Comparative Analysis of Atherogenic Lipoproteins L5 and Lp(a) in Atherosclerotic Cardiovascular Disease.

PURPOSE OF REVIEW: Low-density lipoprotein (LDL) poses a risk for atherosclerotic cardiovascular disease (ASCVD). As LDL comprises various subtypes differing in charge, density, and size, understanding their specific impact on ASCVD is crucial. Two highly atherogenic LDL subtypes-electronegative LDL (L5) and Lp(a)-induce vascular cell apoptosis and atherosclerotic changes independent of plasma cholesterol levels, and their mechanisms warrant further investigation. Here, we have compared the roles of L5 and Lp(a) in the development of ASCVD. RECENT FINDINGS: Lp(a) tends to accumulate in artery walls, promoting plaque formation and potentially triggering atherosclerosis progression through prothrombotic or antifibrinolytic effects. High Lp(a) levels correlate with calcific aortic stenosis and atherothrombosis risk. L5 can induce endothelial cell apoptosis and increase vascular permeability, inflammation, and atherogenesis, playing a key role in initiating atherosclerosis. Elevated L5 levels in certain high-risk populations may serve as a distinctive predictor of ASCVD. L5 and Lp(a) are both atherogenic lipoproteins contributing to ASCVD through distinct mechanisms. Lp(a) has garnered attention, but equal consideration should be given to L5.

PON-1 carbamylation is enhanced in HDL of uremia patients.

High-density lipoprotein (HDL) carbamylation has been known in uremia patients. Paraoxonase-1 (PON-1) is an important HDL protein responsible for HDL anti-oxidant, arylesterase and lactonase activities. PON-1 carbamylation in uremic HDL has never been explored. We isolated HDL from uremia patients and control healthy subjects for study. Sandwich ELISA was used to estimate carbamylated PON-1 protein expression in HDL, and nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) was applied to identify the amino acid in PON-1 carbamylated. PON-1 enzyme activities were estimated by substrates conversion method. HDL anti-oxidant activity was gauged by fluorescence changes of indicator dye in the presence of H2O2. Our study results proved that the degree of PON-1 carbamylation was higher in uremic HDL than in control HDL. Sandwich ELISA study showed that carbamylated PON-1 concentration in uremic HDL was 1.49 ± 0.08 fold higher than that in HDL from controls (p < 0.05). The nanoLC-MS/MS showed that the carbamylation of lysine 290 (K290) of PON-1, a residue adjacent to PON-1 activity determining site, was detected in uremic HDL but not detected in control HDL. K290 carbamylation leads to local conformation changes that reduce accessible solvent accessibility. The HDL paraoxonase, arylesterase, and lactonase activities were all significantly lower in uremia patients than in control subjects. Additionally, HDL anti-antioxidant ability was also lower in uremia patients. Carbamylation of PON-1 in uremia patients could be one of the factors in impairing PON-1 enzyme activities and HDL anti-oxidation function.

Increased electronegativity of high-density lipoprotein in uremia patients impairs its functional properties and is associated with the risk of coronary artery disease.

BACKGROUND AND AIMS: Uremia patients have impaired high-density lipoprotein (HDL) function and a high risk of coronary artery disease (CAD). Increased lipoprotein electronegativity can compromise lipoprotein function, but the effect of increased HDL electronegativity on HDL function and its association with CAD in uremia patient are not clear. We aimed to assess HDL electronegativity and various properties of HDL in uremia patients and investigate whether electronegative HDL is a risk factor for CAD in these individuals. METHODS: HDL from 60 uremia patients and 43 healthy controls was separated into 5 subfractions (H1H5) with increasing electronegativity by using anion-exchange chromatography. Lipoprotein content was analyzed by gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry. HDL anti-oxidant, anti-apoptosis and cholesterol efflux activities were examined by fluorescence-based assays. RESULTS: The percentage of H5 HDL (H5%) was significantly higher in uremia patients than in controls (p < 0.001). The concentration of apolipoprotein (Apo) AI was lower and apolipoprotein modifications were more prevalent in uremia HDL subfractions than in control HDL subfractions. Carbamylation of ApoAI and ApoCIII was increased in more electronegative HDL subfractions from uremia patients. Anti-oxidant activity, anti-apoptotic activity, and cholesterol efflux capability were reduced in HDL subfractions from uremia patients when compared with control HDL subfractions. Multiple logistic regression analysis showed that H5% was associated with CAD risk in uremia patients. CONCLUSIONS: In HDL of uremia patients, increased electronegativity is accompanied by compositional changes and impaired function. Our findings indicate that increased H5% is associated with increased CAD risk in uremia patients.

Range of L5 LDL levels in healthy adults and L5's predictive power in patients with hyperlipidemia or coronary artery disease.

Electronegative L5 low-density lipoprotein (LDL) level may be a useful biomarker for predicting cardiovascular disease. We determined the range of plasma L5 levels in healthy adults (n = 35) and examined the power of L5 levels to differentiate patients with coronary artery disease (CAD; n = 40) or patients with hyperlipidemia (HLP) without evidence of CAD (n = 35) from healthy adults. The percent L5 in total LDL (L5%) was quantified by using fast-protein liquid chromatography with an anion-exchange column. Receiver operating characteristic curve analysis was performed to determine cut-off values for L5 levels. The mean L5% and plasma concentration of L5 (ie, [L5]) were significantly higher in patients with HLP or CAD than in healthy adults (P < 0.001). The ranges of L5% and [L5] in healthy adults were determined to be <1.6% and <1.7 mg/dL, respectively. In individuals with L5% >1.6%, the odds ratio was 9.636 for HLP or CAD. In individuals with [L5] >1.7 mg/dL, the odds ratio was 17.684 for HLP or CAD. The power of L5% or [L5] to differentiate patients with HLP or CAD from healthy adults was superior to that of the LDL/high-density lipoprotein ratio. The ranges of L5% and [L5] in healthy adults determined here may be clinically useful in preventing and treating cardiovascular disease.

Electronegative Low-density Lipoprotein Increases Coronary Artery Disease Risk in Uremia Patients on Maintenance Hemodialysis.

Electronegative low-density lipoprotein (LDL) is a recognized factor in the pathogenesis of coronary artery disease (CAD) in the general population, but its role in the development of CAD in uremia patients is unknown. L5 is the most electronegative subfraction of LDL isolated from human plasma. In this study, we examined the distribution of L5 (L5%) and its association with CAD risk in uremia patients.The LDL of 39 uremia patients on maintenance hemodialysis and 21 healthy controls was separated into 5 subfractions, L1-L5, with increasing electronegativity. We compared the distribution and composition of plasma L5 between uremia patients and controls, examined the association between plasma L5% and CAD risk in uremia patients, and studied the effects of L5 from uremia patients on endothelial function.Compared to controls, uremia patients had significantly increased L5% (P < 0.001) and L5 that was rich in apolipoprotein C3 and triglycerides. L5% was significantly higher in uremia patients with CAD (n = 10) than in those without CAD (n = 29) (P < 0.05). Independent of other major CAD risk factors, the adjusted odds ratio for CAD was 1.88 per percent increase in plasma L5% (95% CI, 1.01-3.53), with a near-linear dose-response relationship. Compared with controls, uremia patients had decreased flow-mediated vascular dilatation. In ex vivo studies with preconstricted rat thoracic aortic rings, L5 from uremia patients inhibited acetylcholine-induced relaxation. In cultured human endothelial cells, L5 inhibited endothelial nitric oxide synthase activation and induced endothelial dysfunction.Our findings suggest that elevated plasma L5% may induce endothelial dysfunction and play an important role in the increased risk of CAD in uremia patients.

Mass Spectrometry-Based Proteomic Study Makes High-Density Lipoprotein a Biomarker for Atherosclerotic Vascular Disease.

High-density lipoprotein (HDL) is a lipid and protein complex that consists of apolipoproteins and lower level HDL-associated enzymes. HDL dysfunction is a factor in atherosclerosis and decreases patient survival. Mass spectrometry- (MS-) based proteomics provides a high throughput approach for analyzing the composition and modifications of complex HDL proteins in diseases. HDL can be separated according to size, surface charge, electronegativity, or apoprotein composition. MS-based proteomics on subfractionated HDL then allows investigation of lipoprotein roles in diseases. Herein, we review recent developments in MS-based quantitative proteomic techniques, HDL proteomics and lipoprotein modifications in diseases, and HDL subfractionation studies. We also discuss future directions and perspectives in MS-based proteomics on HDL.

Early prediction of severe acute pancreatitis by urinary ß-2 microglobulin/saposin B peak ratios on MALDI-TOF.

The current methods for predicting severe acute pancreatitis (severe AP) are either complicated or lack efficient sensitivity and specificity. In this study, a simple and practical approach was developed to predict severe AP by using peak intensity ratio of urinary ß-2 microglobulin (B2M) to saposin B (SB) on MALDI-TOF MS. Patients with B2M/SB ratio higher than 1.127 present severe AP symptom with a higher Ranson score, computed tomography (CT) grade and longer hospitalization with a sensitivity of 83.7% and specificity of 74.3%. Label-free quantitative proteomics by nanoLC-MS/MS was applied to urine of severe AP patients and found that severe AP is accompanied with kidney injury and inflammation. The measurement of B2M/SB ratios by MALDI-TOF MS could be a simple, accurate and rapid method to diagnose severe AP as well as to monitor AP progression.

Biochemical and functional characterization of charge-defined subfractions of high-density lipoprotein from normal adults.

High-density lipoprotein (HDL) is regarded as atheroprotective because it provides antioxidant and anti-inflammatory benefits and plays an important role in reverse cholesterol transport. In this paper, we outline a novel methodology for studying the heterogeneity of HDL. Using anion-exchange chromatography, we separated HDL from 6 healthy individuals into five subfractions (H1 through H5) with increasing charge and evaluated the composition and biologic activities of each subfraction. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that apolipoprotein (apo) AI and apoAII were present in all 5 subfractions; apoCI was present only in H1, and apoCIII and apoE were most abundantly present in H4 and H5. HDL-associated antioxidant enzymes such as lecithin-cholesterol acyltransferase, lipoprotein-associated phospholipase A2, and paraoxonase 1 were most abundant in H4 and H5. Lipoprotein isoforms were analyzed in each subfraction by using matrix-assisted laser desorption-time-of-flight mass spectrometry. To quantify other proteins in the HDL subfractions, we used the isobaric tags for the relative and absolute quantitation approach followed by nanoflow liquid chromatography-tandem mass spectrometry analysis. Most antioxidant proteins detected were found in H4 and H5. The ability of each subfraction to induce cholesterol efflux from macrophages increased with increasing HDL electronegativity, with the exception of H5, which promoted the least efflux activity. In conclusion, anion-exchange chromatography is an attractive method for separating HDL into subfractions with distinct lipoprotein compositions and biologic activities. By comparing the properties of these subfractions, it may be possible to uncover HDL-specific proteins that play a role in disease.

Oxidized ApoC1 on MALDI-TOF and glycated-ApoA1 band on gradient gel as potential diagnostic tools for atherosclerotic vascular disease.

BACKGROUND: Atherosclerotic vascular disease (ASVD), including coronary artery disease, ischemic stroke, and peripheral vascular disease, is the most common cause of death both in the general population and in high-risk patients; patients with diabetes mellitus (DM), uremia (UM), primary hyperlipidemia (HP) have increased risks for developing ASVD. METHODS: To identify new biomarkers for early prediction of ASVD, HDL samples from patients with disease (ASVD) and patients with increased risks but no documented ASVD (non-ASVD) were collected for Bis-Tris gradient gel and MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) analyses. RESULTS: Oxidation of ApoC1 was detected specifically in ASVD samples by MALDI-TOF. On the Bis-Tris gradient gel, non-ASVD ApoA1 was displayed into 2 distinct bands A and B. An additional C band of ApoA1 appeared exclusively in ASVD patients. The extraordinary C band of ApoA1 was characterized by high levels of glycation and oxidation. MALDI-TOF analyses of ApoA1 peptides after trypsin digestion confirmed higher levels of glycation and oxidation levels in the ASVD than non-ASVD samples. CONCLUSIONS: Gel identification of the highly-glycated and oxidized C band of ApoA1 and MALDI-TOF detection of oxidized ApoC1 in HDL may provide a new approach for early ASVD diagnosis.

Identification of the HDL-ApoCIII to VLDL-ApoCIII ratio as a predictor of coronary artery disease in the general population: the Chin-Shan Community Cardiovascular Cohort (CCCC) study in Taiwan.

BACKGROUND: Apolipoprotein (Apo) levels are considered more reliable than plasma lipoprotein levels for predicting coronary artery disease (CAD). However, a unanimous Apo marker for CAD has not been identified. In the Chin-Shan Community Cardiovascular Cohort (CCCC), we sought to identify a common Apo marker for predicting CAD in the general population. METHODS: We examined the cross-sectional association between Apo markers and CAD in the CCCC from 1990 to 2001. Among 3,602 subjects, 90 had angiographically proven CAD (>50% stenosis in ≥1 vessel), and 200 did not have CAD. These subjects were divided into the following 4 groups for analysis: normolipidemic (total cholesterol [TC] <200 mg/dL, triglyceride [TG] <150 mg/dL), hypertriglyceridemic (TC <200 mg/dL, TG ≥150 mg/dL), hypercholesterolemic (TC ≥200 mg/dL, TG <150 mg/dL), and hyperlipidemic (TC ≥200 mg/dL, TG ≥150 mg/dL). RESULTS: Compatible with findings in other populations, our results showed that CAD patients in the CCCC had higher ApoB and lower high-density lipoprotein (HDL) cholesterol and ApoAI concentrations than non-CAD subjects, but the differences were not significant in all groups. Plasma concentrations of ApoE and lipoprotein (a) were not consistently correlated with CAD. In contrast, the ratio of HDL-ApoCIII to very-low-density lipoprotein (VLDL)-ApoCIII was the only universal determinant for CAD in the normolipidemic group (P=0.0018), the hypertriglyceridemic group (P=0.0001), the hypercholesterolemic group (P=0.0001), and the hyperlipidemic group (P=0.0001). Overall, a high HDL-ApoCIII/VLDL-ApoCIII ratio was observed in all CAD patients, including those with a normal lipid profile. In multivariate analyses, the HDL-ApoCIII/VLDL-ApoCIII ratio was the strongest predictor for CAD among all lipid factors investigated (odds ratio, 2.04; 95% confidence interval, 1.46-2.84; P<0.0001). CONCLUSIONS: A high HDL-ApoCIII to VLDL-ApoCIII ratio is a better marker for predicting CAD than are the conventional lipid markers or ApoAI and ApoB. High HDL-ApoCIII and low VLDL-ApoCIII values in CAD, irrespective of lipid variations, suggest that ApoCIII is markedly transported from VLDL to HDL in this disease. Measurement of plasma ApoCIII may improve CAD prediction in the general population.

The most negatively charged low-density lipoprotein L5 induces stress pathways in vascular endothelial cells.

BACKGROUND/AIMS: L5, the most negatively charged species of low-density lipoprotein (LDL), has been implicated in atherogenesis by inducing apoptosis of endothelial cells (ECs) and inhibiting the differentiation of endothelial progenitor cells. In this study, we compared the effects of LDL charge on cellular stress pathways leading to atherogenesis. METHODS: We isolated L5 and L1 (the least negatively charged LDL) from the plasma of patients with familial hypercholesterolemia and used JC-1 staining to examine the effects of L5 and L1 on the mitochondrial membrane potential (DCm) in human umbilical vein ECs (HUVECs). Additionally, we characterized the gene expression profiles of 7 proteins involved in various types of cellular stress. RESULTS: The DCm was severely compromised in HUVECs treated with L5. Furthermore, compared with L1, L5 induced a decrease in mRNA and protein expression of the endoplasmic reticulum (ER) chaperone proteins ORP150, Grp94, and Grp58, mitochondrial proteins Prdx3 and ATP synthase, and an increase in the expression of the pro-inflammatory protein hnRNP C1/C2. CONCLUSIONS: Our work suggests that L5, but not L1, may promote the destruction of ECs that occurs during atherogenesis by causing mitochondrial dysfunction and modulating the expression of key proteins to promote inflammation, ER dysfunction, oxidative stress, and apoptosis.

Electronegative low-density lipoprotein induces cardiomyocyte apoptosis indirectly through endothelial cell-released chemokines.

Cardiomyocyte apoptosis has a critical role in the pathogenesis of heart failure. L5, the most negatively charged subfraction of human plasma low-density lipoprotein (LDL), induces several atherogenic responses in endothelial cells (ECs), including apoptosis. We hypothesized that L5 also contributes to cardiomyocyte apoptosis and studied whether it does so indirectly by inducing the secretion of factors from ECs. We examined apoptosis of rat cardiomyocytes treated with culture-conditioned medium (CCM) of rat ECs that were exposed to L5 or L1 (the least negatively charged LDL subfraction). Apoptosis at early and late time points was twofold greater in cardiomyocytes treated with L5 CCM than in those treated with L1 CCM. The indirect effect of L5 on cardiomyocyte apoptosis was significantly reduced by pretreating ECs with inhibitors of phosphatidylinositol 3-kinase (PI3K) or CXC receptor 2 (CXCR2). Studies with cytokine protein arrays revealed that L5 CCM, but not L1 CCM, contained high levels of ELR(+) CXC chemokines, including lipopolysaccharide-induced chemokine (LIX) and interleukin (IL)-8. The L5-induced release of these chemokines from ECs was abolished by inhibiting the lectin-like oxidized LDL receptor-1 (LOX-1). Addition of recombinant LIX or IL-8 to CCM-free cardiomyocyte cultures increased apoptosis and enhanced production of tumor necrosis factor (TNF)-α and IL-1ß by increasing the translocation of NF-κB into the nucleus; these effects were attenuated by inhibiting PI3K and CXCR2. In conclusion, L5 may indirectly induce cardiomyocyte apoptosis by enhancing secretion of ELR(+) CXC chemokines from ECs, which in turn activate CXCR2/PI3K/NF-κB signaling to increase the release of TNF-α and IL-1ß.

PTEN overexpression attenuates angiogenic processes of endothelial cells by blockade of endothelin-1/endothelin B receptor signaling.

Arteriovenous (AV) graft is frequently used as vascular access in hemodialysis patients. However, clotting or thrombosis of AV grafts often occurs and requires surgical removal. At present, the molecular pathogenesis underlying thrombosis of AV graft is not clear. The PTEN/Akt signaling has been implicated in the pathogenesis of vascular diseases. In this study, elevated PTEN expression and concomitant Akt inactivation was observed in endothelium of atherosclerotic brachial arteries from hemodialysis patients. To investigate whether PTEN upregulation affects endothelial function, adenovirus-mediated PTEN (Ad-PTEN) overexpression was performed in aorta rings and cultured endothelial cells. It was found that PTEN overexpression potently inhibited the microvessel sprouting in aorta rings and the angiogenic activities of endothelial cells including migration and tube formation. On the contrary, PTEN knockdown by RNA interference promoted the endothelial migration and reversed the Ad-PTEN-induced inhibition of endothelial migration. Expression analysis showed that PTEN overexpression attenuated the expression of endothelin-1 (ET-1) and endothelin B receptor (ETBR) in endothelial cells at transcriptional levels. However, exogenous ET-1 supply only partially reversed the PTEN-induced inhibition of migration and tube formation. This was delineated due to that PTEN overexpression also perturbed endothelial nitric oxide synthase (eNOS) activation and vascular endothelial growth factor (VEGF) release. In summary, PTEN upregulation induces endothelial dysfunction by attenuating the availability and signaling of multiple angiogenic pathways in endothelial cells, thereby may contribute to thrombosis of AV graft.

Effects of electronegative VLDL on endothelium damage in metabolic syndrome.

OBJECTIVE: Biochemical heterogeneity governs functional disparities among lipoproteins. We examined charge-defined VLDL subfractions in metabolic syndrome (MetS) to determine whether their increased electronegativity is associated with increased cytotoxicity and whether high concentrations of highly electronegative subfractions render VLDL harmful to the vascular endothelium. RESEARCH DESIGN AND METHODS: Plasma VLDL of normal individuals (control subjects) (n = 13) and of those with MetS (n = 13) was resolved into subfractions with increasing negative charge (V1-V5) by anion-exchange chromatography. Human aortic endothelial cells were treated with V1-V5 or unfractionated VLDL. RESULTS: Compared with the control subjects, individuals with MetS had a significantly higher percentage of V5 VLDL (V5/VLDL%) (34 ± 20 vs. 39 ± 11%, respectively; P < 0.05) and plasma V5 concentration ([V5]) (5.5 ± 4.4 vs. 15.2 ± 8.5 mg/dL, respectively; P < 0.001). Apolipoprotein (apo)B100 levels decreased and apoC levels increased from V1 to V5, indicating that V5 is apoC-rich VLDL. Regression analyses of all 26 individuals showed that [V5] was positively correlated with total cholesterol (P = 0.016), triglyceride (P < 0.000001), and V5/VLDL% (P = 0.002). Fasting plasma glucose, but not waist circumference, exhibited a positive trend (P = 0.058); plasma HDL cholesterol exhibited a weak inverse trend (P = 0.138). V5 (10 µg/mL) induced apoptosis in ~50% of endothelial cells in 24 h. V5 was the most rapidly (<15 min) internalized subfraction and induced the production of reactive oxygen species (ROS) in endothelial cells after 20 min. Unfractionated MetS VLDL, but not control VLDL, also induced ROS production and endothelial cell apoptosis. CONCLUSIONS: In populations with increased risk of diabetes, the vascular endothelium is constantly exposed to VLDL that contains a high proportion of V5. The potential impact of V5-rich VLDL warrants further investigation.

Iron-catalyzed oxidation of Trp residues in low-density lipoprotein.

The mechanisms of oxidation of low-density lipoproteins (LDLs) are not well defined, but epidemiological and experimental studies suggest that iron-catalyzed processes may contribute to atherogenesis. The aim of this study was to test the hypothesis that iron-catalyzed oxidations of LDLs in vitro produce diagnostic biomarkers of oxidation of the apolipoprotein that could be applied to studies in vivo. LDLs were oxidized in the presence of Fe2+, EDTA, and ascorbic acid for up to 40 h. Following delipidation and trypsin digestion, the peptides were separated by HPLC, with four peaks detected at 365 nm, whereas none were observed in peptides from unoxidized LDLs. The peptides were identified by MALDI-QTOF mass spectrometry as IVQILP(W+4) EQNEQVK, IYSL(W+4)EHSTK, FEGLQE(W+4)EGK, and YH(W+4)EHTGLTLR, with (W+4) rather than the W residues of the unoxidized protein. The mass gains (+4 increase in m/z in tryptophan, W) and absorbance at 365 nm indicate kynurenines, which were trypsin-releasable peptides that are on the surface of LDL particles. All four peptides thus characterized share the sequence of WE. The preferential oxidation of W residues in WE sequences suggest contributions from the C-proximate glutamate residues in chelation of the iron species, thereby influencing site selectivities of oxidation. These kynurenine-containing peptides might serve as biomarkers of iron-mediated oxidations in vivo.

Chemical composition-oriented receptor selectivity of L5, a naturally occurring atherogenic low-density lipoprotein.

Anion-exchange chromatography resolves human plasma low-density lipoprotein (LDL) into 5 subfractions, with increasing negative surface charge in the direction of L1 to L5. Unlike the harmless L1 to L4, the exclusively atherogenic L5 is rejected by the normal LDL receptor (LDLR) but endocytosed into vascular endothelial cells through the lectin-like oxidized LDL receptor-1 (LOX-1). Analysis with SDS-PAGE and 2-dimensional electrophoresis showed that the protein framework of L1 was composed mainly of apolipoprotein (apo) B100, with an isoelectric point (pI) of 6.620. There was a progressively increased association of additional proteins, including apoE (pI 5.5), apoAI (pI 5.4), apoCIII (pI 5.1), and apo(a) (pI 5.5), from L1 to L5. LC/MSE was used to quantify protein distribution in all subfractions. On the basis of weight percentages, L1 contained 99% apoB-100 and trace amounts of other proteins. In contrast, L5 contained 60% apoB100 and substantially increased amounts of apo(a), apoE, apoAI, and apoCIII. The compositional characteristics contribute to L5's electronegativity, rendering it unrecognizable by LDLR. LOX-1, which has a high affinity for negatively charged ligands, is known to mediate the signaling of proinflammatory cytokines. Thus, the chemical composition-oriented receptor selectivity hinders normal metabolism of L5, enhancing its atherogenicity through abnormal receptors, such as LOX-1.

Impact of adventitial neovascularisation on atherosclerotic plaque composition and vascular remodelling in a porcine model of coronary atherosclerosis.

AIMS: There is little in vivo data in regards to the impact of adventitial neovascularisation on vascular remodelling and plaque composition. Using a porcine model of coronary atherosclerosis, we aimed to determine the impact of adventitial neovascularisation on plaque composition and vascular remodelling evaluated by IVUS. METHODS AND RESULTS: Coronary atherosclerosis was induced by adventitial delivery of lipids and a high cholesterol diet. At termination all vessels were analysed using IVUS to determine the degree of remodelling of each individual segment containing atherosclerotic lesions. Then, each segment was correlated with its correspondent histological frame for plaque composition and neovessel density. A total of 57 atherosclerotic lesions at different stages of development were analysed. The total neovessel count (TNC) correlated to the degree of plaque burden (15.6+/-7.2 TNC in <40% stenosis versus 35.7+/-14.0 TNC in >60% stenosis, p<0.01) and to the amount of intra-plaque collagen (32.4+/-14.1%, lower TNC tertile versus 47.5+/-8.9% upper TNC tertile, p< 0.01). The amount of intra-plaque SMC content inversely correlated with the TNC (49.7+/-18.9% versus 36.4+/-14.4%, lower versus upper tertiles, p<0.05). Plaques with the highest TNC showed higher remodelling indexes by IVUS (0.89+/-0.32 in lower TNC tertile versus 1.36+/-0.73 in upper TNC tertile, p<0.05) and higher macrophage cell content (161.42+/-157.6 in lower TNC tertile versus 340.6+/-127.2 in upper TNC tertile, p<0.05) compared to non-remodelled segments. CONCLUSIONS: Adventitial neovascularisation is more prominent in positively remodelled segments and appears to be associated to SMC loss, increase collagen deposition and localised macrophage infiltration.

Mediation of electronegative low-density lipoprotein signaling by LOX-1: a possible mechanism of endothelial apoptosis.

The lectin-like oxidized LDL receptor LOX-1 mediates endothelial cell (EC) uptake of experimentally prepared copper-oxidized LDL (oxLDL). To confirm the atherogenic role of this receptor cloned against copper-oxLDL, we examined whether it mediates EC uptake of L5, an electronegative LDL abundant in dyslipidemic but not normolipidemic human plasma. Hypercholesterolemic (LDL-cholesterol, >160 mg/dL) human LDL was fractionated into L1-L5, increasingly electronegative, by ion-exchange chromatography. In cultured bovine aortic ECs (BAECs), L5 upregulated LOX-1 and induced apoptosis. Transfection of BAECs with LOX-1-specific small interfering RNAs (siLOX-1) minimized baseline LOX-1 production and restrained L5-induced LOX-1 upregulation. Internalization of labeled L1-L5 was monitored in BAECs and human umbilical venous ECs by fluorescence microscopy. LOX-1 knockdown with siLOX-1 impeded the endocytosis of L5 but not L1-L4. In contrast, blocking LDL receptor with RAP (LDL receptor-associated protein) stopped the internalization of L1-L4 but not L5. Although chemically different, L5 and oxLDL competed for EC entry through LOX-1. Via LOX-1, L5 signaling hampered Akt phosphorylation and suppressed EC expression of fibroblast growth factor-2 and Bcl-2. L5 also selectively inhibited Bcl-xL expression and endothelial nitric oxide synthase phosphorylation but increased synthesis of Bax, Bad, and tumor necrosis factor-alpha. Blocking Akt phosphorylation with wortmannin increased LOX-1 expression, suggesting a modulatory role of Akt in LOX-1 synthesis; L5 upregulated LOX-1 by dephosphorylating Akt. Because endothelial nitric oxide synthase and Bcl-2 activities are Akt-dependent, L5 impairs Akt-mediated growth and survival signals in vascular ECs by way of LOX-1. Thus, the L5/LOX-1 complex may play a critical role in atherogenesis and illuminate important targets for disease intervention.

Electronegative LDL circulating in smokers impairs endothelial progenitor cell differentiation by inhibiting Akt phosphorylation via LOX-1.

Endothelial progenitor cells (EPCs), important for endothelial regeneration and vasculogenesis, are reduced by cigarette smoking. To elucidate the mechanisms, we examined the effects of electronegative LDL, circulating in chronic smokers, on EPC differentiation. Using ion-exchange chromatography, we purified smoker LDL into five subfractions, L1-L5. In matched, nonsmoking healthy subjects, L5, the most electronegative subfraction, was either absent or scanty. Sustained L5 treatment inhibited CD31 and KDR expression and EPC differentiation, whereas L1-L4 had no effect. L5 also inhibited telomerase activity to accelerate EPC senescence in correlation with reduced Akt phosphorylation. Transfection of day 3 EPCs with dominant negative Akt constructs inhibited CD31 and KDR expression, stalled EPC differentiation, and promoted early senescence. In contrast, transfection with constitutively active Akt rendered the EPCs resistant to L5, allowing normal maturation. L5 upregulated the lectin-like oxidized low density lipoprotein receptor 1 (LOX-1), and pretreatment of EPCs with TS20, a LOX-1-neutralizing antibody, blocked internalization of L5 by EPCs and prevented L5-mediated inhibition of EPC differentiation. Mixing L5 with L1 to physiological L5/L1 ratios did not attenuate L5's effects. These findings suggest that cigarette smoking is associated with the formation of L5, which inhibits EPC differentiation by impairing Akt phosphorylation via the LOX-1 receptor.

Electronegative LDL impairs vascular endothelial cell integrity in diabetes by disrupting fibroblast growth factor 2 (FGF2) autoregulation.

OBJECTIVE: L5, a circulating electronegative LDL identified in patients with hypercholesterolemia or type 2 diabetes, induces endothelial cell (EC) apoptosis by suppressing fibroblast growth factor (FGF)2 expression. FGF2 plays a pivotal role in endothelial regeneration and compensatory arteriogenesis. It is likely that vasculopathy and poor collateralization in diabetes is a result of FGF2 dysregulation. RESEARCH DESIGN AND METHODS: To investigate this mechanism, we isolated L5 from type 2 diabetic patients. In cultured bovine aortic ECs (BAECs), L5 inhibited FGF2 transcription and induced apoptosis. Because FGF2 stimulates the phosphatidylinositol 3-kinase (PI3K)-Akt pathway, we examined whether FGF2 transcription is regulated by Akt through a feedback mechanism. RESULTS: Diabetic L5 reduced FGF2 release to the medium but enhanced caspase-3 activity, with resultant apoptosis. Inhibition of PI3K with wortmannin or suppression of Akt activation with dominant-negative Akt inhibited FGF2 expression. Transfection of BAECs with FGF2 antisense cDNA depleted endogenous FGF2 protein. In these cells, not only was Akt phosphorylation inhibited, but FGF2 transcription was also critically impaired. In contrast, transfecting BAECs with FGF2 sense cDNA augmented Akt phosphorylation. Treatment with constitutively active Akt enhanced FGF2 expression. Augmentation of either FGF2 transcription or Akt phosphorylation rendered BAECs resistant to L5. CONCLUSIONS: These findings suggest that FGF2 is the primary initiator of its own expression, which is autoregulated through a novel FGF2-PI3K-Akt loop. Thus, by disrupting FGF2 autoregulation in vascular ECs, L5 may impair reendothelialization and collateralization in diabetes.