly visit their general practitioner to learn about and optimise their cholesterol levels via appropriate treatment and monitoring. There is a need for urgent public education and management by individuals and the health community. Strategies to address 'cholesterol complacency', in the sense of a willingness to accept sub-optimal standards of cholesterol control at both the patient and healthcare system levels (general practitioners in particular), are urgently needed to truncate an anticipated rising tide of cardiovascular disease in Australia.

The Clinical Value of Modified Low Density Lipoprotein-Cholesterol in Patients Who Underwent Percutaneous Coronary Intervention

Background and Objectives: It is well known that atherosclerosis is characterized by chronic inflammation of an injured intima and the pathological processes are initiated by an accumulation of morphologically distinct, modified forms of low density lipoprotein (LDL)-cholesterol. However, it is not well known whether the level of modified LDL-cholesterol has clinical significance for the patients who underwent percutaneous coronary intervention (PCI). Subjects and Methods: Eighty seven patients (mean age: 63.0±11.1 years, 58 men) who underwent PCI were enrolled. The patients with stable or unstable angina pectoris were classified as group I (n=44, mean age: 62.4±9.3 years), and the patients with acute myocardial infarction were classified as group II (n=43, mean age: 63.6±12.7 years). Modified LDL-cholesterol was expressed semiquantitatively by agarose gel electrophoresis with using the charge modification frequency (CMF). The clinical and coronary angiographic data was analyzed. Results: The clinical diagnosis was stable angina in 13 patients, unstable angina in 31 patients, non-ST elevation myocardial infarction in 5 patients and ST elevation myocardial infarction in 38 patients. There were no significant differences of the CMF between two groups (3.0±7.9 vs. 2.1±10.9, respectively, p=0.671). The diameter stenosis was severe in the patients with a CMF greater than 10 (84.0±10.4% vs. 78.6±13.7%, respectively, p=0.047). The six-month major adverse cardio-vascular events (MACEs) had no relationship with the CMF in group I. However, in group II, the 6-month MACEs developed more frequently in the patients with a CMF higher than 10 {2 (28%) for group ll vs. 2 (5%) for group 1, p=0.031}. The patients with acute myocardial infarction and whose CMF was higher than 10 had in-stent restenosis observed on their follow-up coronary angiography (p=0.003). Conclusion: A higher level of modified LDL-cholesterol is associated with severe angiographic findings and a poor prognosis for patients with acute myocardial infarction. (Korean Circ J 2008;38:475-482)

Modified low density lipoproteins differentially bind and activate the C1 complex of complement

Several studies suggest that complement plays an important role in atherogenesis. To further investigate this question, we have studied the ability of native and modified forms of low density lipoprotein (LDL) to bind and activate C1, the complex that triggers the classical pathway of complement. For this purpose, LDL was both obtained commercially and purified according to an established procedure, and oxidized (oxLDL) and enzymatically modified (E-LDL) derivatives were generated from each preparation. Whereas the unmodified LDL and oxLDL samples did not activate C1 in the presence of excess C1 inhibitor, the E-LDL derivatives obtained by sequential treatment of LDL with a protease and then with cholesterol esterase triggered efficient C1 activation under these conditions, with activation levels ∼60% upon incubation with 1 μM E-LDL for 90 min at 37 °C. In agreement with these findings, as shown by surface plasmon resonance spectroscopy (SPR), the C1q recognition subunit of C1 showed no interaction with unmodified LDL but bound to both E-LDL samples with high affinity ( K D = 58–75 nM). More unexpectedly, although they did not trigger direct C1 activation, both oxLDL samples were also efficiently recognized by C1q. Whereas the oxLDL derivative of commercial LDL activated C1 to a significant extent (∼30%) in the presence of C-reactive protein (CRP), much lower activation levels (<10%) were obtained using the oxLDL derivative of purified LDL. As measured by SPR, CRP bound equally well to the oxLDL and E-LDL derivatives obtained from purified LDL. These data provide the first experimental evidence that E-LDL triggers efficient C1 activation under conditions close to the physiological situation, suggesting that activation of the classical complement pathway by this derivative may be a crucial factor in the pathogenesis of atherosclerosis. In contrast, it appears unlikely that oxLDL significantly activates C1 directly or in a CRP-dependent manner in vivo.

Metabolism of Myeloperoxidase-derived 2-Chlorohexadecanal

Numerous studies have suggested relationships between myeloperoxidase (MPO), inflammation, and atherosclerosis. MPO-derived reactive chlorinating species attack membrane plasmalogens releasing alpha-chloro fatty aldehydes including 2-chlorohexadecanal (2-ClHDA), which have been found to accumulate in activated neutrophils, activated monocytes, infarcted myocardium and human atheromas. The present study employed synthetically prepared 2-Cl-[3H]-HDA as well as stable isotope-labeled 2-ClHDA to elucidate the metabolism of 2-ClHDA. The results herein demonstrate that human coronary artery endothelial cells oxidize and reduce 2-ClHDA to its respective chlorinated fatty acid (alpha-ClFA) and chlorinated fatty alcohol (alpha-ClFOH). Within the first hour of incubations of human coronary artery endothelial cells with 2-Cl-[3H]-HDA, the label was incorporated into the alpha-ClFOH and alpha-ClFA pools. After 1 h, the radiolabel was predominantly found in the alpha-ClFOH pool. Cell-derived alpha-ClFOH and alpha-ClFA were also released into the cell culture medium. Additionally, chlorinated fatty acid was incorporated into complex endothelial cell glycerolipids, including monoglycerides, triglycerides, phosphatidylcholine, and phosphatidylethanolamine. The oxidation and reduction of 2-ClHDA to alpha-ClFA and alpha-ClFOH, respectively, was further supported by mass spectrometric analyses of human coronary artery endothelial cells incubated with either 2-ClHDA or stable isotope-labeled 2-ClHDA (2-Cl-[d4]-HDA). 2-ClHDA was also oxidized to alpha-ClFA and reduced to alpha-ClFOH in both control and phorbol 12-myristate 13-acetate-stimulated neutrophils. Taken together, these results show that a family of chlorinated lipidic metabolites is produced from alpha-chloro fatty aldehydes derived from reactive chlorinating species targeting of plasmalogens. These metabolites are incorporated into complex lipids and their biological roles may provide new insights into MPO-mediated disease.

Troglitazone Inhibits Long-Term Glycation and Oxidation of Low-Density Lipoprotein

Troglitazone (T) is a member of a new class of antidiabetic drugs termed thiazolidinediones (TZDs), which has previously been used as an anti-diabetic agent. In this study we investigated the influence of T, a ligand for PPAR-gamma receptor, on copper-catalyzed or cell-mediated oxidation of native, glycated, and glycoxidated low-density lipoprotein (LDL). A dose-dependent inhibition of copper-mediated low-density lipoprotein-oxidation, as monitored by the formation of oxidation-specific fluorescence, was observed for both native and glycated low-density lipoprotein. At the concentration of 20 microg/mL the inhibition amounted from 14.7% to 64.7% by all low-density lipoprotein forms. For glycated low-density lipoprotein we obtained the highest oxidation rate, but the most pronounced inhibition by T was found for glycoxidated low-density lipoprotein (goLDL). Inhibitory effects of T were also investigated by measurement of relative electrophoretic mobility (REM) in the concentration range of 0 to 20 microg/mL. The inhibition of 4h oxidation of native low-density lipoprotein was found in the entire concentration range, but significance was seen at 10 microg/mL. The long-term glycation and glycoxidation of low-density lipoprotein as measured by 5-hydroxymethyl-2-furaldehyde (5-HMF) formation and binding of fructosamine was found to be inhibited by T. In endothelial cell-mediated oxidation of low-density lipoprotein cytotoxicity of T in the concentration range of 0 to 160 microg/mL during 2 to 24 h oxidation was investigated. In the non-cytotoxic concentration range of 5 to 20 microg/mL, a significantly reduced liberation of isoprostane 8-epi-PGF2alpha during 24 h cell-mediated oxidation of low-density lipoprotein and its modifications was found. This inhibitory action of T was most significant in the case of goLDL and amounted to approximately 20% to 60% inhibition at 5 to 20 microg/mL T, respectively. In the concentration range of 40 to 160 microg/mL, however, T showed an increasing cytotoxic action, as evidenced by loss of cell adhesion, loss of cellular protein, morphological changes, and cell disintegration as well as by strongly enhanced troglitazone-mediated isoprostane 8-IP liberation (fivefold to sixfold). T may be used as a model to explore the thiazolidinediones' mechanism on oxidation in a more general aspect for treatment for T2DM, because T is not clinically available.