A dot-blot assay for the low density lipoprotein receptor.

We describe a new method for detecting the interaction of low density lipoprotein with its receptor using unmodified nitrocellulose as support for membrane protein. The method is specific and sensitive down to 3 micrograms of membrane protein. Unlabeled LDL, but not HDL, competes with 125I-labeled LDL for binding, and binding is abolished by pretreatment of the membranes with pronase and is dependent upon the presence of Ca2+. Furthermore, modification of arginine or lysine residues on LDL abolishes the lipoprotein interaction with the receptor protein supported on the nitrocellulose. When the membranes are solubilized with octyl glucoside, purification steps of the receptor can be directly followed with no interference of the detergent, therefore eliminating the need for its removal. The increased expression of LDL receptors on liver membranes from estradiol-treated rats was also demonstrated. We suggest, therefore, that this method can be used to detect the presence of LDL receptors on minute amounts of membrane protein

Molecular Mechanisms Responsible for the Antiinflammatory and Protective Effect of HDL on the Endothelium

In addition to their role in reverse cholesterol transport, high-density lipoproteins (HDL) exert several beneficial effects, including the prevention and correction of endothelial dysfunction. HDL promote endothelium proliferation and diminish endothelial apoptosis; they play a key role in vasorelaxation by increasing the release of nitric oxide and prostacyclin through the induction of the expression and the activity of endothelial nitric oxide synthase and the coupling of cyclooxygenase 2 and prostacyclin synthase. In addition, HDL affect coagulation, fibrynolisis, platelet adhesion, adhesion molecules, and protease expression, and they exert antioxidant activity. These effects are achieved at the gene expression level and are dependent on the activation of several intracellular signaling pathways, including PI3K/Akt, ERK1/2, PKC, and p38MAPK. The complexity of the signaling pathways modulated by HDL reflects the different effects of the components of this class of lipoproteins such as apolipoproteins or lipids on endothelial cell gene expression and the subsequent modulation of endothelial function observed. The in vivo relevance of these findings to endothelial recovery during physiological or pathological conditions remains to be addressed; nevertheless, the results of clinical studies with synthetic HDL, ApoA-I mimetics, and drugs that are becoming available that selectively affect HDL plasma levels and biological functions support the importance of the correction of endothelial function by HDL

'Diet and lifestyle' in the management of dyslipidaemia and prevention of CVD - Understanding the level of knowledge and interest of European Atherosclerosis Society members.

Abstract To better understand the level of knowledge and interest in 'diet and lifestyle' for cholesterol management and CVD prevention, European Atherosclerosis Society (EAS) members were invited to take part in an online survey. In total, 269 EAS members participated of which 64 (24%) were students/postdocs, 102 (38%) researchers involved with CVD-related research and 103 (38%) doctors and clinicians who directly interact with patients. All (99%) of the participants either agreed or strongly agreed that 'diet and lifestyle' have a role to play in cholesterol management, with 80% indicating that 'diet and lifestyle' is very or extremely important. Of the clinicians, 75% indicated that their patients voluntarily ask for 'diet and lifestyle' advice and over 80% said they continuously provide 'diet and lifestyle advice' to their patients. Of the surveyed clinicians, 91% feel sufficiently educated and confident to provide expert advice and over 90% recommend medication, diet change, frequent exercise and smoking cessation to their patients. In view of more specific dietary advice, clinicians reportedly recommend a 'Mediterranean diet', and advise to avoid high-fat foods, and to increase intake of high-fibre foods. Interestingly, smoking cessation and alcohol avoidance were mentioned less frequently. In view of educational needs, over half of the surveyed EAS members use the internet and 'guidelines' to learn about 'diet and lifestyle' in relation to cholesterol and CVD risk management. Clinicians tend to use 'guidelines' more often, while students/postdocs tend to use the internet significantly more than clinicians and CVD researchers. Regarding unmet needs for educational tools addressing specifically 'diet and lifestyle', clinicians feel that patient-oriented leaflets and pocket guidelines would be most beneficial materials to introduce, while students/postdocs would prefer an app. In summary, the role of 'diet and lifestyle' as a cornerstone of cholesterol management and CVD risk prevention seems well recognised amongst EAS members surveyed

Combination therapy in cholesterol reduction: focus on ezetimibe and statins

Although widely used in lipid lowering therapy, HMG CoA reductase inhibitors (even when administered at high doses) are frequently insufficient to achieve guideline-recommended LDL-C goals for many patients with hypercholesterolemia in everyday clinical practice. Many patients do not achieve LDL-C goal on the initial dose of statin and the majority of these patients does not reach their goal after 6 months. As a consequence, a wide therapeutic gap exists between target LDL-C levels and those typically achieved in clinical practice. A recent and more effective therapeutic hypocholesterolemic strategy is to treat the two main sources of cholesterol simultaneously (production of cholesterol, mainly in the liver, and absorption of cholesterol in the intestine) with a complementary mechanism of action, by co-administering ezetimibe, a novel agent inhibiting cholesterol absorption, with a statin, which inhibits cholesterol production in the liver. Ezetimibe can be effectively and safely co-administered with any dose of any statin and, compared with the single inhibition of cholesterol production, afforded by statins alone, provides consistently greater reductions in LDL-C through dual inhibition of both cholesterol production and absorption. We summarize the pivotal role of both the liver and intestine in the overall balance of cholesterol in the body and describe the clinical impact and relevance of using ezetimibe either alone or co-administered with statins in controlling elevated levels of plasma LDL cholesterol

Subfractionation of human very low density lipoproteins by heparin-Sepharose affinity chromatography.

Very low density lipoproteins obtained from normolipidemic subjects were fractionated into subclasses by means of affinity chromatography on a heparin-Sepharose column in the presence of MnCl2. The four subfractions eluted at 0.05, 0.12, 0.20, and 0.38 M NaCl and they differed in chemical composition and apoprotein pattern. The relative amounts of apoB and apoE in subfractions increased with increasing concentrations of the NaCl eluant. Modification of the arginyl residues with 1-2 cyclohexadione demonstrated that arginine plays an important role in determining the elution pattern of VLDL. In vitro studies indicated that only fractions eluted at 0.2 and 0.5 M NaCl compete with LDL for cellular receptors. These data suggest that the various subfractions may represent VLDL at different stages of catabolism

Lifestyle interventions and nutraceuticals: Guideline-based approach to cardiovascular disease prevention

Abstract Lowering low-density lipoprotein cholesterol (LDL-C) levels is associated with a well-documented reduction in cardiovascular (CV) disease (CVD) risk. Current guidelines and literature support lifestyle interventions as the primary strategy for reducing CV risk. Association of dietary modifications (such as the Mediterranean diet), physical activity and the cessation of smoking with reduced CV morbidity and mortality has been evidenced. Where lifestyle interventions are not adequate for lowering LDL-C levels and CV risk, pharmacological therapies, most commonly statins, may also be considered. The benefits of lifestyle and pharmacological interventions in the prevention of CVD are widely known, but poor adherence and persistence to these necessitate an approach that aims to improve LDL-C lowering for CVD prevention. Nutraceuticals (targeted functional foods or dietary supplements of plant or microbial origin) are included in EU guidelines as lifestyle interventions and may provide an additional approach to controlling LDL-C levels when a pharmaceutical intervention is not (yet) indicated. However, among different nutraceuticals, the level of clinical evidence supportive of efficacy for lipid lowering needs to be considered. Meta-analyses of randomised clinical trials have demonstrated that some nutraceuticals (e.g. red yeast rice and berberine) and some nutraceutical combinations improve lipid profiles, including lowering of LDL-C, total cholesterol and triglyceride levels. Therefore, nutraceuticals may be considered in specific patient groups where there is appropriate evidence to support the efficacy and safety

Atherogenic markers in predicting cardiovascular risk and targeting residual cardiovascular risk

Abstract Low-density lipoprotein (LDL) cholesterol (LDL-C) is the primary target in cardiovascular (CV) disease prevention and is commonly used in estimating CV risk; however, alternative markers may be needed when LDL-C is not an appropriate marker (e.g. in the presence of low LDL-C levels or elevated triglyceride [TG] levels). Non-high-density lipoprotein cholesterol (non-HDL-C) and apolipoprotein B (apoB) are markers of atherogenic lipoproteins with evidenced associations with CV risk and are, therefore, recommended as secondary targets, appropriate for use in the presence of elevated TG levels. The reported strength of the associations of non-HDL-C and apoB in comparison to LDL-C is conflicting between studies, potentially due to discordance of the markers which can alter their predictive pattern. Although LDL-C levels are commonly managed with statin treatment, a residual risk of CV events still remains, and an abnormal lipid profile can persist. Combination therapy to further reduce LDL-C levels can be beneficial; a statin therapy combined with other LDL-C-lowering therapy further reduced the number of CV events. In addition, targeting other markers, including non-HDL-C, apoB, total cholesterol and TGs may also be beneficial, specifically in patients with low HDL-C and elevated TG levels. More clinical evidence is required before definitive recommendations can be made; however, a statin–fenofibrate combination demonstrated favourable reductions in major CV events in these specific patients

Pharmaceutical strategies for reducing LDL-C and risk of cardiovascular disease

Abstract A key strategy in preventing cardiovascular (CV) disease is the reduction of low-density lipoprotein cholesterol (LDL-C). Statins are a crucial therapy for achieving LDL-C reductions, with the highest tolerated dose often prescribed, especially for patients who are at the greatest risk of CV disease. However, statin intolerance, heterogeneous responses to statins and non-adherence make alternative therapies necessary in some cases. Statins can be combined with a multitude of therapies with synergistic mechanisms of action to effectively manage lipid profiles, while improving safety and tolerability profiles. Addition of a cholesterol absorption inhibitor, bile acid sequestrant or fibrate to statin therapy leads to greater numbers of patients achieving and maintaining LDL-C goals. Furthermore, combination therapies can alter the plasma profiles of other molecules involved in hypercholesterolaemia, including triglycerides and high-density lipoprotein cholesterol. An additional strategy is proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition therapy, for use in patients who are statin intolerant, patients with heterozygous or homozygous familial hypercholesterolaemia, and patients at very high CV risk, as a potential means for achieving large LDL-C reductions and maintaining LDL-C goals. Clinical trials have demonstrated that PCSK9 inhibition therapy is not only effective but can also be combined with statin therapy to ensure greater reductions in LDL-C. Current, ongoing studies are investigating the efficacy of novel therapies, including selective peroxisome proliferator-activated receptor (PPAR) alpha modulators, PCSK9-specific ribonucleic acid (RNA) interference and anti-inflammatory therapies