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Aims: Low-density lipoprotein (LDL) particles cause atherosclerotic cardiovascular disease (ASCVD) through their retention, modification, and accumulation within the arterial intima. High plasma concentrations of LDL drive this disease, but LDL quality may also contribute. Here, we focused on the intrinsic propensity of LDL to aggregate upon modification. We examined whether inter-individual differences in this quality are linked with LDL lipid composition and coronary artery disease (CAD) death, and basic mechanisms for plaque growth and destabilization. Methods and results: We developed a novel, reproducible method to assess the susceptibility of LDL particles to aggregate during lipolysis induced ex vivo by human recombinant secretory sphingomyelinase. Among patients with an established CAD, we found that the presence of aggregation-prone LDL was predictive of future cardiovascular deaths, independently of conventional risk factors. Aggregation-prone LDL contained more sphingolipids and less phosphatidylcholines than did aggregation-resistant LDL. Three interventions in animal models to rationally alter LDL composition lowered its susceptibility to aggregate and slowed atherosclerosis. Similar compositional changes induced in humans by PCSK9 inhibition or healthy diet also lowered LDL aggregation susceptibility. Aggregated LDL in vitro activated macrophages and T cells, two key cell types involved in plaque progression and rupture. Conclusion: Our results identify the susceptibility of LDL to aggregate as a novel measurable and modifiable factor in the progression of human ASCVD.

Approximately 35 million people worldwide suffer from heterozygous familial hypercholesterolaemia (HeFH), a condition characterized by genetically determined life-long elevation of plasma low-density lipoprotein cholesterol (LDL-C). One in three of these patients also inherit an elevated plasma concentration of lipoprotein (a) [Lp(a)], a lipoprotein particle with atherogenic, inflammatory and prothrombotic properties. Accordingly, the combination of high plasma LDL-C and Lp(a) can markedly accelerate premature atherosclerotic cardiovascular disease (ASCVD). Neither statin nor ezetimibe lowers Lp(a), so that FH patients with high Lp(a) remain at high residual risk of ASCVD. PCSK9 monoclonal antibodies are indicated for HeFH patients not at guideline-recommended LDL-C target, but only lower Lp(a) concentration by 15-30%. Recent trials employing apo(a) antisense therapy show more potent (up to 90%) reductions in plasma Lp(a). The combination of PCSK9 inhibitor and apo(a) antisense therapy appears the optimal strategy for mitigating residual risk of ASCVD in HeFH patients with high Lp(a).

AIMS: The first aim was to critically evaluate the extent to which familial hypercholesterolaemia (FH) is underdiagnosed and undertreated. The second aim was to provide guidance for screening and treatment of FH, in order to prevent coronary heart disease (CHD).METHODS AND RESULTS: Of the theoretical estimated prevalence of 1/500 for heterozygous FH, CONCLUSION: Owing to severe underdiagnosis and undertreatment of FH, there is an urgent worldwide need for diagnostic screening together with early and aggressive treatment of this extremely high-risk condition.

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) CONCLUSION: We recommend screening for elevated Lp(a) in those at intermediate or high CVD/CHD risk, a desirable level

According to current estimates, as many as 4.5 million people in Europe suffer from the heterozygous form of familial hypercholesterolemia (HeFH).1 Their plasma concentration of low-density lipoprotein cholesterol (LDL-C) is two to three times above normal from birth, and accordingly, if untreated, the risk of coronary heart disease (CHD) and acute myocardial infarction (AMI) is markedly increased compared with non-HeFH populations.1 Indeed, in the pre-statin era, a clinical study on the incidence of CHD in patients with molecularly defined HeFH showed that the mean age of onset of symptomatic CHD was 42 ± 7 (SD) years for men and 48 ± 11 years for women, and that the corresponding ages at the time of first AMI were 47 ± 12 and 59 ± 13 years, respectively ( Figure 1 ).2 The early onset of clinically significant CHD in the pre-statin era is understandable, since the plasma concentration of LDL-C strongly elevated from birth. It also explains the early onset of subclinical coronary atherosclerosis in these patients. Thus, extrapolation of coronary angiographic data from HeFH males and HeFH females predicted that, on average, coronary stenosis starts at 17 and 25 years of age, respectively ( Figure 2 ).3 Corresponding non-invasive data have been obtained recently by assessing accumulation of coronary plaque burden with computed tomography angiography in a HeFH patient population on suboptimal statin therapy, which had been initiated in adulthood.4 When the plaque burden was extrapolated to age, the data suggested that atherosclerotic plaques may start to develop in HeFH males and females at a mean age of ∼20 and 30 years, respectively. Moreover, the total plaque burden significantly predicted future coronary …

This article is based on the concluding remarks by the author at the Ninth Paavo Nurmi Symposium on 'Lipoproteins and the Pathobiology of the Arterial Intima'. . . . Circulating cholesterol is carried into the arterial intima, the site of atherogenesis, in low-density lipoprotein (LDL) particles, and from the intima back into the circulation in high-density lipoprotein (HDL) particles. At affected sites in the intima, cholesterol accumulates in deposits known as atheromas. These local accumulations are due to disturbances in the cholesterol flow through the intima, resulting in imbalance between inflow and outflow of cholesterol. The rate of cholesterol accumulation depends ultimately on the severity of the imbalance. The factor primarily responsible for this cholesterol imbalance appears to be local modification of LDL particles. Hence, to prevent accumulation of cholesterol in the intima, the production of modified LDL particles must be prevented. This can best be achieved by reducing the inflow of LDL particles into the intima. This, in turn, can be achieved by lowering the concentration of circulating LDL particles. In addition, increasing the concentration of circulating HDL particles should accelerate the rate of removal of cholesterol from the intima, so further improving the disturbed cholesterol balance at the atheromatous sites.

Aims In aortic stenosis (AS), adverse remodelling of the valves may depend on altered local regulation of pro- and antifibrotic systems. We have recently shown that angiotensin-converting enzyme (ACE), which generates profibrotic angiotensin II and inactivates antifibrotic bradykinin (BK), is upregulated in stenotic aortic valves. Here, we analyse the expression of neutral endopeptidase (NEP), another profibrotic and BK-degrading enzyme, and of BK receptors in aortic valves in AS. Methods and results Stenotic aortic valves ( n = 86) were obtained at valve replacement surgery and control valves ( n = 13) at cardiac transplantation. Expression levels of NEP and BK type 1 and 2 receptors (BK-1R and BK-2R) in aortic valves and in isolated valvular myofibroblasts were analysed by real-time PCR and immunohistochemistry, and NEP activity was quantified by autoradiography. NEP, BK-1R, and BK-2R mRNA levels were higher in stenotic than in non-stenotic valves ( P < 0.05 for each) and the respective proteins localized to valvular endothelial cells and myofibroblasts. In stenotic valves, the proteolytic activity of NEP was significantly increased (4.5-fold, P < 0.001), and tumour necrosis factor-α induced the expression of NEP in cultured myofibroblasts. Finally, treatment of cultured myofibroblasts with an NEP inhibitor (phosphoramidon) downregulated the expression of profibrotic transforming growth factor-β1, whereas addition of BK decreased the expression of collagens I and III which was reversed by a BK-2R antagonist. Conclusion NEP activity is increased in stenotic aortic valves in parallel with increased expression of BK-receptors. The upregulation of NEP and BK-1R have the potential to promote valvular fibrosis and remodelling while the increase in BK-2R may represent a compensatory antifibrotic response. These findings add novel pathogenic insight and raise potential new therapeutic targets in AS.

AIMS Aortic stenosis (AS) is characterized by extensive remodelling of the valves, including infiltration of inflammatory cells, extracellular matrix degradation, and fibrosis. The molecular mechanisms behind this adverse remodelling have remained obscure. In this article, we study whether cathepsin G, an angiotensin II (Ang II)-forming elastolytic enzyme, contributes to progression of AS. METHODS AND RESULTS Stenotic aortic valves (n = 86) and control valves (n = 17) were analysed for cathepsin G, transforming growth factor-beta1 (TGF-beta1), and collagens I and III with RT-PCR and immunohistochemistry. Valvular collagen/elastin ratio was quantified by histochemistry. In stenotic valves, cathepsin G was present in mast cells and showed increased expression (P < 0.001), which correlated positively (P < 0.001) with the expression levels of TGF-beta1 and collagens I and III. TGF-beta1 was also present in mast cell-rich areas and cathepsin G induced losartan-sensitive TGF-beta1 expression in cultured fibroblasts. Collagen/elastin ratio was increased in stenotic valves (P < 0.001) and correlated positively with smoking (P = 0.02). Nicotine in cigarette smoke activated mast cells and induced TGF-beta1 expression in cultured fibroblasts. Fragmented elastin was observed in stenotic valves containing activated cathepsin G-secreting mast cells and in normal valves treated with cathepsin G. CONCLUSION In stenotic aortic valves, mast cell-derived cathepsin G may cause adverse valve remodelling and AS progression.

Rat serosal mast cells contain secretory granules composed of a heparin proteoglycan matrix in which neutral proteases are embedded. Stimulation of the mast cells leads to granule exocytosis and formation of two pools of granules located extracellularly, firstly, granules expelled into the 'free' extracellular space and ultimately phagocytosed by the scavenging cells in the vicinity of mast cells and, secondly, granules which remain associated with their parent mast cells, and become internalized by them during recovery from stimulation. If mast cells are stimulated in the presence of macrophages in a low density lipoprotein (LDL)-containing medium, LDL is bound to the heparin proteoglycan component of the exocytosed granules whether they are expelled into the 'free' extracellular space or remain associated with the mast cells. The granules located in the 'free' extracellular space degrade, by the action of their neutral proteases, the apolipoprotein B component of the bound LDL. The proteolytic degradation of the granule-bound LDL results in its modification such that large fused LDL particles are formed on the granule surface. Phagocytosis, by macrophages, of the granules containing fused LDL particles leads to lysosomal degradation of LDL and cholesterol accumulation in macrophages as non-membrane-bound cholesteryl ester droplets, typical of foam cells. In contrast, the rapid internalization of the LDL-bearing, mast-cell-associated granules by recovering mast cells is not followed by lysosomal processing of LDL. Instead, it leads to cholesterol accumulation in mast cells, in the form of large, partially degraded, modified LDL particles, in the granule compartment.