High ox-LDL Concentrations Research Articles

Oxidative and Antioxidative Stress Status in Children with Inflammatory Bowel Disease as a Result of a Chronic Inflammatory Process.

Oxidative stress (OS) has been recently implicated in the disease pathogenesis in inflammatory bowel disease (IBD). The aim of the study was to evaluate oxidative and antioxidative stress status and the risk of the atherosclerotic process in children with IBD and functional gastrointestinal disorders (FGID). The prospective study included a group of 71 children during a period of 2 years. In all children, laboratory tests were performed and intima-media complex in the carotid artery was measured (IMC). Low values of OS were more frequent in children with IBD than in the FGID group. The average concentration of oxidized lipoprotein with average density (oxLDL) was lower in patients with IBD. Among patients with IBD, higher concentrations of oxLDL were recorded in patients with longer-duration disease and with higher concentrations of total cholesterol. In the IBD group, more often, higher concentrations of anti-oxLDL were recorded among patients with longer-duration disease. The obtained results did not support the hypothesis of total antioxidant capacity depletion and greater overall OS in patients with IBD. Patients with IBD with a longer duration of the disease have higher concentrations of oxLDL and anti-oxLDL.

Athero-inflammatory nanotherapeutics: Ferulic acid-based poly(anhydride-ester) nanoparticles attenuate foam cell formation by regulating macrophage lipogenesis and reactive oxygen species generation.

Future development of anti-oxidant formulations for atherosclerosis applications is essential to deliver an efficacious dose while limiting localized concentrations of pro-oxidants. In this study, we illustrate the potential of degradable ferulic acid-based polymer nanoparticles to control macrophage foam cell formation by significantly reducing oxLDL uptake through downregulation of scavenger receptors, CD-36, MSR-1, and LOX-1. Another critical finding is the ability of the degradable ferulate-based polymer nanoparticles to lower macrophage reactive oxygen species (ROS) levels, a precursor to apoptosis and plaque escalation. The degradable ferulic acid-based polymer nanoparticles hold significant promise as a means to alter the treatment and progression of atherosclerosis.

The biphasic effects of oxidized-low density lipoprotein on the vasculogenic function of endothelial progenitor cells.

Late-outgrowth endothelial progenitor cells (EPCs) are stress-resistant and responsible for reparative functions in the cardiovascular system. Oxidized-LDL (oxLDL) plays a critical role in cardiovascular disease pathogenesis. However, it is largely unknown what the impacts of oxLDL are on late-outgrowth EPCs. This study aimed to investigate the concentration-related effects of oxLDL on EPC functions and related angiogenesis, in vitro and in vivo. In this study, early and late-outgrowth EPCs were generated from circulating human mononuclear cells. oxLDL may regulate EPC vasculogenic function via the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). Lower concentrations (5 μg/mL) of oxLDL can potentiate EPC tube formation in vitro and in vivo by activating eNOS mechanisms, which are mediated by p38 MAPK- and SAPK/JNK-related pathways. Higher concentrations of oxLDL (10-50 μg/mL) impaired EPC function via the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase pathways and consequent inhibition of eNOS activity, which could be reversed by anti-oxidants (diphenylene iodonium and apocynin) and gp91phox siRNA. In conclusion, oxLDL has concentration-dependent biphasic effects on human late-outgrowth EPC tube formation in vitro and in vivo.

Oxidized Low Density Lipoprotein and High Sensitive C-Reactive Protein in Non-Diabetic, Pre-Diabetic and Diabetic Patients in the Acute Phase of the First Myocardial Infarction Treated by Primary Percutaneous Coronary Intervention.

SummaryBackgroundOxidized low density lipoprotein (ox-LDL) and high-sensitive C-reactive protein (hs-CRP) are elevated in diabetes mellitus (DM) and associated with accelerated atherosclerosis. Little is known about their dynamics in the acute phase of ST segment elevation myocardial infarction (STEMI), especially in relation to the presence of DM and pre-diabetes (pre-DM). This study aimed to analyze time-dependent changes in ox-LDL and hs-CRP regarding the presence of pre-DM and DM in STEMI patients treated by primary percutaneous coronary intervention (pPCI).MethodsIn 103 consecutive patients with the first anterior STEMI ox-LDL and hs-CRP were measured before pPCI, on day 2 and day 7 after pPCI.ResultsPatients were classified into: non-diabetics, pre-diabetics and diabetics. In each group the maximal ox-LDL concentration was found on admission, decreased on day 2 and reached the lowest values on day 7 (p<0.001). Diabetics had the highest ox-LDL concentrations compared to pre-diabetics and non-diabetics (on admission: p=0.028, on day 2: p=0.056, on day 7: p=0.004). hs-CRP concentration rose from admission, reached its peak on day 2 and decreased on day 7, in each group (p<0.001). Significant differences in hs-CRP concentrations were found between non-diabetics and pre-diabetics on admission (p=0.018) and day 2 (p=0.026). In a multivariate analysis DM was an independent determinant of high ox-LDL concentrations. Both ox-LDL and hs-CRP significantly correlated with Killip class, left ventricular ejection fraction, NT-proBNP and peak troponin I.ConclusionsIn patients with the first STEMI treated by pPCI there were significant differences in ox-LDL and hs-CRP concentrations between non-diabetics, pre-diabetics and diabetics. Ox-LDL and hs-CRP concentrations were related to heart failure parameters.

Oxidized low-density lipoprotein in postmenopausal women.

Oxidized low-density lipoprotein (oxLDL) leads to atherosclerosis and cardiovascular disease, the most frequent causes of death worldwide. After menopause, lipid and lipoprotein metabolism changes and women are at greater risk of cardiovascular disease compared to fertile women. The aim of this study was to determine the prevalence of serum oxLDL in postmenopausal women and to identify possible associations of clinical and laboratory features with oxLDL in these patients. After clinical examination and completing a clinical questionnaire, an ultrasound examination of both carotid arteries was conducted and blood was drawn from 533 postmenopausal women. oxLDL concentration was determined using proton NMR spectroscopy. Oxidized LDL was detected in 12.4% (95% confidence interval 9.7-15.5) of postmenopausal women with a median of 0.18 mg/dl (interquartile range 0.10-0.43). Although intima-media thickness did not differ, postmenopausal women with serous oxLDL had more often atherosclerotic plaques compared to women without oxLDL (6/66 vs. 0/467; P < 0.01). Higher concentrations of high-density lipoprotein, impaired glucose intolerance, and DBP were independently associated with the occurrence of oxLDL. If oxLDL was present, higher high-density lipoprotein and glucose intolerance were associated with higher concentrations of oxLDL. In contrast, higher blood urea concentrations were associated with lower concentrations of oxLDL. This study presents the prevalence and concentration of oxLDL in postmenopausal women and demonstrates that oxLDL concentration can be quantified by proton NMR spectroscopy in large patient samples. The data suggest that oxLDL may be a biomarker for incipient atherosclerotic changes in postmenopausal women. In contrary to the association of dyslipoproteinemia and diabetes, higher blood urea concentrations were associated with lower concentrations of oxLDL.

Dendritic Cells in Cardiovascular Diseases

Cardiovascular diseases (CVDs) are one of the leading causes of mortality worldwide.1 It has been so for decades, notwithstanding a wide array of – mostly preventive – treatment modalities targeting known risk factors, such as hyperlipidemia, type 2 diabetes mellitus, hypertension, or obesity. Recent technical and conceptual advances have unveiled important contributions of the immune system in the pathophysiology of a variety of CVDs such as atherosclerosis, ischemic stroke, chronic heart failure, and other myocardial conditions like myocardial ischemia and reperfusion, viral myocarditis, and cardiac transplantation.2–4 In many of these disorders, so-called danger-associated molecular patterns (DAMPs), released from necrotic tissue and dying cells, can lead to the activation of certain immune cell populations such as monocytes/ macrophages, granulocytes, and T cells, thus aggravating ongoing inflammatory processes at the lesion site. Dendritic cells (DCs) are key modulators of immunity, pivotal in directing innate and adaptive immune responses against microbial, viral, but also modified self-antigens present at the sites of injury. Given the tissue trauma underlying various CVDs, it is not surprising that recent observations have allocated a regulatory role for DCs in CVD-associated immune responses. Interestingly, nondiseased arteries of young individuals were seen to host a network of resident vascular DCs (CD1a+ S100+ lag+ CD31− CD83− CD86−),5 representing a phenotype related to Langerhans cells in the skin. In agreement, monocyte-derived CD11c+ CD68+ dendritic cells could be detected in the atherosclerosis-prone lesser curvature and aortic sinus in inbred atherosclerosis-susceptible (C57Bl/6), but not resistant mouse strains (balb/c).6 Murine vascular resident DCs express an immature phenotype with low expression of costimulatory molecules, and are present in the subendothelial space with occasional probing into the vascular lumen. DCs have …

Abdominal adiposity, insulin resistance, and oxidized low-density lipoproteins in Latino adolescents

Abdominal obesity and insulin resistance (IR) place youth at higher risk for premature cardiovascular disease (CVD), but the underlying mechanisms are not clear. In adults, abdominal obesity and IR contribute to the oxidation of low-density lipoprotein (LDL). Whether similar mechanisms are operational in Latino adolescents is unknown. Therefore, we determined whether IR and abdominal adiposity are associated with higher oxLDL concentrations in Latino adolescents. Data from 123 Latino adolescents (16.3 ± 2.5 years; female = 74) were used for the present analysis. Participants were assessed for waist circumference, fasting serum oxLDL, and insulin sensitivity by the whole body insulin sensitivity index. In separate linear regression models adjusting for age and sex, both waist circumference and insulin sensitivity were significant predictors of oxLDL (β = 1.9; p = 0.002; R2 = 0.13, β = -1.7; p = 0.006; R2 = 0.11, respectively). When insulin sensitivity and waist circumference were included in the same model, both remained independent predictors of oxLDL (β = 1.7; p = 0.016 and, β = -1.5; p = 0.055, respectively; R2 = 0.16). These results suggest that insulin resistance and abdominal adiposity are associated with higher levels of LDL oxidation which may be a mechanism contributing to increased CVD risk in Latino adolescents.

Abstract 179: Atheroprone Hemodynamics and Oxidized Phospholipids Enhance Endothelial and Smooth Muscle Inflammatory Signaling

Atherosclerosis is a focal inflammatory disease marked by activation of NFkB within regions where low and oscillating shear stresses (Atheroprone) “prime” the endothelium for an inflammatory response. Interestingly, oxPAPC, a major component of oxLDL, has not been shown to act through the canonical NFkB pathway, and although oxLDL is capable of increasing the expression of NFkB-dependent genes it often requires higher concentrations of oxLDL (&gt;100ug/ml). Further, these studies were all performed within static monocultures, where the endothelium was not a “primed” physiologic phenotype. Therefore, we wanted to test whether the addition of oxLDL within Atheroprone hemodynamics (where NFkB signaling was already activated) would further enhance inflammatory signals downstream of NFkB in an endothelial cell (EC) and smooth muscle cell (SMC) co-culture. After preconditioning cells with Atheroprone shear stress, NFkB activity and gene expression is significantly increased compared to cells exposed to shear stress patterns from regions protected from atherosclerosis. Adding oxLDL to this system further enhances inflammatory gene expression in a dose-dependent fashion above Atheroprone conditions treated with native LDL (nLDL). Performing gene arrays we found that oxLDL regulated over 350 genes in ECs compared to cells treated with nLDL. These gene panels were enriched with shear stress-regulated, pro- (VCAM, E-Selectin) and anti-(KLF2, eNOS) inflammatory genes. Though not directly exposed to either Atheroprone shear stress or LDL, over 50 genes were regulated by these conditions in the underlying SMCs in our co-culture system, including inflammatory genes. Lastly, we measured changes in protein secretion due to the addition of oxLDL compared to nLDL. Many NFkB-dependent, pro-inflammatory cytokine levels were increased, including IL8, MCP-1, and IL6. Investigating the role of oxLDL within the context of physiologic shear stress was found to enhance the Atheroprone-regulated gene profile towards a more pro-inflammatory phenotype. These conditions mimic vessel wall inflammation found in human arteries and provide an ideal environment for testing drugs intended for treating advanced atherosclerosis.

Coordination of Id1 and p53 Activation by Oxidized LDL Regulates Endothelial Cell Proliferation and Migration

Considering that oxidized low-density lipoprotein (ox-LDL) may inhibit endothelial cell (EC) migration and proliferation during endothelialization, we hypothesize that the Id1 protein promotes endothelialization exposed to ox-LDL. Cell proliferation was evaluated by cell counts, and cell migration was evaluated by wound closure assay. The role of Id1 in the cell migration and proliferation was appraised through building Id1 overexpression and silencing ECs. Here, we report that Id1 in human umbilical vascular ECs (HUVECs) was up-regulated by ox-LDL in a dose- and time-dependent manner. Low concentrations of ox-LDL increased the proliferation and migration of EC. High concentrations of ox-LDL suppressed HUVECs proliferation and migration, whose inhibitory effects were abolished by Id1 over-expression. Attenuated proliferation and migration of ECs exposed to high concentrations of ox-LDL may be correlated with the nuclear localization of p53, which was obviously weakened by over-expression of Id1 and strengthened by silencing Id1. Collectively, changes in EC, comprising proliferation and migration, upon exposure to various concentrations of ox-LDL are, at least in part, attributed to the modulatory effect of the Id1 protein, which suggests that manipulating Id1 protein activity may offer therapeutic opportunities to promote re-endothelialization under high concentrations of ox-LDL.

The effect of olive oil polyphenols on antibodies against oxidized LDL. A randomized clinical trial

Oxidized LDL (oxLDL) is a highly immunogenic particle that plays a key role in the development of atherosclerosis. Some data suggest a protective role of OxLDL autoantibodies (OLAB) in atherosclerosis. Our aim was to assess the effect of olive oil polyphenols on the immunogenicity of oxLDL to autoantibody generation. In a crossover, controlled trial, 200 healthy men were randomly assigned to 3-week sequences of 25mL/day of 3 olive oils with high (366mg/kg), medium (164mg/kg), and low (2.7mg/kg) phenolic content. Plasma OLAB concentration was inversely associated with oxLDL (p<0.001). Olive oil phenolic content increased OLAB generation, with the effect being stronger at higher concentrations of oxLDL (p=0.020 for interaction). A direct relationship was observed between OLAB and the total olive oil phenol content in LDL (r=0.209; p=0.014). OLAB concentrations, adjusted for oxLDL, increased directly in a dose-dependent manner with the polyphenol content of the olive oil administered (p=0.023). Olive oil polyphenols promote OLAB generation. This effect is stronger at higher concentrations of lipid oxidative damage.

Mathematical modelling of atherosclerosis as an inflammatory disease

Atherosclerosis is an inflammatory disease. The atherosclerosis process starts when low-density lipoproteins (LDLs) enter the intima of the blood vessel, where they are oxidized (ox-LDLs). The anti-inflammatory response triggers the recruitment of monocytes. Once in the intima, the monocytes are transformed into macrophages and foam cells, leading to the production of inflammatory cytokines and further recruitment of monocytes. This auto-amplified process leads to the formation of an atherosclerotic plaque and, possibly, to its rupture. In this paper we develop two mathematical models based on reaction-diffusion equations in order to explain the inflammatory process. The first model is one-dimensional: it does not consider the intima's thickness and shows that low ox-LDL concentrations in the intima do not lead to a chronic inflammatory reaction. Intermediate ox-LDL concentrations correspond to a bistable system, which can lead to a travelling wave that can be initiated by certain conditions, such as infection or injury. High ox-LDL concentrations correspond to a monostable system, and even a small perturbation of the non-inflammatory case leads to travelling-wave propagation, which corresponds to a chronic inflammatory response. The second model we suggest is two-dimensional: it represents a reaction-diffusion system in a strip with nonlinear boundary conditions to describe the recruitment of monocytes as a function of the cytokines' concentration. We prove the existence of travelling waves and confirm our previous results, which show that atherosclerosis develops as a reaction-diffusion wave. The results of the two models are confirmed by numerical simulations. The latter show that the two-dimensional model converges to the one-dimensional one if the thickness of the intima tends to zero.

‘Danger’ effect of low-density lipoprotein (LDL) and oxidized LDL on human immature dendritic cells

Dendritic cell (DC) maturation may accelerate autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, and may contribute to accelerated atherosclerosis seen in these patients. The immune system responds to both exogenous and endogenous 'dangerous' signals that can induce dendritic cell maturation. We have found that autologous plasma contains danger signals that induce up-regulation of major histocompatibility complex (MHC) class II and co-stimulatory molecules in immature DCs (iDCs). The objective of this study was to determine whether low-density lipoprotein (LDL) and/or oxidized LDL (oxLDL) constitute danger signals, and to assess the effect of exposure to LDL and oxLDL following monocyte differentiation into iDCs in lipoprotein-deficient serum (LPDS). IDCs were generated in the presence of autologous plasma or LPDS. Expression of maturation and migration molecules was evaluated using flow cytometry, and morphology was assessed by light microscopy. Pro- or anti-apoptotic effect was determined using annexin V and propidium iodide binding. Phagocytosis of apoptotic cells was evaluated using autologous plasma or LPDS. LDL and oxLDL were clearly able to slightly up-regulate levels of HLA-DR and co-stimulatory molecule CD86. High oxLDL concentrations (50-100 microg/ml) were associated with expression of additional maturation molecules. Moreover, iDCs that were prepared in LPDS showed partial maturation following exposure to LDL and oxLDL, and improved tolerogenic apoptotic cell uptake. This study suggests that oxLDL, and to some extent LDL, are at least partly responsible for the iDC 'danger' response induced by autologous plasma.

Atherosclerosis Initiation Modeled as an Inflammatory Process

In this work we study the in∞ammatory process resulting in the development of atherosclerosis. We develop a one- and two-dimensional models based on reaction-difiusion systems to describe the set up of a chronic in∞ammatory response in the intima of an artery vessel wall. The concentration of the oxidized low density lipoproteins (ox-LDL) in the intima is the critical parameter of the model. Low ox-LDL concentrations do not lead to a chronic in∞ammatory reaction. Intermediate ox-LDL concentrations correspond to a bistable system and can lead to a travelling wave propagation corresponding to a chronic in∞ammatory reaction. In this case the disease development depends on the initial condition. If the concentration of monocytes in the intima is su-ciently high, which can be caused by an in∞ammation related to other factors, then the development of atherosclerosis can start. Otherwise, the system returns to the stable disease free equilibrium. High ox-LDL concentrations correspond to a monostable system and even a small perturbation of the non in∞ammatory case leads to a travelling wave propagation which corresponds to a chronic in∞ammatory response.

Concentration of Circulating Oxidized LDL in HIV-Infected Patients Treated with Antiretroviral Agents: Relation to HIV-Related Lipodystrophy

Background: Circulating oxidized LDL (ox-LDL) is associated with clinical manifestations of atherosclerosis. The aim of the study was to investigate the concentrations of ox-LDL in HIV-infected patients under antiretroviral therapy with (HIV-LD) or without (HIV-nLD) HIV-related lipodystrophy. Method: A total of 44 HIV-infected men were enrolled in the study. Half of them had HIV-LD. The control group included 12 age- and body mass index (BMI)-matched HIV-uninfected men. Ox-LDL concentration and C-reactive protein level were determined. Insulin sensitivity was measured using the homeostasis model assessment (HOMA-IR). LD was assessed by using a validated score calculated from clinical and biological data. Results: HIV-infected patients had significantly higher ox-LDL concentrations when compared to HIV-negative controls (0.8 ±0.3 mg/dL vs. 0.60 ±0.1 mg/dL; p = .007). HIV-LD patients had significantly higher ox-LDL concentrations than HIV-nLD patients (0.91 ±0.38 and 0.69 ±0.16; p = .04). In HIV-LD patients, current therapy with protease inhibitors (PIs); duration of PI therapy; HOMA-IR; and time exposure to stavudine, efavirenz, ritonavir, saquinavir, and amprenavir were significantly higher than in HIV-nLD patients. In multivariate analysis, time exposures to stavudine and ox-LDL concentration were independently related to lipodystrophy. Conclusion: The high concentration of ox-LDL was found in HIV-infected patients under antiretroviral therapy, especially in those with lipodystrophy.

Concentration of Circulating Oxidized LDL in HIV-Infected Patients Treated with Antiretroviral Agents: Relation to HIV-Related Lipodystrophy

Background: Circulating oxidized LDL (ox-LDL) is associated with clinical manifestations of atherosclerosis. The aim of the study was to investigate the concentrations of ox-LDL in HIV-infected patients under antiretroviral therapy with (HIV-LD) or without (HIV-nLD) HIV-related lipodystrophy. Method: A total of 44 HIV-infected men were enrolled in the study. Half of them had HIV-LD. The control group included 12 age- and body mass index (BMI)-matched HIV-uninfected men. Ox-LDL concentration and C-reactive protein level were determined. Insulin sensitivity was measured using the homeostasis model assessment (HOMA-IR). LD was assessed by using a validated score calculated from clinical and biological data. Results: HIV-infected patients had significantly higher ox-LDL concentrations when compared to HIV-negative controls (0.8 ±0.3 mg/dL vs. 0.60 ±0.1 mg/dL; p = .007). HIV-LD patients had significantly higher ox-LDL concentrations than HIV-nLD patients (0.91 ±0.38 and 0.69 ±0.16; p = .04). In HIV-LD patients, current therapy with protease inhibitors (PIs); duration of PI therapy; HOMA-IR; and time exposure to stavudine, efavirenz, ritonavir, saquinavir, and amprenavir were significantly higher than in HIV-nLD patients. In multivariate analysis, time exposures to stavudine and ox-LDL concentration were independently related to lipodystrophy. Conclusion: The high concentration of ox-LDL was found in HIV-infected patients under antiretroviral therapy, especially in those with lipodystrophy.

Circulating oxidized LDL is associated with increased waist circumference independent of body mass index in men and women

Obesity is associated with oxidative stress, and the oxidation of LDL is thought to play a crucial role in the generation of atherosclerotic lesions. The objective was to describe the association of waist circumference (WC) and body mass index (BMI; in kg/m2) with plasma circulating oxidized LDL (ox-LDL) and C-reactive protein (CRP). This cross-sectional study included data for circulating ox-LDL and CRP from a subpopulation of 586 men and women enrolled in a population-based survey conducted in 2000 in Girona, Spain. Multivariate analysis was performed to describe the independent association of WC and BMI with ox-LDL and CRP. Multivariate logistic regression analysis adjusted for lifestyle, educational level, and dietary confounders showed a direct association of WC (quartile distribution) and BMI categories with ox-LDL (P for linear trend = 0.002) and CRP (P for linear trend = 0.004). Subjects in the top quartile of WC and with a BMI > 29.9 were at high risk of elevated circulating concentrations of ox-LDL and CRP. Further adjustment for cardiovascular disease risk factors did not substantially modify these associations. The risk of high ox-LDL concentrations in overweight (BMI = 25.0-29.9) or obese (BMI > or = 30) subjects with a WC < 102 cm (men) or < 88 cm (women) was not significantly different from that in normal-weight subjects with these WCs. In contrast, overweight or obese subjects with higher WCs (WC > or = 102 cm for men and > or = 88 cm for women) were at significantly higher risk of increased ox-LDL. High WC was associated with high concentrations of ox-LDL independently of BMI in the study population.

Induced expression of manganese superoxide dismutase by non-toxic concentrations of oxidized low-density lipoprotein (oxLDL) protects against oxLDL-mediated cytotoxicity.

Oxidized low-density lipoprotein (oxLDL) affects macrophages and plays a critical role in the development of atherosclerosis. In the present paper, we demonstrate that high concentrations of oxLDL provoked apoptosis of human Mono-Mac-6 cells, which was blocked by diphenylene-iodonium (DPI), an inhibitor of flavin-containing enzymes, such as NADPH oxidase, suggesting the involvement of reactive oxygen species (ROS). Importantly, pre-treatment of cells with low concentrations of oxLDL prevented apoptosis in response to high concentrations of oxLDL by up-regulating manganese superoxide dismutase (MnSOD). DPI prevented expression of MnSOD by oxLDL, whereas inhibitors of cytochrome P450 (methoxalen) or xanthine oxidase (allopurinol) did not, thus pointing to a role of NADPH-oxidase-derived ROS in oxLDL-induced MnSOD expression. Transfection of cells with MnSOD antisense, but not scrambled antisense, oligonucleotides significantly attenuated oxLDL-mediated MnSOD expression and hindered cytoprotective effects of non-toxic oxLDL concentrations. Our findings suggest that up-regulation of MnSOD by low concentrations of oxLDL is critical for protection towards oxLDL-mediated cytotoxicity.

Oxidized low-density lipoproteins bind to the scavenger receptor, CD36, of hepatic stellate cells and stimulate extracellular matrix synthesis

Cumulating evidence suggests that oxidative stress resulting in lipid peroxidation and protein modification is involved in the pathogenesis of chronic liver injury and fibrogenesis. We investigated the effects of oxidized low-density lipoproteins (oxLDL) on collagen and fibronectin synthesis of cultured human and rat hepatic stellate cells (HSC). As shown on protein and mRNA levels, oxLDL dose-dependently stimulated the synthesis of collagen types I and III and fibronectin of cultured HSC. The effect was biphasic, with a maximum between 5 and 25 microg/mL oxLDL (c-fibronectin concentration in HSC supernatants increased 3.9-fold; collagen type I increased 4-fold). Higher oxLDL concentrations were cytotoxic. LDL modified with malondialdehyde (MDA) was not toxic, but stimulated extracellular matrix synthesis as well. As demonstrated by immunofluorescence microscopy (double staining of CD36 and iso-alpha-smooth muscle actin [iso-alpha-sm actin]), immunoblot, and reverse-transcription polymerase chain reaction (RT-PCR), respectively, cultured human HSC express the oxLDL receptor, CD36 (glycoprotein IIIb). Colocalization of CD36 and iso-alpha-sm actin on sinusoidal lining cells was further demonstrated using sections of human fibrotic liver. Preincubation of cultured human HSC with the monoclonal antibody, OKM5, known to block CD36-mediated oxLDL uptake, resulted in a reduction of the oxLDL-stimulated collagen type I synthesis by 56%. In summary, our results demonstrate that low concentrations of modified lipoproteins (oxLDL and MDA-LDL) represent fibrogenic mediators that bind to CD36 and stimulate matrix synthesis of HSC.

Role of endothelial cell adhesion molecules and chemoattractants in atherosclerosis

OxLDL primarily due to its lipid moiety, may potentially activate cytoprotective mechanisms, like a stress response, or lead to cell death, in the forms of necrosis or apoptosis. Incubation of cultured human endothelial cells with OxLDL triggers a stress response, with the synthesis of the inducible form of hsp70. This stress response, which is driven by the lipid moiety of OxLDL, pertains to proliferating cells, is inhibited by simvastatin and specifically involves the biosynthesis of geranylgeraniol. Incubation of endothelial cells with relatively high concentrations of OxLDL can also activate an apoptotic pathway, as demonstrated by the increase of the ratio Bax/Bcl-2 ratio, by the activation of CPP-32 and by labeling of 3~-OH termini of fragmented DNA (TUNEL} in cultured human endothelial cells. Both the above responses to OxLDL, can be elicited also by lipid-derived, water-soluble products of oxidation, an observation which broadens the potential for a role of OxLDL in affecting vessel wall functionality. The biological relevance of these in uitro data is substantiated by ex uiuo observations on early intimal thickening of human and animal aorta, where in fact OxLDL epitopes were found to associate with the inducible form of hsp70, as well as with several markers ofapoptosis. Which factor(s) besides the local concentration of OxLDL. direct toward survival or cell death is presently under investigation. Of possible relevance is also the inhibitory effect of OxLDL on endothelial secretion of MMP-9, an effect which may indirectly favour the sequestration of monocytes/macrophages into the vessel wall in the early phases of atherosclerosis.

Preparation of a human standard for determination of the levels of antibodies to oxidatively modified low-density lipoproteins.

As part of our ongoing effort to develop a standardized competitive enzyme immunoassay for human autoantibodies to oxidized low-density lipoprotein (oxLDL), we have generated a reference human antibody standard and revised some of the conditions of the assay. The preparation of the standard involved purification of human anti-oxLDL antibodies by affinity chromatography using immobilized oxLDL. The total concentration of antibody in this purified human oxLDL antibody was established by adding the concentrations of immunoglobulin G (IgG), IgA, and IgM determined in the standard by radial immunodiffusion. The isolated antibody was used to calibrate a whole human serum standard, which was used to calibrate the assays to detect antibody in serum samples. We also revisited the general conditions for performance of our competitive assay. We determined that 1/20 was the ideal dilution for performing the absorption step, and that 1/20 and 1/40 were optimal dilutions to assay oxLDL antibody in unknown serum samples. We also established that the optimal concentration of oxLDL for absorption of free antibody in serum samples was 200 microgram of oxLDL/ml; no significant decrease in the reactivity of samples with immobilized oxLDL was observed when higher concentrations of oxLDL were used for absorption. The minimum detection level of the assay is 0.65 mg/liter. Because serum samples are diluted 1/20 and 1/40 for the assay, the minimal concentration of antibody detectable in serum is 20-fold higher, i.e., 13 mg/liter. The intraassay coefficient of variation calculated from seven determinations of three samples containing antibody concentrations of 240, 340, and 920 mg/liter ranged from 8 to 6.1%. The interassay coefficients of variation for sera with antibody levels of 100 to 594 mg/liter varied from 9.2 to 7.0%, and for isolated antibodies with concentrations of 52 to 111 mg/liter, the coefficients varied from 5.8 to 3.9%.