Search

Increased plasma concentrations of lipoprotein(a) (Lp(a)) are associated with an increased risk for cardiovascular disease. Lp(a) is composed of apolipoprotein(a) (apo(a)) covalently bound to apolipoprotein B of low-density lipoprotein (LDL). Many of apo(a)'s potential pathological properties, such as inhibition of plasmin generation, have been attributed to its main structural domains, the kringles, and have been proposed to be mediated by their lysine-binding sites. However, available small-molecule inhibitors, such as lysine analogs, bind unselectively to kringle domains and are therefore unsuitable for functional characterization of specific kringle domains. Here, we discovered small molecules that specifically bind to the apo(a) kringle domains KIV-7, KIV-10, and KV. Chemical synthesis yielded compound AZ-05, which bound to KIV-10 with a K(d) of 0.8 μm and exhibited more than 100-fold selectivity for KIV-10, compared with the other kringle domains tested, including plasminogen kringle 1. To better understand and further improve ligand selectivity, we determined the crystal structures of KIV-7, KIV-10, and KV in complex with small-molecule ligands at 1.6–2.1 Å resolutions. Furthermore, we used these small molecules as chemical probes to characterize the roles of the different apo(a) kringle domains in in vitro assays. These assays revealed the assembly of Lp(a) from apo(a) and LDL, as well as potential pathophysiological mechanisms of Lp(a), including (i) binding to fibrin, (ii) stimulation of smooth-muscle cell proliferation, and (iii) stimulation of LDL uptake into differentiated monocytes. Our results indicate that a small-molecule inhibitor targeting the lysine-binding site of KIV-10 can combat the pathophysiological effects of Lp(a).

Inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) reduce low-density lipoprotein (LDL) cholesterol and are used for treatment of dyslipidemia. Current PCSK9 inhibitors are administered via subcutaneous injection. We present a highly potent, chemically modified PCSK9 antisense oligonucleotide (ASO) with potential for oral delivery. Past attempts at oral delivery using earlier-generation ASO chemistries and transient permeation enhancers provided encouraging data, suggesting that improving potency of the ASO could make oral delivery a reality. The constrained ethyl chemistry and liver targeting enabled by N-acetylgalactosamine conjugation make this ASO highly potent. A single subcutaneous dose of 90 mg reduced PCSK9 by >90% in humans with elevated LDL cholesterol and a monthly subcutaneous dose of around 25 mg is predicted to reduce PCSK9 by 80% at steady state. To investigate the feasibility of oral administration, the ASO was coformulated in a tablet with sodium caprate as permeation enhancer. Repeated oral daily dosing in dogs resulted in a bioavailability of 7% in the liver (target organ), about fivefold greater than the plasma bioavailability. Target engagement after oral administration was confirmed by intrajejunal administration of a rat-specific surrogate ASO in solution with the enhancer to rats and by plasma PCSK9 and LDL cholesterol lowering in cynomolgus monkey after tablet administration. On the basis of an assumption of 5% liver bioavailability after oral administration in humans, a daily dose of 15 mg is predicted to reduce circulating PCSK9 by 80% at steady state, supporting the development of the compound for oral administration to treat dyslipidemia.

[Image: see text] In order to assess the potential of sPLA(2)-X as a therapeutic target for atherosclerosis, novel sPLA(2) inhibitors with improved type X selectivity are required. To achieve the objective of identifying such compounds, we embarked on a lead generation effort that resulted in the identification of a novel series of indole-2-carboxamides as selective sPLA2-X inhibitors with excellent potential for further optimization.

Expedited structure-based optimization of the initial fragment hit 1 led to the design of (R)-7 (AZD2716) a novel, potent secreted phospholipase A2 (sPLA2) inhibitor with excellent preclinical pharmacokinetic properties across species, clear in vivo efficacy, and minimized safety risk. Based on accumulated profiling data, (R)-7 was selected as a clinical candidate for the treatment of coronary artery disease.

The present paper shows the non-competitive mechanism by which AZD8797 blocks fractalkine from binding and activating the CX3CR1 receptor. CX3CR1 is involved in many diseases but, lacking non-peptide ligands, it is poorly investigated. Our work can therefore facilitate drug development., The chemokine receptor CX3CR1 has been implicated as an attractive therapeutic target in several diseases, including atherosclerosis and diabetes. However, there has been a lack of non-peptide CX3CR1 inhibitors to substantiate these findings. A selective small-molecule inhibitor of CX3CR1, AZD8797, was recently reported and we present here an in-depth in vitro characterization of that molecule. In a flow adhesion assay, AZD8797 antagonized the natural ligand, fractalkine (CX3CL1), in both human whole blood (hWB) and in a B-lymphocyte cell line with IC50 values of 300 and 6 nM respectively. AZD8797 also prevented G-protein activation in a [35S]GTPγS (guanosine 5′-[γ-thio]triphosphate) accumulation assay. In contrast, dynamic mass redistribution (DMR) experiments revealed a weak Gαi-dependent AZD8797 agonism. Additionally, AZD8797 positively modulated the CX3CL1 response at sub-micromolar concentrations in a β-arrestin recruitment assay. In equilibrium saturation binding experiments, AZD8797 reduced the maximal binding of 125I-CX3CL1 without affecting Kd. Kinetic experiments, determining the kon and koff of AZD8797, demonstrated that this was not an artefact of irreversible or insurmountable binding, thus a true non-competitive mechanism. Finally we show that both AZD8797 and GTPγS increase the rate with which CX3CL1 dissociates from CX3CR1 in a similar manner, indicating a connection between AZD8797 and the CX3CR1-bound G-protein. Collectively, these data show that AZD8797 is a non-competitive allosteric modulator of CX3CL1, binding CX3CR1 and effecting G-protein signalling and β-arrestin recruitment in a biased way.

[Image: see text] A lead generation campaign identified indole-based sPLA(2)-X inhibitors with a promising selectivity profile against other sPLA(2) isoforms. Further optimization of sPLA(2) selectivity and metabolic stability resulted in the design of (−)-17, a novel, potent, and selective sPLA(2)-X inhibitor with an exquisite pharmacokinetic profile characterized by high absorption and low clearance, and low toxicological risk. Compound (−)-17 was tested in an ApoE(–/–) murine model of atherosclerosis to evaluate the effect of reversible, pharmacological sPLA(2)-X inhibition on atherosclerosis development. Despite being well tolerated and achieving adequate systemic exposure of mechanistic relevance, (−)-17 did not significantly affect circulating lipid and lipoprotein biomarkers and had no effect on coronary function or histological markers of atherosclerosis.

Objective— High circulating levels of group IIA secretory phospholipase A 2 (sPLA 2 -IIA) activity and mass are independent cardiovascular risk factors. Therefore, inhibition of sPLA 2 -IIA may be a target for the treatment of atherosclerotic cardiovascular disease. The present study evaluated the effects of sPLA 2 -IIA inhibition with varespladib acid in a novel mouse model, human apolipoprotein B (apoB)/human cholesteryl ester transfer protein (CETP)/human sPLA 2 -IIA triple transgenic mice (TTT) fed a Western-type diet. Approach and Results— sPLA 2 -IIA expression increased atherosclerotic lesion formation in TTT compared with human apoB/human CETP double transgenic mice ( P 2 -IIA activity. Surprisingly, however, administration of varespladib acid to TTT had no impact on atherosclerosis, which could be attributed to a proatherogenic plasma lipoprotein profile that appears in response to sPLA 2 -IIA inhibition because of increased plasma CETP activity. In the TTT model, sPLA 2 -IIA decreased CETP activity by reducing the acceptor properties of sPLA 2 -IIA–modified very low-density lipoproteins specifically because of a significantly lower apoE content. Increasing very low-density lipoprotein-apoE content by means of adenovirus-mediated gene transfer in sPLA 2 -IIA transgenic mice restored the acceptor properties for CETP. Conclusions— These data show that in a humanized triple transgenic mouse model with hypercholesterolemia, sPLA 2 -IIA inhibition increases CETP activity via increasing the very low-density lipoprotein-apoE content, resulting in a proatherogenic lipoprotein profile.

Expedited structure-based optimization of the initial fragment hit

Objectives High levels of large HDL (HDL2) reduce cardiovascular disease risks apparently because it mediates reverse cholesterol transport, and it has anti-inflammatory properties. Here we explored the mechanism behind an additional athero-protective HDL effect related to its capacity to interfere with formation of insoluble LDL-proteoglycans associations, a key step in LDL entrapment in the intima and in atherogenesis. Methods and results We found that HDL2 levels from type 2 diabetes patients and controls are inversely correlated with complex formation between serum LDL and the arterial proteoglycans versican. Reconstitution experiments indicate that HDL2 was more efficacious inhibitor of the LDL-versican association than the smaller HDL3. This may explain why serum from patients with dyslipidemia of insulin resistance, with low levels of HDL2, have a higher capacity to form insoluble LDL-proteoglycan complex. ApoE enrichment of HDL2 and HDL3 or addition of copies of an apoE peptide with the proteoglycan-binding sequence of this apolipoprotein increased their inhibition of LDL-versican associations. Conclusions The inhibitory effect of HDL2 and HDL3 on LDL-versican associations was related to formation of apoE-mediated soluble HDL-versican complexes. We speculate that in the intima large, HDL2 subclasses, by forming reversible soluble associations with proteoglycans can compete with formation of irreversible LDL-proteoglycan aggregates. This can contribute to the HDL2 athero-protective effects. In the dyslipidemia of insulin resistance, associated with low levels of HDL2, this athero-protective property may be compromised.

Background. Lipolysis of lipoproteins by secretory phospholipase A2 group V (sPLA2-V) promotes inflammation, lipoprotein aggregation and foam cell formation – all considered as atherogenic mechanisms. Objective. In this study, we compared the susceptibility to sPLA2-V lipolysis of VLDL and LDL from individuals with type 2 diabetes and the metabolic syndrome (T2D-MetS) and from healthy controls. Design. VLDL and LDL were isolated from 38 T2D-MetS subjects and 38 controls, treated pair-wise. Extent of sPLA2-V lipolysis was measured as release of nonesterified free fatty acids (NEFA). In a subset of the subjects, lipoprotein composition was determined as a relationship between lipid and apolipoprotein components. Results. Mean paired increase in sPLA2-V lipolysis after 1 h for T2D-MetS versus control was 2.0 μmol NEFA l−1 for VLDL (P = 0.004) and 0.75 μmol NEFA l−1 for LDL (P = 0.001). There were also substantial differences in lipoprotein composition between the groups. T2D-MetS VLDL had higher triglyceride and cholesterol contents than control VLDL. T2D-MetS LDL was smaller and contained more triglycerides and less cholesterol than control LDL. Both VLDL and LDL from T2D-MetS subjects also contained more apolipoprotein CIII per particle. Conclusion. VLDL and LDL from T2D-MetS individuals were more susceptible to sPLA2-V lipolysis than those from control individuals. This may result in elevated levels of NEFA and lysophosphatidylcholine, both in circulation and in LDL, possibly contributing to the elevated inflammatory state and increased risk of cardiovascular diseases seen in these individuals.

Cerebroside-beta-glucosidase (acid beta-glucosidase) activity was determined in the forebrain from three patients with the infantile type of Gaucher disease, six patients with the Norrbottnian type and one patient with a mixed infantile/Norrbottnian type. The forebrain from ten subjects, aged 3 months to 20 years, served as controls. The activity was assayed with the natural substrate, (glucose-6-3H) nervonoylglucosylsphingosine and the artificial fluorogenic 4-methyl-umbelliferyl-beta-glucoside. The average residual activities of beta-glucosidase were 12% in the Norrbottnian type and 5% in the infantile type when determined with natural or artificial substrate. Optimum discrimination of the two types of Gaucher disease was obtained with the natural substrate.

Objective— We investigated the potential role of ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motif type I) in atherogenesis. Methods and Results— ADAMTS-1 is expressed at the highest levels in the aorta when compared with other human tissues examined. Immunolocalization studies in human aorta and coronary artery indicate that ADAMTS-1 expression is mainly seen at low levels in the medial layer, but upregulated in the intima when plaque is present. We found that ADAMTS-1 mRNA levels are significantly higher in proliferating/migrating cultured primary aortic vascular smooth muscle cells (VSMCs) compared with resting/confluent cells. Using the mouse carotid artery flow cessation model, we show that there are differences in vessel remodeling in ADAMTS-1 transgenic/apoE-deficient mice compared with apoE deficiency alone, particularly a significant increase in intimal hyperplasia. We show that ADAMTS-1 can cleave the large versican containing proteoglycan population purified from cultured human aortic VSMCs. Finally, using versican peptide substrates, we show data suggesting that ADAMTS-1 cleaves versican at multiple sites. Conclusion— We hypothesize that ADAMTS-1 may promote atherogenesis by cleaving extracellular matrix proteins such as versican and promoting VSMC migration.

Macrophages (Mphi) play an important role in the initiation and progress of the atherogenic process. They contribute to the growth of atherosclerotic plaque by affecting lipoprotein metabolism, matrix homeostasis, lipoprotein modification and cholesterol accumulation. Access to in vitro Mphi models is therefore important for understanding the mechanisms involved in the transition of the relatively simple fatty streak to more complex type of lesions. The aim of the present work was to compare the expression profile of macrophages differentiated from the hematopoietic lineage to peritoneal mouse macrophages and two commonly used mouse macrophage cell lines (J774.A1 and RAW264.7). Our results showed that the embryonic stem cell-derived macrophages (ES Mphi) had a more similar expression profile to peritoneal macrophages than the two mouse macrophage cell lines. The ES Mphi had unchanged expression of the majority of cholesterol efflux mediators when compared to mouse peritoneal macrophages, whereas the cell lines showed altered expression of several of these genes. A key gene in this process is apolipoprotein E, which is expressed in ES Mphi but not in macrophage cell lines. In conclusion, ES Mphi are likely to provide a better in vitro model than mouse Mphi cell lines to study macrophage involvement in atherosclerosis.

Objective. This work studied the presence of inflammatory and atherogenic lipoprotein markers that could explain the high incidence of cardiovascular disease (CVD) reported in rheumatoid arthritis (RA) patients. Methods. Inflammatory markers were 1) soluble adhesion molecules (intercellular adhesion molecule [ICAM] and vascular cell adhesion molecule [VCAM]), 2) C-reactive protein (CRP), 3) fibrinogen (Fb), 4) cytokines (interferon-y [IFNy], tumor necrosis factor a [TNFa]), and 5) secretory group IIA phospholipase A 2 (sPLA 2 -IIA). Atherogenic lipoprotein markers were 1) the size distribution of plasma lipoprotein subclasses, and 2) the binding affinity of low-density lipoprotein (LDL) to chondroitin 6-sulfate glycosaminoglycan (GAG). Results. RA patients (n = 31) and matched controls (n = 28) had similar plasma concentrations of total cholesterol, triglycerides, Apo B, Apo A-I, very low-density lipoprotein, intermediate-density lipoprotein, and high-density lipoprotein (HDL). RA patients had significantly higher plasma levels of sPLA 2 -IIA, ICAM, CRP, Fb, TNFa, and IFNy compared with controls. RA patients also had significantly higher levels of small, dense LDL-1 (P < 0.05) and lower levels of small HDL-2 particles (P < 0.001) compared with controls. In addition, LDL from RA patients had a significantly higher binding affinity (K d ) to GAG (mean ± SD K d 204 ± 22.4 nM Apo B) than did LDL from control subjects (K d 312 ± 36 nM Apo B) (P < 0.05). This K d value showed a significant negative correlation with the plasma levels of LDL-1 (r = -0.566, P ≤ 0.004). In RA patients, a significant positive correlation was obtained between sPLA 2 -IIA and CRP, ICAM, and LDL-1. HDL-2 showed a negative correlation with sPLA 2 -IIA. Conclusion. These atherogenic lipoprotein factors combined with the presence of chronic inflammation may contribute to the high CVD-related mortality in RA patients.

Increased expression of secretory non-pancreatic phospholipase A(2) (sPLA(2)-IIA) could be part of the inflammatory reaction in atherosclerosis. However, the factors controlling sPLA(2)-IIA production in human vascular cells are unknown. We investigated regulation of sPLA(2)-IIA expression and secretion by human arterial smooth muscle cells in culture (HASMC). SPLA(2)-IIA was induced after 3-14 days of culture in non-proliferating conditions. SPLA(2)-IIA was co-expressed with heavy caldesmon, a cytoskeleton protein, and p27, a G(1) cyclin inhibitor, proteins characteristically expressed by differentiated cells. Further incubation with 50-500 units/ml of interferon (IFN)-gamma significantly increased sPLA(2)-IIA mRNA and secretion. IFN-gamma-induced sPLA(2)-IIA was found to be active in cell media and associated with cell membrane proteoglycans. IFN-gamma induced sPLA(2)-IIA expression was antagonized by tumor necrosis factor (TNF)-alpha and interleukin (IL)-10. TNF-alpha added individually induced a significant but transient (4 h) increase in sPLA(2)-IIA secretion. IL-10 by itself did not affect sPLA(2)-IIA expression and secretion. IFN-gamma-stimulated sPLA(2)-IIA transcription involved STAT-3 protein. Interestingly, IL-6 but not IFN-gamma up-regulated the sPLA(2)-IIA expression in HepG2 cells, thus sPLA(2)-IIA induction by IFN-gamma response appears to be cell specific. In summary, conditions leading to cell differentiation induced sPLA(2)-IIA expression in HASMC and further exposure to IFN-gamma can up-regulate sPLA(2)-IIA transcription and secretion. This IFN-gamma stimulatory effect can be modulated by other cytokines.

Most models of lipoprotein oxidation by free radi- cals have excluded macromolecular plasma components from the system. This limits their biological significance because oxidation of lipoproteins appears to occur in the intima in the presence of a plasma ultrafiltrate. Hemin, a product of in vivo hemoglobin degradation, binds and oxidizes purified lipo- proteins. However, it is not known whether this occurs in the presence of plasma components that may sequester hemin. We found that hemin in serum diluted to protein levels of the extracellular fluid (10-30%) binds to low and high density lipoproteins (LDL, HDL) with association constants in the nmol/L range. In the presence of H 2 O 2 , hemin oxidizes both lipoproteins in diluted serum with formation of conjugated dienes, thiobarbituric acid reacting substances, and F 2 -iso- prostanes. This appeared to be caused by the high affinity of hemin with LDL and by the Fe 3 1 liberated that remains asso- ciated with the particles after hemin is degraded. Spectropho- tometric and fluorescence experiments and electrophoresis of porphyrins complex with LDL indicated that the heme ring is buried in the lipoprotein surface-monolayer with the carboxylic groups in contact with positive regions of the pro- tein and the solvent. Human macrophages associated and de- graded 3- to 4-times more hemin-oxidized LDL in diluted se- rum than native LDL. It is possible then that at sites of LDL accumulation in the extracellular intima, hemin and H 2 O 2 production could cause oxidation with potential atherogenic consequences for cellular lipoprotein processing. This may occur even when other macromolecules of the extracellular

Secretory nonpancreatic type II phospholipase A 2 (snpPLA 2 ) hydrolyzes fatty acids at the sn-2 position in phospholipids releasing free fatty acids (FFAs) and lysophospholipids. These products may act as intracellular second messengers or can be further metabolized into proinflammatory lipid mediators. The presence of snpPLA 2 in extracellular fluids and serum during inflammation has suggested a role of the enzyme in this process. However, the presence of snpPLA 2 in a variety of normal tissues suggests that snpPLA 2 may also have physiological functions. Atherosclerosis appears to have an inflammatory component. Here we report on the snpPLA 2 localization in normal and atherosclerotic lesions and on the properties of the isolated enzyme. A strong snpPLA 2 immunoreactivity was observed in the arterial media that was colocalized with α-actin–positive vascular smooth muscle cells (SMCs) in both normal and atherosclerotic vessels. In aortic atherosclerotic lesions, snpPLA 2 was observed colocalized with CD68-positive macrophages and HHF-35–positive SMCs and extracellularly in the lipid core. snpPLA 2 was isolated from human normal arteries and from aorta with lesions. The enzyme was isolated by acid extraction of normal arterial tissues followed by immunoaffinity chromatography. The purified snpPLA 2 had an expected molecular weight of 14 kD by polyacrylamide gel electrophoresis and appeared as a single band in immunoblotting. The enzymatic activity was followed by measuring release of fatty acids from phospholipid liposomes or LDL as substrates. The enzymatic activity was inhibited with two specific inhibitors for human snpPLA 2 : (1) monoclonal antibody 187 and (2) LY311727, a synthetic selective inhibitor. The mRNA for snpPLA 2 was detected with reverse transcriptase polymerase chain reaction. These results indicate that snpPLA 2 is present in human arteries and that it is able to hydrolyze phospholipids in LDL. The results support the hypothesis that snpPLA 2 can release proinflammatory lipids at places of LDL deposition in the arterial wall.

High circulating levels of group IIA secretory phospholipase (sPLA 2 -IIA) activity as well as mass are independent cardiovascular risk factors. Therefore, inhibition of sPLA 2 -IIA may be a target for the treatment of atherosclerotic cardiovascular disease (CVD). The present study evaluated the effects of sPLA 2 -IIA inhibition with varespladib acid in a novel mouse model, human apolipoprotein B/human cholesteryl ester transfer protein (CETP)/human sPLA 2 -IIA triple transgenic mice (TTT) fed a Western-type diet. sPLA 2 -IIA expression increased atherosclerotic lesion formation in TTT compared with human apolipoprotein B/human CETP double transgenic mice (p2 -IIA activity. Surprisingly, however, administration of varespladib acid to TTT had no impact on atherosclerosis, which could be attributed to a pro-atherogenic plasma lipoprotein profile that appears in response to sPLA 2 -IIA inhibition due to increased plasma CETP activity. In the TTT model sPLA 2 -IIA decreased CETP activity by reducing the acceptor properties of sPLA 2 -IIA-modified VLDL specifically due to a significantly lower apoE content. Increasing VLDL apoE content by means of adenovirus-mediated gene transfer in sPLA 2 -IIA transgenic mice restored the acceptor properties for CETP. In conclusion, these data show that in a humanized triple transgenic mouse model with hypercholesterolemia, sPLA 2 -IIA inhibition increases CETP activity via increasing the VLDL apoE content resulting in a pro-atherogenic lipoprotein profile.

Phospholipase A2 acting on low density lipoproteins in the extracellular arterial intima may form proinflammatory lipid mediators. Human nonpancreatic secretory phospholipase A2 has three regions that may associate with sulfated glycosaminoglycans. The apoB-100 molecule in low density lipoproteins also has glycosaminoglycan binding regions that could mediate its retention in the arterial intima. Here we report that human nonpancreatic phospholipase A2 isolated from a transfected cell line binds to glycosaminoglycans secreted by cultured human arterial smooth muscle cells. A gel mobility shift assay showed that the affinity of phospholipase A2 for glycosaminoglycans from a heparan sulfate/chondroitin sulfate proteoglycan was higher than for chondroitin sulfate glycosaminoglycans from a larger versican-like proteoglycan. Affinity chromatography confirmed these results. All glycosaminoglycans tested, at concentrations up to 100 μM, increased the activity of phospholipase A2 toward phosphatidylcholine liposomes. Above this concentration, heparan sulfate and heparin inhibited the enzyme. Heparin and chondroitin 6-sulfate increased phospholipase A2 activity on low density lipoproteins up to 4-fold at 100 μM, whereas heparan sulfate had no effect. The results indicate that human nonpancreatic secretory phospholipase A2 interacts with proteoglycans via their glycosaminoglycan moiety and that the enzyme activity may be modulated by the association of the enzyme and its substrate to the sulfated polysaccharides.

Abstract The amino acid sequence of interferon gamma (IFN-γ) has basic amino acid clusters similar to the heparin-binding consensus sequences found in other proteins that bind to proteoglycans (PGs). We investigated whether recombinant human IFN-γ could bind to extracellular matrix (ECM) PGs secreted by human arterial smooth muscle cells (HASMCs) in vitro and whether the interaction affected the cellular response to IFN-γ. As an in vitro model of ECM we used the basement membrane from HASMCs in culture. The binding of 125 I-IFN-γ to ECM was reduced significantly by pretreatment of ECM with chondroitinase ABC, an enzyme that degrades chondroitin-sulfate glycosaminoglycans. IFN-γ binding to ECM was reduced by increasing concentrations of chondroitin-6-sulfate. 125 I-IFN-γ (0.05 to 2 ng/mL) binding data indicated an apparent K d of 2×10 −11 mol/L and a maximum binding of 1.6×10 6 IFN-γ molecules bound per square millimeter of ECM. Experiments with synthetic peptides suggested that residues 127 through 135 (AKTGKRKRS) are involved in the binding. The binding to chondroitin-sulfate PGs was confirmed by affinity chromatography of isolated [ 35 S]chondroitin-sulfate PGs from ECM and cell-culture medium on immobilized IFN-γ. The binding was abolished by treatment with chondroitinase ABC. ECM-bound IFN-γ was more effective in inducing the expression of class II major histocompatibility antigens such as HLA-DR in HASMCs and human arterial endothelial cells than soluble IFN-γ. These results suggest a role for chondroitin-sulfate PGs in immobilizing IFN-γ in the ECM compartment and enhancing the cellular response to IFN-γ.

Association of low-density lipoproteins (LDL) with arterial chondroitin sulfate proteoglycans (CSPG) appears to contribute to their deposition in the extracellular intimal compartment and to its internalization by macrophages. CSPG and LDL interact by ionic bridges with formation of soluble and insoluble complexes. We studied the alterations on LDL structure induced by its association with arterial CSPG and other glycosaminoglycans (GAG). In soluble complexes, at low and at physiological ionic strength, arterial CSPG and sulfated GAG modify the kinetics of apoB-100 proteolysis by trypsin. However, less marked alterations in the peptide patterns were observed with proteinase V8 and almost none with thermolysin. This is indirect evidence that the presence of CSPG and GAG modified the exposure of polar regions of apoB-100 in LDL. Competitive binding experiments with agarose-bound heparin and soluble GAG also suggest that after formation of insoluble complexes with arterial CSPG and resolubilization the exposure of Lys, Arg-rich segments of apoB-100 is increased. Results from differential scanning calorimetry and differential thermal spectrophotometry showed that the CSPG and GAG-induced modifications reduced the thermal stability of the surface and core in LDL. If present in vivo, the structural alterations of polar segments of the LDL protein moiety may influence the outcome of its interaction with the arterial mesenchyma.

Macrophages and arterial chondroitin sulfate proteoglycans (CSPG) are probably associated with extracellular and intracellular lipoprotein deposition during atherogenesis. We found that human arterial CSPG can be used to select subclasses from low density lipoprotein (LDL) with different structural properties and capacities to interact with human monocyte-derived macrophages (HMDM). Four subclasses, LDL(PG)1 to LDL(PG)4, in order of decreasing CSPG-complexing capacity, were prepared and characterized in terms of their ability to interact with HMDM. The LDL subclasses with highest avidity for CSPG, LDL(PG)1 and LDL(PG)2, were bound, internalized, and degraded more efficiently than those of lower avidity for CSPG. From LDL(PG)1 to LDL(PG)4, the gradual decrease in uptake by HMDM and decreasing avidity for CSPG were associated with a gradual decrease in isoelectric point (from 5.93 to 5.68) and an augmented ratio of surface polar lipid to core nonpolar components (from 0.35 to 0.54). Competition experiments indicated that the proteoglycan-selected subfractions shared the binding sites and uptake mechanisms of native LDL. The results suggest the existence of a structurally related gradation in the avidity of LDL subpopulations for cells and matrix components. The presence within LDL subpopulations of a differential capacity to interact with intimal extracellular and cellular elements could be associated with a similar heterogeneity in their atherogenic potential.

There is much interest in the significance of apolipoproteins and proteins that are noncovalently associated with lipoproteins. It is possible that the high ionic strength used for isolation of lipoproteins with KBr and NaI could alter the pattern of associated exchangeable proteins. Here we describe lipoprotein classes fractionation from up to 0.5 ml of serum or plasma with buffers of physiological ionic strength and pH prepared with deuterium oxide (D(2)O) and sucrose. An advantage of the D(2)O/sucrose procedure was that the lipoproteins could be directly analyzed by the techniques described without need for desalting. We compared the isolated lipoproteins with those obtained using ultracentrifugation in KBr from the same plasma pool. Electrophoretic homogeneity of the lipoproteins was very similar using the two methods, as well as their lipid composition evaluated by HPLC. Two-dimensional electrophoresis and surface-enhanced laser adsorption/ionization time-of-flight mass spectrometry indicated that the patterns of exchangeable proteins of VLDL isolated using with the two procedures were very similar. However, significant differences were found in the profiles of LDL and HDL, indicating that the D(2)O/sucrose method allowed a more complete characterization of its exchangeable apolipoproteins and proteins.

Clinical observations strongly support an association of circulating levels of secretory phospholipases A 2 (sPLA 2 ) in atherosclerotic cardiovascular disease (ACVD). Two modes of action can provide causal support for these statistical correlations. One is the action of the enzymes on circulating lipoproteins and the other is direct action on the lipoproteins once in the arterial extracellular intima. In this review we discuss results suggesting a distinct profile of characteristics related to localization, action on plasma lipoproteins and interaction with arterial proteoglycans for sPLA 2 -IIA and sPLA 2 -V. The differences observed indicate that these enzymes may contribute to atherosclerosis through dissimilar pathways. Furthermore, we comment on recent animal studies from our laboratory indicating that the expression of type V enzyme is up-regulated by genetically and nutritionally-induced dyslipidemias but not the group type IIA enzyme, which is well known to be up-regulated by acute inflammation. The results suggest that if similar up-regulation occurs in humans in response to hyperlipidemia, it may create a distinctive link between the group V enzyme and the disease.

Publisher Summary Phospholipases A 2 (PLA 2 ) are enzymes that hydrolyze the sn -2 ester bond in the glyceroacyl phospholipids present in lipoproteins and cell membranes, forming nonesterified fatty acids (NEFA) and lysophosphohpids. PLA 2 activity present in arterial intima-media tissue and in plasma may contribute to atherosclerosis, the main pathological process behind cardiovascular diseases (CVD). The hypothesis of the potential involvement of PLA 2 activity in the pathogenesis of atherosclerosis is reinforced by in vivo data showing that transgenic mice expressing human sPLA-IIA have increased susceptibility to atherosclerosis. This chapter presents evidence supporting the proatherogenic properties of PLA 2 activity contributing to cardiovascular diseases with a main focus on sPLA 2 -IIA enzyme. The chapter discusses the expression of different PLA 2 enzymes in the heart and their function in myocardial lipid metabolism associated with ischemic heart disease.

Several cytokines and growth factors act on cells after their association with the glycosaminoglycan (GAG) moiety of cell surface proteoglycans (PGs). Interferon-gamma (IFN-gamma) binds to GAG; however, the relevance of this interaction for the biological activity of IFN-gamma on human cells remains to be established. Human arterial smooth muscle cells (HASMC), the main cells synthesizing PG in the vascular wall, respond markedly to IFN-gamma. We found that treatment of HASMC with chondroitinase ABC, an enzyme that degrades chondroitin sulfate GAG, reduced IFN-gamma binding by more than 50%. This treatment increased the affinity of 125I-IFN-gamma for cells from a Kd value of about 93 nM to a Kd value of about 33 nM. However, the total binding was reduced from 9. 3 +/- 0.77 pmol/microg to 3.0 +/- 0.23 pmol/mg (n = 4). Interestingly, pretreatment with chondroitinase ABC reduced significantly the cellular response toward IFN-gamma. The interaction of IFN-gamma with chondroitin sulfate GAG was confirmed by affinity chromatography of isolated cell-associated 35S-, 3H-labeled PG on a column with immobilized IFN-gamma. The cell-associated PG that binds to IFN-gamma was a chondroitin sulfate PG (CSPG). This CSPG had a core protein of approximately 110 kDa that was recognized by anti-CD44 antibodies on Western blots. High molecular weight complexes between IFN-gamma and chondroitin 6-sulfate were observed in gel exclusion chromatography. Additions of chondroitin 6-sulfate to cultured HASMC antagonized the antiproliferative effect and expression of major histocompatibility complex II antigens induced by IFN-gamma. These results indicate that IFN-gamma binds with low affinity to the chondroitin sulfate GAG moiety of the cell surface CSPG receptor CD44. This interaction may increase the local concentration of IFN-gamma at the cell surface, thus facilitating its binding to high affinity receptors and modulating the ability of IFN-gamma to signal a cellular response.

The reversible interaction of low density lipoprotein (LDL) with arterial chondroitin sulfate proteoglycans (CSPGs) or glycosaminoglycans (GAGs) selects LDL particles with a high affinity for sulfated GAGs and also induces modifications in apolipoprotein B (apo B) and the lipid organization of the lipoprotein. In the present work we studied the effect that the reversible interaction with sulfated polysaccharides has on the susceptibility of LDL to in vitro oxidation. For this purpose soluble, nonaggregated CSPG- or GAG-treated LDL was subjected to oxidation in the presence of 5 microM CuSO4 for as long as 48 hours. The rate of formation of thiobarbituric acid-reactive substances, the decrease in isoelectric point, the increase in relative electrophoretic mobility of LDL, the higher degradation rate by human macrophages, and the lower degradation rate by human arterial smooth muscle cells showed that LDLs exposed to CSPGs and GAGs were significantly more susceptible to oxidation than native LDL. Results from competition experiments indicate that C6S-treated LDL after 4 hours of oxidation is taken up via the acetylated LDL receptor in human macrophages. Coincubation of lipoproteins with human macrophages or human arterial smooth muscle cells for 24 hours also indicated that C6S-treated LDL was more susceptible to cell-induced modifications than native LDL. The occurrence in vivo of similar processes may contribute to focal retention, increased rate oxidation of LDL in the arterial intima, and foam cell formation during atherogenesis.

Chondroitin sulfate proteoglycans (CSPG) appear to contribute to retention of low density lipoproteins (LDL) in atherosclerotic lesions. In vitro, CSPG and glycosaminoglycans (GAG) modify LDL structure and increase its uptake by macrophages. This latter effect appears related to increased exposure of arginine- and lysine-rich segments of apoB-100. We explored whether alterations of LDL induced by human arterial CSPG and purified GAG alter the lipoprotein susceptibility to transition metals-catalyzed oxidation. Human LDL was complexed with human arterial CSPG and dissociated by raising the ionic strength. The nonaggregated, CSPG- and GAG-treated LDL was subjected to oxidation by micromolar amounts of Cu+, Cu2+, Fe2+, and Fe3+. This treatment increased LDL susceptibility to Cu2+ oxidation 3- to 5-times, as indicated by the degradation rate of phospholipids and cholesteryl esters and formation rates of dienes and thiobarbituric acid-reacting substances (TBARS). Also, human macrophages degraded the CSPG-treated, Cu2+-oxidized LDL 3- to 6-times faster than native LDL similarly treated. No enhancement of oxidation was observed with Fe2+, Fe3+, and Cu+. Quenching of the LDL intrinsic fluorescence by Cu2+ showed that heparin, CSPG, and chondroitin-6-SO4 pretreatment increased the access of Cu2+ to hydrophobic chromophores, probably tryptophan, 6- to 7-, 3- to 4-, and 2- to 3-fold, respectively. Also, the affinity constant (Ka) of LDL for Cu2+ was increased from 0.12 microM to 0.20 microM by the treatment with CSPG and GAG. These results and evaluation of the fraction of surface-accessible LDL chromophores to acrylamide quenching suggest that the increased susceptibility to oxidation may be associated with an increase in the access of Cu2+ to hydrophobic regions in LDL caused by treatment with CSPG and GAG. This effect was not detected with Cu+, Fe2+, or Fe3+. The phenomenon may contribute to acceleration of the oxidative modifications of LDL in cell culture models and in vivo.

Modifications of low density lipoproteins (LDL) that enter the arterial intima appear to be responsible for their eventual extracellular and intracellular accumulation during atherogenesis. Some of these modifications seem to be the result of LDL msociation with intirnal chondroitin sulphate-rich proteoglycans (CSPG). We have used frontal elution affinity chromatography, binding and competition experiments with synthetic segments of apoB-100 to better define the ligand regions for the LDL-CSPG complexes. The minimum structural requirement for recognition by the CSPG appears to be a hydrophilic nine-residue amino-acid segment with five lysine and arginine residues. Analysis of other similar regions in apoB-100 and other glycosaminoglycan-binding proteins suggest that besides a cluster of positively charged amino-acids, the presence of hydroxyl-containing residues favours the association with sulphated proteoglycans. With controlled proteolytic hydrolysis, we found that the interaction of LDL with CSPG modifies the surface accessibility of a apoB-100 segments containing arginine and lysine. Because these apoB-100 domains may also be involved in cell-receptor binding, the CSPG-induced modifications could be the structural explanation for the observed increase in cellular uptake of proteoglycan modified LDL.

The glycosphingolipid pattern was examined in three cases of late infantile metachromatic leukodystrophy (MLD): one with a relatively short (2.5 years), one with a long (7.8 years), and one with a very long (13.2 years) survival time. All values were compared with those of age-matched normal controls. The cerebroside concentration was reduced to 25, 12, and 4%, respectively, in the MLD white matter, whereas the sulfatide concentration was increased up to 200% of the control value. The yield of myelin was reduced to less than 15% in the early case and to less than 3 and 1%, respectively, in the two later cases. There was no sign of increased sulfatide proportion in the myelin. The ganglioside pattern was normal in cerebral gray matter, but in the white matter, contents of gangliosides of the lacto series were significantly increased, in particular, the ganglioside suggested by us as being characteristic of reactive astrocytosis. For the first time, lysosulfatide was identified in MLD and normal human brains by mass spectrometry and radioimmunoaffinity TLC using specific monoclonal antibody. Its quantity was found to be similar in normal and MLD brains. These findings support our postulation that the lysoglycosphingolipids are synthesized de novo from sphingosine and that they do not play a key role in pathogenetic mechanisms.

The major mono- and disialogangliosides of the extensively characterized established human glioma line D54MG were isolated and purified from subcutaneous solid xenografts grown in athymic (nu/nu) mice. Structural determination showed that they belonged to the lactotetraosylceramide series. The sialyllactotetraosylceramide contained 90% N-glycolyl- and 10% N-acetylneuraminic acid linked in an alpha 2-3 linkage (IV3NeuGc-LcOse4Cer, IV3NeuAc-LcOse4Cer). The disialogangliosides had a previously undescribed type of structure with sialic acids linked to the terminal galactose in an alpha 2-3 linkage and to N-acetylglucosamine in an alpha 2-6 linkage. Not only did species with NeuAc or NeuGc occur, but also species with mixtures of the two sialic acids, e.g. NeuAc and NeuGc. The schematic structures of the new disialogangliosides are (Formula:see text).

A new type of strong anion-exchange resin, Mono Q, has been used in the separation of brain gangliosides. The resin consists of monodisperse particles (9.8 micron) and was used in prepacked columns with a bed volume of 1 ml. The gangliosides were separated into mono-, di-, tri- and tetrasialoganglioside fractions by a discontinuous gradient of potassium acetate in methanol. The separation was complete in a volume of 50 ml. The major advantages of the new procedure compared to conventional methods are the shorter separation time, higher loading capacity and recovery of separated ganglioside fractions in small solvent volumes. The procedure was applied to the separation of gangliosides from normal human and GM2-gangliosidosis brain.

The developmental profiles of the four major brain gangliosides, GM1, GD1a, GD1b, and GT1b, were examined in human frontal lobe covering the period from 10 fetal weeks to 80 years of age. The ganglioside concentration increased approx. 3-fold from the 10th gestational week to the age of about 5 years. Gangliosides GM1 and GD1a increased 12–15-fold during the same period. The most rapid increase of GM1 and GD1a occurred around term, during the period for dendrite arborization, outgrowth of axons and synaptogenesis. GT1b showed a quite different developmental curve. It was the major ganglioside during the 3rd to 5th gestational month, whereafter its concentration dropped rapidly to term, from which time the concentration then increased up to 50 years of age. Similar curves were found for the other gangliosides of the b-series, GD3, GD2, GD1b and GQ1b. Ganglioside 3′-isoLM1 was a characteristic early fetal ganglioside which dropped rapidly to the 5th gestational month, reached a small peak around term and then disappeared during adulthood. The concentration of gangliosides of the neolacto series was larger than that of the lacto series during the whole developmental period. In the beginning of the second trimester, 3′-LM1 constituted 2% and LD1 10% of total ganglioside sialic acid. The new findings demonstrate more dynamic changes of the ganglioside patterns during development than noted in previous studies.

The ganglioside composition of the brain from an individual with classical Tay-Sachs disease and from an individual with Sandhoff disease was examined using our new quantitative methods for ganglioside content determination and compared with that of age-matched control brains. The concentration of GM2 was found to be 12.2 and 13.0 mumol/g of fresh tissue in Tay-Sachs disease and in Sandhoff disease cerebral gray matter, respectively. GM2 was 86 and 87% respectively, of total gangliosides. The concentration of GM1 and, in particular, GM3 ganglioside was also found to be increased, whereas the concentration of the major di- and trisialogangliosides (GD1a, GD1b, and GT1b) had diminished markedly. There was no significant increase in level of any other ganglioside than lyso-GM2. Its concentration was 12 and 16 nmol/g in cerebral gray matter of two Tay-Sachs disease brains and 43 nmol/g in Sandhoff disease brain. The Sandhoff disease brain also differed from the classical Tay-Sachs disease brain by having a much higher concentration of gangliotriaosylceramide and globotetraosylceramide. The structures of relevant gangliosides and neutral glycolipids were established by fast atom bombardment-mass spectrometry and permethylation studies.

Human plasma lipoproteins, fractionated by density gradient ultracentrifugation, and very low density lipoproteins, subfractionated by cumulative rate centrifugation, were subjected to agarose isoelectric focusing in small format thin gels prepared in the laboratory for the commercially available PhastSystem (Pharmacia). From preparation of the gels to their staining, the procedure took less than 3 h. The pH gradient was found reproducible and the apparent average pI of individual low density lipoproteins could be measured with a coefficient of variation of less than 5% between and less than 2% within the same run. The method appears especially suitable for the exploration of charge properties of multiple lipoprotein samples, or other large macromolecules as low density lipoproteins and very low density lipoproteins, with considerable economy of time and reagents.