Lipoprotein (a) and coronary artery calcification: prospective study assessing interactions with other risk factors.

BACKGROUND: Elevated plasma lipoprotein (a) [Lp(a)] and coronary artery calcification (CAC) are established cardiovascular risk factors that correlate with each other. We hypothesized that other cardiovascular risk factors could affect their relationship. METHODS: We tested for interactions of 24 study variables related to dyslipidemia, diabetes, insulin resistance, hypertension, inflammation and coagulation with baseline Lp(a) on change in CAC volume and density over 9.5 years in 5975 Multi-Ethnic Study of Atherosclerosis (MESA) participants, free of apparent cardiovascular disease at baseline. RESULTS: Elevated Lp(a) was associated with larger absolute increase in CAC volume (3.21 and 4.45 mm3/year higher for Lp(a) ≥30 versus <30 mg/dL, and Lp(a) ≥50 versus <50 mg/dL, respectively), but not relative change in CAC volume. No association was found with change in CAC density when assessing continuous ln-transformed Lp(a). The association between elevated Lp(a) (≥30 mg/dL) and absolute change in CAC volume was greater in participants with higher circulating levels of interleukin-2 soluble receptor α, soluble tumor necrosis factor alpha receptor 1 and fibrinogen (15.33, 11.81 and 7.02 mm3/year in quartile 4, compared to -3.44, -0.59 and 1.91 mm3/year in quartile 1, respectively). No significant interaction was found for other study variables. Similar interactions were seen when assessing Lp(a) levels ≥50 mg/dL. CONCLUSIONS: Elevated Lp(a) was associated with an absolute increase in CAC volume, especially in participants with higher levels of selected markers of inflammation and coagulation. These results suggest Lp(a) as a potential biomarker for CAC volume progression.

Lipoprotein (a) and the risk of elevated depressive symptoms: The Multi-Ethnic Study of Atherosclerosis.

Previous studies suggested a potential relationship between plasma lipoprotein (a) [Lp(a)] and elevated depressive symptoms. We aimed to investigate any such relationship in the Multi-Ethnic Study of Atherosclerosis participants who were free of cardiovascular events. Analysis included 4938 participants without elevated depressive symptoms and with Lp(a) levels measured at baseline. Participants were examined at four clinic visits over a 10-year period. Elevated depressive symptoms were assessed by the Center for Epidemiologic Studies Depression Scale (CES-D) and were defined as a CES-D score ≥16 or use of anti-depressants. Lp(a) level was measured with a latex-enhanced turbidimetric immunoassay. After adjusting for demographics, socioeconomic factors and other confounding factors in Cox regression analyses, a higher ln-transformed Lp(a) level was associated with new elevated depressive symptoms since baseline (hazard ratio [95% CI] = 1.09 [1.02-1.16] per SD increment in ln-transformed level, P = 0.01). However, no association was found when elevated Lp(a) levels were assessed using clinical cut-off point (≥30 or 50 mg/dL), nor in sensitivity analyses using alternative definitions of elevated depressive symptoms. No significant interaction with race/ethnicity was found for all the above analyses. Also, no significant association was found between baseline Lp(a) levels and absolute or relative changes in CES-D score between baseline and last follow-up visits. Our study suggests a potential association between Lp(a) level and new elevated depressive symptoms, but such association was not robust in the sensitivity analyses. Future studies are warranted to investigate the role of Lp(a) in depressive symptoms in other cohorts.

High density lipoprotein-associated miRNA is increased following Roux-en-Y gastric bypass surgery for severe obesity.

Roux-en-Y gastric bypass (RYGB) is one of the most commonly performed weight-loss procedures, but how severe obesity and RYGB affect circulating HDL-associated microRNAs (miRNAs) remains unclear. Here, we aim to investigate how HDL-associated miRNAs are regulated in severe obesity and how weight loss after RYGB surgery affects HDL-miRNAs. Plasma HDLs were isolated from patients with severe obesity (n = 53) before and 6 and 12 months after RYGB by immunoprecipitation using goat anti-human apoA-I microbeads. HDLs were also isolated from 18 healthy participants. miRNAs were extracted from isolated HDL and levels of miR-24, miR-126, miR-222, and miR-223 were determined by TaqMan miRNA assays. We found that HDL-associated miR-126, miR-222, and miR-223 levels, but not miR-24 levels, were significantly higher in patients with severe obesity when compared with healthy controls. There were significant increases in HDL-associated miR-24, miR-222, and miR-223 at 12 months after RYGB. Additionally, cholesterol efflux capacity and paraoxonase activity were increased and intercellular adhesion molecule-1 (ICAM-1) levels decreased. The increases in HDL-associated miR-24 and miR-223 were positively correlated with an increase in cholesterol efflux capacity (r = 0.326, P = 0.027 and r = 0.349, P = 0.017, respectively). An inverse correlation was observed between HDL-associated miR-223 and ICAM-1 at baseline. Together, these findings show that HDL-associated miRNAs are differentially regulated in healthy participants versus patients with severe obesity and are altered after RYGB. These findings provide insights into how miRNAs are regulated in obesity before and after weight reduction and may lead to the development of novel treatment strategies for obesity and related metabolic disorders.

microRNA-367-3p regulation of GPRC5A is suppressed in ischemic stroke.

Ischemic stroke is a major cause of mortality and long-term disability with limited treatment options, and a greater understanding of the gene regulatory mechanisms underlying ischemic stroke-associated neuroinflammation is required for new therapies. To study ischemic stroke in vivo, mice were subjected to sustained ischemia by intraluminal filament-induced middle cerebral artery occlusion (MCAo) for 24 h without reperfusion or transient ischemia for 30 min followed by 23.5 h reperfusion, and brain miRNA and mRNA expression changes were quantified by TaqMan OpenArrays and gene (mRNA) expression arrays, respectively. Sustained ischemia resulted in 18 significantly altered miRNAs and 392 altered mRNAs in mouse brains compared to Sham controls; however, the transient ischemic condition was found to impact only 6 miRNAs and 126 mRNAs. miR-367-3p was found to be significantly decreased in brain homogenates with sustained ischemia. G protein-coupled receptor, family C, group 5, member A (Gprc5a), a miR-367-3p target gene, was found to be significantly increased with sustained ischemia. In primary neurons, inhibition of endogenous miR-367-3p resulted in a significant increase in Gprc5a expression. Moreover, miR-367-3p was found to be co-expressed with GPRC5A in human neurons. Results suggest that loss of miR-367-3p suppression of GPRC5A may contribute to neuroinflammation associated with ischemic stroke.

High-density lipoproteins induce miR-223-3p biogenesis and export from myeloid cells: Role of scavenger receptor BI-mediated lipid transfer.

BACKGROUND AND AIMS: We recently showed that miR-223-3p on high-density lipoproteins (HDL) is exported to endothelial cells, where it inhibits inflammation. However, the origin of miR-223-3p on HDL is unknown. We hypothesize that HDL-associated miR-223-3p originates in myeloid cells and is exported to HDL in a scavenger receptor BI (SR-BI)-dependent manner. METHODS: Polymorphonuclear neutrophils (PMNs) and human monocyte derived macrophages (HMDMs) were incubated with native HDL (nHDL) or discoidal reconstituted HDL (rHDL). Total RNA was isolated before and after incubation. Mature and primary miR-223-3p (pri-mir-223-3p) levels were quantified by real-time PCR. RESULTS: Incubation with nHDL and rHDL increased miR-223-3p export from PMNs and HMDMs. In PMNs, nHDL but not rHDL, increased mature and pri-mir-223-3p. Incubation with HDL also increased Dicer mRNA, a critical regulator of miRNA biogenesis. Incubation of HMDMs with nHDL did not increase cellular levels of mature miR-223-3p, but significantly increased pri-mir-223 levels. Incubation with rHDL had no effect on either mature or pri-mir-223-3p levels. Activated PMNs increased miR-223-3p export to HDL and the production of reactive oxygen species and activated protein kinase C. Blocking HDL binding to SR-BI increased miR-223-3p export to HDL in both PMNs and HMDMs, but did not affect mature and primary miR-223-3p levels. Chemical inhibition of cholesterol flux by Block Lipid Transport (BLT)-1 inhibited HDL-induced pri-mir-223 expression in PMNs. CONCLUSIONS: HDL-associated miR-223-3p originates in PMNs and macrophages. HDL stimulates miR-223-3p biogenesis in PMNs in a process that is regulated by SR-BI-mediated lipid flux.

Biology, pathophysiology and current therapies that affect lipoprotein (a) levels.

Lipoprotein (a) [Lp(a)] has recently emerged as a causal, independent and genetic risk factor for cardiovascular disease and calcific aortic valve disease. Given the high incidence of elevated Lp(a) among the general population, significant gaps in the knowledge of Lp(a) biology, pathophysiology and current therapies affecting Lp(a) reduction exist. As plasma Lp(a) levels are genetically determined and insensible to diet, exercise and lifestyle changes, lipid-lowering therapies seem to be the solution to lower elevated Lp(a) levels. This review summarises the current knowledge of Lp(a) structure, metabolism, catabolism, pathophysiology, and Lp(a) response to statins, lipid apheresis, proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, cholesterol esterase transferase protein (CETP) inhibitors and antisense oligonucleotides (ASOs).

Apolipoprotein A-I improves pancreatic ß-cell function independent of the ATP-binding cassette transporters ABCA1 and ABCG1.

Apolipoprotein A-I (apoA-I), the main protein constituent of HDLs, increases insulin synthesis and insulin secretion in pancreatic ß cells. ApoA-I also accepts cholesterol that effluxes from cells expressing ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1). Mice with conditional deletion of ABCA1 and ABCG1 in ß cells [ß-double knockout (DKO) mice] have increased islet cholesterol levels and reduced glucose-stimulated insulin secretion (GSIS). The project asks whether metabolic pathways are dysregulated in ß-DKO mouse islets and whether this can be corrected, and GSIS improved, by treatment with apoA-I. ß-DKO mice were treated with apoA-I or PBS, and islets were isolated for determination of GSIS. Total RNA was extracted from ß-DKO and control mouse islets for microarray analysis. Metabolic pathways were interrogated by functional enrichment analysis. ApoA-I treatment improved GSIS in ß-DKO but not control mouse islets. Plasma lipid and lipoprotein levels and islet cholesterol levels were also unaffected by treatment with apoA-I. Cholesterol metabolism, glucose metabolism, and inflammation pathways were dysregulated in ß-DKO mouse islets. This was not corrected by treatment with apoA-I. In summary, apoA-I treatment improves GSIS by a cholesterol-independent mechanism, but it does not correct metabolic dysregulation in ß-DKO mouse islets.-Hou, L., Tang, S., Wu, B. J., Ong, K.-L., Westerterp, M., Barter, P. J., Cochran, B. J., Tabet, F., Rye, K.-A. Apolipoprotein A-I improves pancreatic ß-cell function independent of the ATP-binding cassette transporters ABCA1 and ABCG1.

Apolipoprotein A-I enhances insulin-dependent and insulin-independent glucose uptake by skeletal muscle.

Therapeutic interventions that increase plasma high density lipoprotein (HDL) and apolipoprotein (apo) A-I levels have been reported to reduce plasma glucose levels and attenuate insulin resistance. The present study asks if this is a direct effect of increased glucose uptake by skeletal muscle. Incubation of primary human skeletal muscle cells (HSKMCs) with apoA-I increased insulin-dependent and insulin-independent glucose uptake in a time- and concentration-dependent manner. The increased glucose uptake was accompanied by enhanced phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), the serine/threonine kinase Akt and Akt substrate of 160 kDa (AS160). Cell surface levels of the glucose transporter type 4, GLUT4, were also increased. The apoA-I-mediated increase in glucose uptake by HSKMCs was dependent on phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt, the ATP binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-B1). Taken together, these results establish that apoA-I increases glucose disposal in skeletal muscle by activating the IR/IRS-1/PI3K/Akt/AS160 signal transduction pathway. The findings suggest that therapeutic agents that increase apoA-I levels may improve glycemic control in people with type 2 diabetes.

Transcoronary gradients of HDL-associated MicroRNAs in unstable coronary artery disease.

AIMS: MicroRNAs (miRNAs) are transported on high-density lipoproteins (HDLs) and HDL-associated miRNAs are involved in intercellular communication. We explored HDL-associated miRNAs concentration gradients across the coronary circulation in stable and unstable coronary artery disease patients and whether changes in the transcoronary gradient were associated with changes in HDL composition and size. METHODS: Acute coronary syndrome (ACS, n=17) patients, those with stable coronary artery disease (stable CAD, n=19) and control subjects without CAD (n=6) were studied. HDLs were isolated from plasma obtained from the coronary sinus (CS), aortic root (arterial blood) and right atrium (venous blood). HDL-associated miRNAs (miR-16, miR-20a, miR-92a, miR-126, miR-222 and miR-223) were quantified by TaqMan miRNA assays. HDL particle sizes were determined by non-denaturing polyacrylamide gradient gel electrophoresis. HDL composition was measured immunoturbidometrically or enzymatically. RESULTS: A concentration gradient across the coronary circulation was observed for all the HDL-associated miRNAs. In ACS patients, there was a significant inverse transcoronary gradient for HDL-associated miR-16, miR-92a and miR-223 (p<0.05) compared to patients with stable CAD. Changes in HDL-miRNA transcoronary gradients were not associated with changes in HDL composition or size. CONCLUSION: HDLs are depleted of miR-16, miR-92a and miR-223 during the transcoronary passage in patients with ACS compared to patients with stable CAD.

Effect of atorvastatin, cholesterol ester transfer protein inhibition, and diabetes mellitus on circulating proprotein subtilisin kexin type 9 and lipoprotein(a) levels in patients at high cardiovascular risk.

BACKGROUND: Proprotein subtilisin kexin type 9 (PCSK9) and lipoprotein (a) [Lp(a)] levels are causative risk factors for coronary heart disease. OBJECTIVES: The objective of the study was to determine the impact of lipid-lowering treatments on circulating PCSK9 and Lp(a). METHODS: We measured PCSK9 and Lp(a) levels in plasma samples from Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events trial patients with coronary heart disease and/or type II diabetes (T2D) mellitus. Patients received atorvastatin, which was titrated (10, 20, 40, or 80 mg/d) to achieve low-density lipoprotein cholesterol levels <100 mg/dL (baseline) and were subsequently randomized either to atorvastatin + torcetrapib, a cholesterol ester transfer protein inhibitor, or to atorvastatin + placebo. RESULTS: At baseline, both plasma PCSK9 and Lp(a) were dose-dependently increased with increasing atorvastatin doses. Compared with patients without T2D, those with T2D had higher PCSK9 (357 ± 123 vs 338 ± 115 ng/mL, P = .0012) and lower Lp(a) levels (28 ± 32 vs 32 ± 33 mg/dL, P = .0005). Plasma PCSK9 levels significantly increased in patients treated with torcetrapib (+13.1 ± 125.3 ng/mL [+3.7%], P = .005), but not in patients treated with placebo (+2.6 ± 127.9 ng/mL [+0.7%], P = .39). Plasma Lp(a) levels significantly decreased in patients treated with torcetrapib (-3.4 ± 10.7 mg/dL [-11.1%], P < .0001), but not in patients treated with placebo (+0.3 ± 9.4 mg/dL [+0.1%], P = .92). CONCLUSION: In patients at high cardiovascular disease risk, PCSK9 and Lp(a) are positively and dose-dependently correlated with atorvastatin dosage, whereas the presence of T2D is associated with higher PCSK9 but lower Lp(a) levels. Cholesterol ester transfer protein inhibition with torcetrapib slightly increases PCSK9 levels and decreases Lp(a) levels.

Impact of Perturbed Pancreatic ß-Cell Cholesterol Homeostasis on Adipose Tissue and Skeletal Muscle Metabolism.

Elevated pancreatic ß-cell cholesterol levels impair insulin secretion and reduce plasma insulin levels. This study establishes that low plasma insulin levels have a detrimental effect on two major insulin target tissues: adipose tissue and skeletal muscle. Mice with increased ß-cell cholesterol levels were generated by conditional deletion of the ATP-binding cassette transporters, ABCA1 and ABCG1, in ß-cells (ß-DKO mice). Insulin secretion was impaired in these mice under basal and high-glucose conditions, and glucose disposal was shifted from skeletal muscle to adipose tissue. The ß-DKO mice also had increased body fat and adipose tissue macrophage content, elevated plasma interleukin-6 and MCP-1 levels, and decreased skeletal muscle mass. They were not, however, insulin resistant. The adipose tissue expansion and reduced skeletal muscle mass, but not the systemic inflammation or increased adipose tissue macrophage content, were reversed when plasma insulin levels were normalized by insulin supplementation. These studies identify a mechanism by which perturbation of ß-cell cholesterol homeostasis and impaired insulin secretion increase adiposity, reduce skeletal muscle mass, and cause systemic inflammation. They further identify ß-cell dysfunction as a potential therapeutic target in people at increased risk of developing type 2 diabetes.

Inhibition of inflammatory signaling pathways in 3T3-L1 adipocytes by apolipoprotein A-I.

Activation of inflammatory signaling pathways links obesity with metabolic disorders. TLR4-mediated activation of MAPKs and NF-κB are 2 such pathways implicated in obesity-induced inflammation. Apolipoprotein A-I (apoA-I) exerts anti-inflammatory effects on adipocytes by effluxing cholesterol from the cells via the ATP binding cassette transporter A1 (ABCA1). It is not known if these effects involve inhibition of inflammatory signaling pathways by apoA-I. This study asks if apoA-I inhibits activation of MAPKs and NF-κB in mouse 3T3-L1 adipocytes and whether this inhibition is ABCA1 dependent. Incubation of differentiated 3T3-L1 adipocytes with apoA-I decreased cell surface expression of TLR4 by 16 ± 2% and synthesis of the TLR4 adaptor protein, myeloid differentiation primary response 88, by 24 ± 4% in an ABCA1-dependent manner. ApoA-I also inhibited downstream activation of MAPKs, such as ERK, p38MAPK, and JNK, as well as expression of proinflammatory adipokines in bacterial LPS-stimulated 3T3-L1 adipocytes in an ABCA1-dependent manner. ApoA-I, by contrast, suppressed nuclear localization of the p65 subunit of NF-κB by 30 ± 3% in LPS-stimulated 3T3-L1 adipocytes in an ABCA1-independent manner. In conclusion, apoA-I inhibits TLR4-mediated inflammatory signaling pathways in adipocytes by preventing MAPK and NF-κB activation.-Sultana, A., Cochran, B. J., Tabet, F., Patel, M., Cuesta Torres, L., Barter, P. J., Rye, K.-A. Inhibition of inflammatory signaling pathways in 3T3-L1 adipocytes by apolipoprotein A-I.

High-Density Lipoprotein-Associated miR-223 Is Altered after Diet-Induced Weight Loss in Overweight and Obese Males.

BACKGROUND AND AIMS: microRNAs (miRNAs) are small, endogenous non-coding RNAs that regulate metabolic processes, including obesity. The levels of circulating miRNAs are affected by metabolic changes in obesity, as well as in diet-induced weight loss. Circulating miRNAs are transported by high-density lipoproteins (HDL) but the regulation of HDL-associated miRNAs after diet-induced weight loss has not been studied. We aim to determine if HDL-associated miR-16, miR-17, miR-126, miR-222 and miR-223 levels are altered by diet-induced weight loss in overweight and obese males. METHODS: HDL were isolated from 47 subjects following 12 weeks weight loss comparing a high protein diet (HP, 30% of energy) with a normal protein diet (NP, 20% of energy). HDL-associated miRNAs (miR-16, miR-17, miR-126, miR-222 and miR-223) at baseline and after 12 weeks of weight loss were quantified by TaqMan miRNA assays. HDL particle sizes were determined by non-denaturing polyacrylamide gradient gel electrophoresis. Serum concentrations of human HDL constituents were measured immunoturbidometrically or enzymatically. RESULTS: miR-16, miR-17, miR-126, miR-222 and miR-223 were present on HDL from overweight and obese subjects at baseline and after 12 weeks of the HP and NP weight loss diets. The HP diet induced a significant decrease in HDL-associated miR-223 levels (p = 0.015), which positively correlated with changes in body weight (r = 0.488, p = 0.032). Changes in miR-223 levels were not associated to changes in HDL composition or size. CONCLUSION: HDL-associated miR-223 levels are significantly decreased after HP diet-induced weight loss in overweight and obese males. This is the first study reporting changes in HDL-associated miRNA levels with diet-induced weight loss.

Apolipoprotein A-I Limits the Negative Effect of Tumor Necrosis Factor on Lymphangiogenesis.

OBJECTIVE: Lymphatic endothelial dysfunction underlies the pathogenesis of many chronic inflammatory disorders. The proinflammatory cytokine tumor necrosis factor (TNF) is known for its role in disrupting the function of the lymphatic vasculature. This study investigates the ability of apolipoprotein (apo) A-I, the principal apolipoprotein of high-density lipoproteins, to preserve the normal function of lymphatic endothelial cells treated with TNF. APPROACH AND RESULTS: TNF decreased the ability of lymphatic endothelial cells to form tube-like structures. Preincubation of lymphatic endothelial cells with apoA-I attenuated the TNF-mediated inhibition of tube formation in a concentration-dependent manner. In addition, apoA-I reversed the TNF-mediated suppression of lymphatic endothelial cell migration and lymphatic outgrowth in thoracic duct rings. ApoA-I also abrogated the negative effect of TNF on lymphatic neovascularization in an ATP-binding cassette transporter A1-dependent manner. At the molecular level, this involved downregulation of TNF receptor-1 and the conservation of prospero-related homeobox gene-1 expression, a master regulator of lymphangiogenesis. ApoA-I also re-established the normal phenotype of the lymphatic network in the diaphragms of human TNF transgenic mice. CONCLUSIONS: ApoA-I restores the neovascularization capacity of the lymphatic system during TNF-mediated inflammation. This study provides a proof-of-concept that high-density lipoprotein-based therapeutic strategies may attenuate chronic inflammation via its action on lymphatic vasculature.

MicroRNA-223 coordinates cholesterol homeostasis.

MicroRNAs (miRNAs) regulate a wide variety of biological processes and contribute to metabolic homeostasis. Here, we demonstrate that microRNA-223 (miR-223), an miRNA previously associated with inflammation, also controls multiple mechanisms associated with cholesterol metabolism. miR-223 promoter activity and mature levels were found to be linked to cellular cholesterol states in hepatoma cells. Moreover, hypercholesterolemia was associated with increased hepatic miR-223 levels in athero-prone mice. miR-223 was found to regulate high-density lipoprotein-cholesterol (HDL-C) uptake, through direct targeting and repression of scavenger receptor BI, and to inhibit cholesterol biosynthesis through the direct repression of sterol enzymes 3-hydroxy-3-methylglutaryl-CoA synthase 1 and methylsterol monooxygenase 1 in humans. Additionally, miR-223 was found to indirectly promote ATP-binding cassette transporter A1 expression (mRNA and protein) through Sp3, thereby enhancing cellular cholesterol efflux. Finally, genetic ablation of miR-223 in mice resulted in increased HDL-C levels and particle size, as well as increased hepatic and plasma total cholesterol levels. In summary, we identified a critical role for miR-223 in systemic cholesterol regulation by coordinated posttranscriptional control of multiple genes in lipoprotein and cholesterol metabolism.

HDL-transferred microRNA-223 regulates ICAM-1 expression in endothelial cells.

High-density lipoproteins (HDL) have many biological functions, including reducing endothelial activation and adhesion molecule expression. We recently reported that HDL transport and deliver functional microRNAs (miRNA). Here we show that HDL suppresses expression of intercellular adhesion molecule 1 (ICAM-1) through the transfer of miR-223 to endothelial cells. After incubation of endothelial cells with HDL, mature miR-223 levels are significantly increased in endothelial cells and decreased on HDL. However, miR-223 is not transcribed in endothelial cells and is not increased in cells treated with HDL from miR-223(-/-) mice. HDL inhibit ICAM-1 protein levels, but not in cells pretreated with miR-223 inhibitors. ICAM-1 is a direct target of HDL-transferred miR-223 and this is the first example of an extracellular miRNA regulating gene expression in cells where it is not transcribed. Collectively, we demonstrate that HDL's anti-inflammatory properties are conferred, in part, through HDL-miR-223 delivery and translational repression of ICAM-1 in endothelial cells.

Elevated plasma PCSK9 level is equally detrimental for patients with nonfamilial hypercholesterolemia and heterozygous familial hypercholesterolemia, irrespective of low-density lipoprotein receptor defects.

OBJECTIVES: Do elevated proprotein convertase subtilisin/kexin type 9 (PCSK9) levels constitute an even greater risk for patients who already have reduced low-density lipoprotein receptor (LDLR) levels, such as those with heterozygous familial hypercholesterolemia (HeFH)? BACKGROUND: As a circulating inhibitor of LDLR, PCSK9 is an attractive target for lowering LDL-cholesterol (LDL-C) levels. METHODS: Circulating PCSK9 levels were measured by enzyme-linked immunosorbent assay in nontreated patients with HeFH carrying a D206E (n = 237), V408M (n = 117), or D154N (n = 38) LDLR missense mutation and in normolipidemic controls (n = 152). Skin fibroblasts and lymphocytes were isolated from a subset of patients and grown in 0.5% serum and mevastatin with increasing amounts of recombinant PCSK9. LDLR abundance at the cell surface was determined by flow cytometry. RESULTS: PCSK9 reduced LDLR expression in a dose-dependent manner in control and FH fibroblasts to similar extents, by up to 77 ± 8% and 82 ± 7%, respectively. Likewise, PCSK9 reduced LDLR abundance by 39 ± 8% in nonfamilial hypercholesterolemia (non-FH) and by 45 ± 10% in HeFH lymphocytes, irrespective of their LDLR mutation status. We found positive correlations of the same magnitude between PCSK9 and LDL-C levels in controls (beta = 0.22; p = 0.0003), D206E (beta = 0.20; p = 0.0002), V408M (beta = 0.24; p = 0.0002), and D154N (beta = 0.25; p = 0.048) patients with HeFH. The strengths of these associations were all similar. CONCLUSIONS: Elevated PCSK9 levels are equally detrimental for patients with HeFH or non-FH: a 100-ng/ml increase in PCSK9 will lead to an increase in LDL-C of 0.20 to 0.25 mmol/l in controls and HeFH alike, irrespective of their LDLR mutation. This explains why patients with non-FH or HeFH respond equally well to monoclonal antibodies targeting PCSK9.

MicroRNAs in the onset and development of cardiovascular disease.

Physiological and pathological roles for small non-encoding miRNAs (microRNAs) in the cardiovascular system have recently emerged and are now widely studied. The discovery of widespread functions of miRNAs has increased the complexity of gene-regulatory processes and networks in both the cardiovascular system and cardiovascular diseases. Indeed, it has recently been shown that miRNAs are implicated in the regulation of many of the steps leading to the development of cardiovascular disease. These findings represent novel aspects in miRNA biology and, therefore, our understanding of the role of these miRNAs during the pathogenesis of cardiovascular disease is critical for the development of novel therapies and diagnostic interventions. The present review will focus on understanding how miRNAs are involved in the onset and development of cardiovascular diseases.