Philippe Lesnik

Lipoprotein(a) as a cardiovascular risk factor: current status

Aims The aims of the study were, first, to critically evaluate lipoprotein(a) [Lp(a)] as a cardiovascular risk factor and, second, to advise on screening for elevated plasma Lp(a), on desirable levels, and on therapeutic strategies. Methods and results The robust and specific association between elevated Lp(a) levels and increased cardiovascular disease (CVD)/coronary heart disease (CHD) risk, together with recent genetic findings, indicates that elevated Lp(a), like elevated LDL-cholesterol, is causally related to premature CVD/CHD. The association is continuous without a threshold or dependence on LDL- or non-HDL-cholesterol levels. Mechanistically, elevated Lp(a) levels may either induce a prothrombotic/anti-fibrinolytic effect as apolipoprotein(a) resembles both plasminogen and plasmin but has no fibrinolytic activity, or may accelerate atherosclerosis because, like LDL, the Lp(a) particle is cholesterol-rich, or both. We advise that Lp(a) be measured once, using an isoform-insensitive assay, in subjects at intermediate or high CVD/CHD risk with premature CVD, familial hypercholesterolaemia, a family history of premature CVD and/or elevated Lp(a), recurrent CVD despite statin treatment, ≥3% 10-year risk of fatal CVD according to European guidelines, and/or ≥10% 10-year risk of fatal + non-fatal CHD according to US guidelines. As a secondary priority after LDL-cholesterol reduction, we recommend a desirable level for Lp(a) <80th percentile (less than ∼50 mg/dL). Treatment should primarily be niacin 1–3 g/day, as a meta-analysis of randomized, controlled intervention trials demonstrates reduced CVD by niacin treatment. In extreme cases, LDL-apheresis is efficacious in removing Lp(a). Conclusion We recommend screening for elevated Lp(a) in those at intermediate or high CVD/CHD risk, a desirable level <50 mg/dL as a function of global cardiovascular risk, and use of niacin for Lp(a) and CVD/CHD risk reduction.

Triglyceride-rich lipoproteins and high-density lipoprotein cholesterol in patients at high risk of cardiovascular disease: evidence and guidance for management

Even at low-density lipoprotein cholesterol (LDL-C) goal, patients with cardiometabolic abnormalities remain at high risk of cardiovascular events. This paper aims (i) to critically appraise evidence for elevated levels of triglyceride-rich lipoproteins (TRLs) and low levels of high-density lipoprotein cholesterol (HDL-C) as cardiovascular risk factors, and (ii) to advise on therapeutic strategies for management. Current evidence supports a causal association between elevated TRL and their remnants, low HDL-C, and cardiovascular risk. This interpretation is based on mechanistic and genetic studies for TRL and remnants, together with the epidemiological data suggestive of the association for circulating triglycerides and cardiovascular disease. For HDL, epidemiological, mechanistic, and clinical intervention data are consistent with the view that low HDL-C contributes to elevated cardiovascular risk; genetic evidence is unclear however, potentially reflecting the complexity of HDL metabolism. The Panel believes that therapeutic targeting of elevated triglycerides (≥1.7 mmol/L or 150 mg/dL), a marker of TRL and their remnants, and/or low HDL-C (<1.0 mmol/L or 40 mg/dL) may provide further benefit. The first step should be lifestyle interventions together with consideration of compliance with pharmacotherapy and secondary causes of dyslipidaemia. If inadequately corrected, adding niacin or a fibrate, or intensifying LDL-C lowering therapy may be considered. Treatment decisions regarding statin combination therapy should take into account relevant safety concerns, i.e. the risk of elevation of blood glucose, uric acid or liver enzymes with niacin, and myopathy, increased serum creatinine and cholelithiasis with fibrates. These recommendations will facilitate reduction in the substantial cardiovascular risk that persists in patients with cardiometabolic abnormalities at LDL-C goal.

Adenovirus-Mediated Overexpression of Tissue Inhibitor of Metalloproteinase-1 Reduces Atherosclerotic Lesions in Apolipoprotein E–Deficient Mice

BACKGROUND To define the role of metalloproteinases (MMPs) in the development of lipid-rich atherosclerotic lesions in relation to the balance between proteolytic and antiproteolytic activities, we investigated the impact of adenovirus-mediated elevation in the circulating levels of human tissue inhibitor of MMP (TIMP-1) in atherosclerosis-susceptible apolipoprotein E-deficient (apoE(-/-)) mice. METHODS AND RESULTS Infusion of apoE(-/-) mice fed a lipid-rich diet with rAd.RSV.TIMP-1 (1x10(11) viral particles) resulted in high hepatic expression of TIMP-1. At 2 weeks after injection, plasma TIMP-1 levels ranged from 7 to 24 micrograms/mL (mean 14.8+/-6.8). Marked overexpression of TIMP-1 was transient, with levels of TIMP-1 decreasing to 2.5 to 8 micrograms/mL (mean 4.3+/-2.1) at 4 weeks. Plasma lipid and lipoprotein levels in mice treated with rAd.RSV.TIMP-1 were similar to those treated with rAd.RSV.betaGal. However, rAd.RSV.TIMP-1-infused mice displayed a marked reduction (approximately 32%; P<0.05) in mean lesion area per section (512+/-121 micrometers(2)x10(3); n=12 sections from 4 animals) as compared with rAd.RSV.betaGal-infused mice (750+/-182 micrometers(2)x10(3); n=12 sections from 4 animals). Similarly, marked reduction in macrophage deposition as well as MMP-2, MMP-3, and MMP-13 antigens was observed. CONCLUSIONS Histological and immunohistologic analyses of atherosclerotic lesions revealed increases in collagen, elastin, and smooth muscle alpha-actin content in mice treated with rAd.RSV.TIMP-1. These qualitative and quantitative features were the consequence of TIMP-1 infiltration from plasma to arterial intima, as immunohistochemical analyses revealed an abundance of TIMP-1 specifically in lesions of rAd.RSV. TIMP-1-treated mice.

Macrophage Apoptosis Exerts Divergent Effects on Atherogenesis as a Function of Lesion Stage

Background— Because apoptotic cell clearance appears to be defective in advanced compared with early atherosclerotic plaques, macrophage apoptosis may differentially affect plaque progression as a function of lesion stage. Methods and Results— We first evaluated the impact of targeted protection of macrophages against apoptosis at both early and advanced stages of atherosclerosis. Increased resistance of macrophages to apoptosis in early atherosclerotic lesions was associated with increased plaque burden; in contrast, it afforded protection against progression to advanced lesions. Conversely, sustained induction of apoptosis in lesional macrophages of advanced lesions resulted in a significant increase in lesion size. Such enhanced lesion size occurred as a result not only of apoptotic cell accumulation but also of elevated chemokine expression and subsequent intimal recruitment of circulating monocytes. Conclusions— Considered together, our data suggest that macrophage apoptosis is atheroprotective in fatty streak lesions, but in contrast, defective clearance of apoptotic debris in advanced lesions favors arterial wall inflammation and enhanced recruitment of monocytes, leading to enhanced atherogenesis.

Conventional Dendritic Cells at the Crossroads Between Immunity and Cholesterol Homeostasis in Atherosclerosis

Background— Immunoinflammatory mechanisms are implicated in the atherogenic process. The polarization of the immune response and the nature of the immune cells involved, however, are major determinants of the net effect, which may be either proatherogenic or antiatherogenic. Dendritic cells (DCs) are central to the regulation of immunity, the polarization of the immune response, and the induction of tolerance to antigens. The potential role of DCs in atherosclerosis, however, remains to be defined. Methods and Results— We created a mouse model in which the lifespan and immunogenicity of conventional DCs are enhanced by specific overexpression of the antiapoptotic gene hBcl-2 under the control of the CD11c promoter. When studied in either low-density lipoprotein receptor–deficient or apolipoprotein E–deficient backgrounds, DC-hBcl2 mice exhibited an expanded DC population associated with enhanced T-cell activation, a T-helper 1 and T-helper 17 cytokine expression profile, and elevated production of T-helper 1–driven IgG2c autoantibodies directed against oxidation-specific epitopes. This proatherogenic signature, however, was not associated with acceleration of atherosclerotic plaque progression, because expansion of the DC population was unexpectedly associated with an atheroprotective decrease in plasma cholesterol levels. Conversely, depletion of DCs in hyperlipidemic CD11c–diphtheria toxin receptor/apolipoprotein E–deficient transgenic mice resulted in enhanced cholesterolemia, thereby arguing for a close relationship between the DC population and plasma cholesterol levels. Conclusions— Considered together, the present data reveal that conventional DCs are central to the atherosclerotic process, because they are directly implicated in both cholesterol homeostasis and the immune response.

Knockdown expression and hepatic deficiency reveal an atheroprotective role for SR-BI in liver and peripheral tissues

Scavenger receptor SR-BI has been implicated in HDL-dependent atheroprotective mechanisms. We report the generation of an SR-BI conditional knockout mouse model in which SR-BI gene targeting by loxP site insertion produced a hypomorphic allele (hypomSR-BI). Attenuated SR-BI expression in hypomSR-BI mice resulted in 2-fold elevation in plasma total cholesterol (TC) levels. Cre-mediated SR-BI gene inactivation of the hypomorphic SR-BI allele in hepatocytes (hypomSR-BI-KO(liver)) was associated with high plasma TC concentrations, increased plasma free cholesterol/TC (FC/TC) ratio, and a lipoprotein-cholesterol profile typical of SR-BI-/- mice. Plasma TC levels were increased 2-fold in hypomSR-BI and control mice fed an atherogenic diet, whereas hypomSR-BI-KO(liver) and SR-BI-/- mice developed severe hypercholesterolemia due to accumulation of FC-rich, VLDL-sized particles. Atherosclerosis in hypomSR-BI mice was enhanced (2.5-fold) compared with that in controls, but to a much lower degree than in hypomSR-BI-KO(liver) (32-fold) and SR-BI-/- (48-fold) mice. The latter models did not differ in either plasma lipid levels or in the capacity of VLDL-sized lipoproteins to induce macrophage cholesterol loading. However, reduced atherosclerosis in hypomSR-BI-KO(liver) mice was associated with decreased lesional macrophage content as compared with that in SR-BI-/- mice. These data imply that, in addition to its major atheroprotective role in liver, SR-BI may exert an antiatherogenic role in extrahepatic tissues.

Anticoagulant activity of tissue factor pathway inhibitor in human plasma is preferentially associated with dense subspecies of LDL and HDL and with Lp(a).

Human plasma contains a multivalent, Kunitz-type proteinase inhibitor termed tissue factor pathway inhibitor (TFPI), which specifically inhibits the action of the factor VII(a)-tissue factor complex in coagulation. In the present study, we examined the distribution and anticoagulant activity of TFPI among plasma lipoprotein subspecies separated by isopycnic density gradient ultracentrifugation; this procedure permitted the simultaneous fractionation of the major lipoprotein classes (very-low-density lipoprotein [VLDL], intermediate-density lipoprotein [IDL], low-density lipoprotein [LDL], high-density lipoprotein [HDL] 2 and 3, and very-high-density lipoprotein [VHDL]). Studies of eight normolipidemic subjects revealed two major lipoprotein carriers of TFPI activity: dense LDL subspecies (d = 1.039 to 1.063 g/mL) and both dense HDL particles and VHDL (d = 1.133 to 1.190 g/mL), representing 33.8% and 35.9%, respectively, of the total lipoprotein-associated TFPI activity in plasma. TFPI activity was also associated with lipoprotein(a) (Lp[a]), whose density distribution (d = 1.044 to 1.100 g/mL) overlapped that of LDL and HDL2; such association was related to Lp(a)s particle size and phenotype. VLDL, IDL, and LDL1 through LDL3 (d = 1.019 to 1.039 g/mL), HDL2 (d = 1.063 to 1.100 g/mL), and light subfractions of HDL3 (d = 1.100 to 1.167 g/mL) conveyed only 1.8%, 10%, and 18.5%, respectively, of lipoprotein-associated TFPI activity. Such anticoagulant activity was dependent on the presence of TFPI protein. The dense subspecies of HDL3 (d = 1.133 to 1.167 g/mL) with which TFPI was preferentially associated were small, displayed a cholesteryl ester to protein ratio of approximately 0.2, and were deficient in phospholipid (13.6% to 18.3%). HDL subspecies of d = 1.110 to 1.167 g/mL mainly contained the higher relative molecular mass form of TFPI of 41 kD (a form that is known to be covalently associated with apolipoprotein [apo] A-II) and minor bands of the 35- and 52-kD forms. The second major localization of TFPI was within the hydrated density range of small, dense LDL particles (d = 1.033 to 1.063 g/mL), which in comparison with light LDL (d = 1.019 to 1.033 g/mL) exhibited a markedly lower proportion of triglyceride and enrichment in cholesteryl ester.(ABSTRACT TRUNCATED AT 400 WORDS)

Local apoptosis mediates clearance of macrophages from resolving inflammation in mice.

Chronic inflammatory diseases such as atherosclerosis are characterized by an accumulation of macrophages. To design therapies that would reduce macrophage burden during disease, understanding the cellular and molecular mechanisms that regulate macrophage removal from sites of resolving inflammation is critical. Although past studies have considered the local death of macrophages or the possibility that they emigrate out of inflammatory foci, methods to quantify death or emigration have never been employed. Here, we applied quantitative competition approaches and other methods to study resolution of thioglycollate-induced peritonitis, the model in which earlier work indicated that emigration to lymph nodes accounted for macrophage removal. We show that migration to lymph nodes occurred in a CC chemokine receptor 7-independent manner but, overall, had a quantitatively minor role in the removal of macrophages. Blocking migration did not significantly delay resolution. However, when macrophages resistant to death were competed against control macrophages, contraction of the macrophage pool was delayed in the apoptosis-resistant cells. These data refute the concept that macrophages are dominantly cleared through emigration and indicate that local death controls macrophage removal. This finding alters the emphasis on which cellular processes merit targeting in chronic diseases associated with accumulation of macrophages.

Tissue factor pathway inhibitor is expressed by human monocyte-derived macrophages : relationship to tissue factor induction by cholesterol and oxidized LDL.

Lipid-laden macrophages express tissue factor (TF), which may activate the extrinsic coagulation pathway on rupture of the atherosclerotic plaque. Tissue factor pathway inhibitor (TFPI) is a major regulator of TF-induced coagulation. We evaluated the possibility that monocyte-derived macrophages express this protein, thereby contributing to regulation of TF activity (TFact). Equally, we investigated the effect of cholesterol and of oxidized LDL (Ox-LDL) on the expression of TFPI and TF by human monocyte-derived macrophages (HMDMs). Northern blot analysis of TFPI mRNA from cultured HMDMs revealed a single band at 4.2 kb with weak intensity; this finding was confirmed by reverse transcription-polymerase chain reaction. Gel filtration of HMDM supernatants showed the presence of an active 100-kDa form of TFPI, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions; under reducing conditions, however, the immunoblot revealed a 40-kDa form of TFPI. The TFPI in HMDM supernatants possessed heparin-binding affinity, suggesting potential interaction of TFPI with heparan sulfate proteoglycans. Stimulation of foam cell formation by incubation of macrophages for 48 hours with exogenous free cholesterol indicated that neither the biological activity nor the de novo synthesis of TFPI protein was affected. In contrast, cholesterol loading with exogenous free cholesterol induced significant upregulation of total TFact (2.6-fold: 25.0 versus 9.4 mU/mg cell protein, cholesterol-treated versus control cells; P<0. 05); such induction was not correlated with an elevation in TF antigen (8.5 versus 7.8 ng/mg cell protein, cholesterol-treated versus control cells). Similarly, cholesterol-rich Ox-LDL induced an increase in TFact (1.9-fold: 18.9 versus 10.0 mU/mg cell protein, Ox-LDL-treated versus control cells; P<0.05); by contrast, the amount of TF antigen remained unchanged (7.1 versus 7.9 ng/mg cell protein, Ox-LDL-treated versus control cells). Our data indicate that enhancement of the procoagulant activity of TF in macrophage-derived foam cells is not counterbalanced by upregulation of TFPI activity, suggesting that lesion foam cells are in a procoagulant state; they may therefore contribute to thrombus generation on plaque rupture.

Interleukin-6 Protects Human Macrophages from Cellular Cholesterol Accumulation and Attenuates the Proinflammatory Response

Cholesterol-laden monocyte-derived macrophages are phagocytic cells characteristic of early and advanced atherosclerotic lesions. Interleukin-6 (IL-6) is a macrophage secretory product that is abundantly expressed in atherosclerotic plaques but whose precise role in atherogenesis is unclear. The capacity of macrophages to clear apoptotic cells, through the efferocytosis mechanism, as well as to reduce cellular cholesterol accumulation contributes to prevent plaque progression and instability. By virtue of its capacity to promote cellular cholesterol efflux from phagocyte-macrophages, ABCA1 was reported to reduce atherosclerosis. We demonstrated that lipid loading in human macrophages was accompanied by a strong increase of IL-6 secretion. Interestingly, IL-6 markedly induced ABCA1 expression and enhanced ABCA1-mediated cholesterol efflux from human macrophages to apoAI. Stimulation of ABCA1-mediated cholesterol efflux by IL-6 was, however, abolished by selective inhibition of the Jak-2/Stat3 signaling pathway. In addition, we observed that the expression of molecules described to promote efferocytosis, i.e. c-mer proto-oncogene-tyrosine kinase, thrombospondin-1, and transglutaminase 2, was significantly induced in human macrophages upon treatment with IL-6. Consistent with these findings, IL-6 enhanced the capacity of human macrophages to phagocytose apoptotic cells; moreover, we observed that IL-6 stimulates the ABCA1-mediated efflux of cholesterol derived from the ingestion of free cholesterol-loaded apoptotic macrophages. Finally, the treatment of human macrophages with IL-6 led to the establishment of an anti-inflammatory cytokine profile, characterized by an increased secretion of IL-4 and IL-10 together with a decrease of that of IL-1β. Taken together, our results indicate that IL-6 favors the elimination of excess cholesterol in human macrophages and phagocytes by stimulation of ABCA1-mediated cellular free cholesterol efflux and attenuates the macrophage proinflammatory phenotype. Thus, high amounts of IL-6 secreted by lipid laden human macrophages may constitute a protective response from macrophages to prevent accumulation of cytotoxic-free cholesterol. Such a cellular recycling of free cholesterol may contribute to reduce both foam cell formation and the accumulation of apoptotic bodies as well as intraplaque inflammation in atherosclerotic lesions.