Extracellular vesicles (EVs) facilitate intercellular communication by carrying bioactive molecules such as proteins, messenger RNA, and micro (mi)RNAs. Recently, high-density lipoproteins (HDL) isolated from human plasma were also reported to transport miRNA to other cells. HDL, when isolated from human plasma, ranges in density between 1.063 and 1.21 g/mL, which grossly overlap with the reported density of EVs. Consequently, HDL and EV will be co-isolated when using density gradient ultracentrifugation. Thus, more stringent isolation/separation procedures of EV and HDL are essential to know their relative contribution to the pool of circulating bioactive molecules.

The low-density lipoprotein receptor (LDLR) plays a pivotal role in clearing atherogenic circulating low-density lipoprotein (LDL) cholesterol. Here we show that the COMMD/CCDC22/CCDC93 (CCC) and the Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) complexes are both crucial for endosomal sorting of LDLR and for its function. We find that patients with X-linked intellectual disability caused by mutations in CCDC22 are hypercholesterolaemic, and that COMMD1-deficient dogs and liver-specific Commd1 knockout mice have elevated plasma LDL cholesterol levels. Furthermore, Commd1 depletion results in mislocalization of LDLR, accompanied by decreased LDL uptake. Increased total plasma cholesterol levels are also seen in hepatic COMMD9-deficient mice. Inactivation of the CCC-associated WASH complex causes LDLR mislocalization, increased lysosomal degradation of LDLR and impaired LDL uptake. Furthermore, a mutation in the WASH component KIAA0196 (strumpellin) is associated with hypercholesterolaemia in humans. Altogether, this study provides valuable insights into the mechanisms regulating cholesterol homeostasis and LDLR trafficking.

Background-Loss-of-function mutations in the ATP-binding cassette (ABCA)-1 gene locus are the underlying cause for familial hypoalphalipoproteinemia, providing a human isolated low-HDL model. In these familial hypoalphalipoproteinemia subjects, we evaluated the impact of isolated low HDL on endothelial function and the vascular effects of an acute increase in HDL. Methods and Results-In 9 ABCA1 heterozygotes and 9 control subjects, vascular function was assessed by venous occlusion plethysmography. Forearm blood flow responses to the endothelium-dependent and -independent vasodilators serotonin (5HT) and sodium nitroprusside, respectively, and the inhibitor of nitric oxide synthase N G -monomethyl-Larginine (L-NMMA) were measured. Dose-response curves were repeated after systemic infusion of apolipoprotein A-I/phosphatidylcholine (apoA-I/PC) disks. At baseline, ABCA1 heterozygotes had decreased HDL levels (0.4Ϯ0.2 mmol/L; PϽ0.05), and their forearm blood flow responses to both 5HT (maximum, 49.0Ϯ10.4%) and L-NMMA (maximum, Ϫ22.8Ϯ22.9%) were blunted compared with control subjects (both PՅ0.005). Infusion of apoA-I/PC disks increased plasma HDL to 1.3Ϯ0.4 mmol/L in ABCA1 heterozygotes, which resulted in complete restoration of vasomotor responses to both 5HT and L-NMMA (both PՅ0.001). Endothelium-independent vasodilation remained unaltered throughout the protocol. Conclusions-In ABCA1 heterozygotes, isolated low HDL is associated with endothelial dysfunction, attested to by impaired basal and stimulated NO bioactivity. Strikingly, both parameters were completely restored after a single, rapid infusion of apoA-I/PC. These findings indicate that in addition to its long-term role within reverse cholesterol transport, HDL per se also exerts direct beneficial effects on the arterial wall. (Circulation. 2003;107:2944-2948.)

C-reactive protein (CRP) has been postulated to play a causal part in atherosclerosis and its acute complications. We assessed the effects of CRP-infusion on coagulation and inflammatory pathways to determine its role in atherothrombotic disease. Seven male volunteers received an infusion on two occasions, containing 1.25 mg/kg recombinant human CRP (rhCRP) or diluent, respectively. CRP-concentrations rose after rhCRPinfusion from 1.9 (0.3 to 8.5) to 23.9 (20.5 to 28.1) mg/L, and subsequently both inflammation and coagulation were activated. This sequence of events suggests that CRP is not only a well known marker of cardiovascular disease, but is also probably a mediator of atherothrombotic disease. C -reactive protein (CRP) has emerged as an independent predictor of cardiovascular risk in various clinical settings. [1][2][3] Evidence showing direct prothrombotic and inflammatory effects of CRP in vitro 4 -6 has led to the concept that CRP might be an active mediator of atherothrombotic events. Although direct pathophysiological functions of CRP itself are a matter of debate, experimental observations from mice transgenic for human CRP have suggested a contributive role of CRP in the development of cardiovascular complications. 7,8 To date, no in vivo data in humans exist to support a direct atherogenic action of CRP. In the present study, we assessed the effects of rhCRP-infusion on established pathways in cardiovascular disease progression, including inflammation and coagulation in healthy male volunteers. Materials and MethodsSeven healthy, nonsmoking men, aged 33 (26 to 51) years, were included in this study after written informed consent was obtained.None of the volunteers had febrile illness or cardiovascular disease or were on medication. After an overnight fast, a bolus of highly purified rhCRP was given intravenously at a dose of 1.25 mg per kg body weight. Blood was drawn at baseline and 1, 4, 8, and 24 hours after infusion. After 4 weeks, a time-control study was performed using the CRP-free diluent. The study was approved by the institutional review board of the Academic Medical Center Amsterdam.The rhCRP (BiosPacific), derived from Escherichia coli (K12, substrain NM522), was supplied in 20 mmol/L Tris, 140 mmol/L NaCL, 2 mmol/L CaCl 2 , pH 7.5, and 0.05% (wt/vol) sodium azide and revealed a single 23-kDa band (Ͼ99%) after CBBR-staining (1 g; SDS-polyacrylamide gel). Before purification, the host cell protein concentration was 85 ppm, as determined by a high-sensitive ELISA in accordance with manufacturers' instructions (Cygnus Technologies Inc). Subsequently, the rhCRP was purified using size exclusion chromatography to remove contaminants including endotoxin and sodium azide (Univalid bv). Purity as well as stability were evaluated using sequential highperformance liquid chromatography and time-of-flight mass spectrometry, showing no other protein fractions, including the monomeric variant of CRP, besides the CRP pentamer. Endotoxin levels were below 1.5 endotoxin units (EU)/mL as evaluated by Li...