Development of novel aptamers for low-density lipoprotein particle quantification.
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ABSTRACT:
SUBMITTER: Klapak D
PROVIDER: S-EPMC6181373 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Low-density lipoprotein receptor (LDL-R) is a cell surface receptor protein expressed in a variety of solid cancers, including lung, colon, breast, brain, and liver, and therefore it opens up opportunities to deliver lysosome-sensitive anti-cancer agents, especially synthetic nucleic acid-based therapeutic molecules. In this study, we focused on developing novel nucleic acid molecules specific to LDL-R. For this purpose, we performed in vitro selection procedure via systematic evolution of ligands by exponential enrichment (SELEX) methodologies using mammalian cell-expressed human recombinant LDL-R protein as a target. After 10 rounds of selections, we identified a novel DNA oligonucleotide aptamer, RNV-L7, that can bind specifically to LDL-R protein with high affinity and specificity (KD = 19.6 nM). Furthermore, flow cytometry and fluorescence imaging assays demonstrated efficient binding to LDL-R overexpressed human cancer cells, including Huh-7 liver cancer cells and MDA-MB-231 breast cancer cells, with a binding affinity of ?200 nM. Furthermore, we evaluated the functional potential of the developed LDL-R aptamer RNV-L7 by conjugating with a previously reported miR-21 targeting DNAzyme for inhibiting miR-21 expression. The results showed that the miR-21 DNAzyme-RNV-L7 aptamer chimera efficiently reduced the expression of miR-21 in Huh-7 liver cancer cells. As currently there are no reports on LDL-R aptamer development, we think that RNV-L7 could be beneficial toward the development of targeted cancer therapeutics.
| S-EPMC6920325 | biostudies-literature
Project description:BackgroundHypertriglyceridemia is associated with increased remnant-like particle cholesterol (RLP-C) and triglycerides in low-density lipoprotein (LDL-TG). Recent studies have focused on atherogenicity of RLP-C, with few data on LDL-TG.ObjectivesThe aim of this study was to examine associations of RLP-C and LDL-TG with incident cardiovascular disease (CVD) events and genetic variants in the ARIC (Atherosclerosis Risk In Communities) study.MethodsFasting plasma RLP-C and LDL-TG levels were measured in 9,334 men and women without prevalent CVD. Participants were followed for incident CVD events (coronary heart disease and ischemic stroke) for up to 16 years. Associations between LDL-TG and RLP-C levels and genetic variants were assessed by whole-exome sequencing using single-variant analysis for common variants and gene-based burden tests for rare variants; both an unbiased and a candidate gene approach were explored.ResultsRLP-C and LDL-TG levels were correlated with triglyceride levels (r = 0.85 and r = 0.64, p < 0.0001). In minimally adjusted analyses, RLP-C and LDL-TG were associated with CVD risk, but in models adjusted for traditional risk factors including lipids, only LDL-TG was associated with incident CHD (hazard ratio: 1.28; 95% confidence interval: 1.10 to 1.50) and stroke (hazard ratio: 1.47; 95% confidence interval: 1.13 to 1.92). A common APOE variant, rs7412, had the strongest association with LDL-TG and RLP-C (p < 5 × 10-8).ConclusionsRLP-C and LDL-TG levels were predictive of CVD and associated with APOE variants. LDL-TG may represent a marker of dysfunctional remnant lipoprotein metabolism associated with increased CVD risk. Further research is needed to determine whether LDL-TG plays a causal role in CVD and may be a target for therapy.
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Project description:Low-density lipoprotein receptor-related protein 6 (LRP6), a member of the low-density lipoprotein receptor (LDLR) family, displays a unique structure and ligand-binding function. As a co-receptor of the Wnt/β-catenin signaling pathway, LRP6 is a novel therapeutic target that plays an important role in the regulation of cardiovascular disease, lipid metabolism, tumorigenesis, and some classical signals. By using capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX), with recombinant human LRP-6 as the target, four candidate aptamers with a stem-loop structure were selected from an ssDNA library-AptLRP6-A1, AptLRP6-A2, AptLRP6-A3, and AptLRP6-A4. The equilibrium dissociation constant KD values between these aptamers and the LRP6 protein were in the range of 0.105 to 1.279 μmol/L, as determined by CE-LIF analysis. Their affinities and specificities were further determined by the gold nanoparticle (AuNP) colorimetric method. Among them, AptLRP6-A3 showed the highest affinity with LRP6-overexpressed human breast cancer cells. Therefore, the LRP6 aptamer identified in this study constitutes a promising modality for the rapid diagnosis and treatment of LRP6-related diseases.
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Project description:Background and purposeLow-density lipoprotein (LDL) particle size is considered to be one of the more important cardiovascular risk factors, and small LDL particles are known to have atherogenic potential. The aim of this study was to determine whether LDL particle size is associated with stroke severity and functional outcome in patients with atherothrombotic stroke.MethodsBetween January 2009 and May 2011, 248 patients with first-episode cerebral infarction who were admitted to our hospital within 7 days after symptom onset were prospectively enrolled. LDL particle size was measured using the nondenaturing polyacrylamide gradient gel electrophoresis assay. Stroke severity was assessed by applying the National Institutes of Health Stroke Scale (NIHSS) at admission. Functional outcome was investigated at 3 months after the index stroke using the modified Rankin Scale (mRS), and poor functional outcome was defined as an mRS score of ?3.ResultsThe LDL particle size in the 248 patients was 25.9±0.9 nm (mean±SD). LDL particle size was inversely correlated with the degree of cerebral artery stenosis (p=0.010). Multinomial multivariate logistic analysis revealed that after adjustment for age, sex, and variables with p<0.1 in univariate analysis, LDL particle size was independently and inversely associated with stroke severity (NIHSS score ?5; reference, NIHSS score 0-2; odds ratio=0.38, p=0.028) and poor functional outcome (odds ratio=0.44, p=0.038).ConclusionsThe results of this study demonstrate that small LDL particles are independently correlated with stroke outcomes. LDL particle size is thus a potential biomarker for the prognosis of atherothrombotic stroke.
| S-EPMC4302183 | biostudies-literature
Project description:The aim of this study was to investigate the effects of 6-months consumption of green-banana biomass on the LDL particle functionality in subjects with type 2 diabetes. Subjects (n?=?39, mean age 65 years old) of both sexes with diabetes (HbA1c???6·5%) were randomized to receive nutritional support plus green-banana biomass (40 g) (n?=?21) or diet alone (n?=?18) for 6-months. Non-linear optical responses of LDL solutions from these participants were studied by Z-scan technique. UV-visible spectrophotometer was used to measure the absorbance of the LDL samples. Small Angle X-ray Scattering and Dynamic Light Scattering experiments were used to look for any structural changes in LDL samples and to determine their size distribution. The Lipoprint test was used to determine the LDL sub-fractions in terms of distribution and size. Consumption of green-banana biomass, reduced total- (p?=?0.010), non-HDL-cholesterol (p?=?0.043), glucose (p?=?0.028) and HbA1c (p?=?0.0007), and also improved the protection of the LDL particle against oxidation, by the increase in carotenoids content in the particles (p?=?0.007). This higher protection against modifications may decrease the risk of developing cardiovascular disease. These benefits of the green-banana biomass encourage the use of resistant starches with potential clinical applications in individuals with pre-diabetes and diabetes.
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Project description:High-density lipoprotein (HDL) is a mixture of complex particles mediating reverse cholesterol transport (RCT) and several cytoprotective activities. Despite its relevance for human health, many aspects of HDL-mediated lipid trafficking and cellular signaling remain elusive at the mo-lecular level. During HDL’s journey throughout the body, its functions are mediated through in-teractions with cell surface receptors on different cell types. To characterize and better under-stand the functional interplay between HDL particles and tissue, we analyzed the sur-faceome-residing receptor neighborhoods with which HDL potentially interacts. We applied a combination of chemoproteomic technologies including automated cell surface capturing (au-to-CSC) and HATRIC-based ligand–receptor capturing (HATRIC-LRC) on four different cellular model systems mimicking tissues relevant for RCT. The surfaceome analysis of EA.hy926, HEPG2, foam cells, and human aortic endothelial cells (HAECs) revealed the main currently known HDL scavenger receptor B1 (SCRB1), as well as 155 shared cell surface receptors repre-senting potential HDL-receptor interaction candidates . Since vascular endotheli-al growth factor A (VEGF-A) was recently found as a regulatory factor of transendothelial transport of HDL, we next analyzed the VEGF-modulated surfaceome of HAEC using the au-to-CSC technology. VEGF-A treatment led to the remodeling of the surfaceome of HAEC cells, including the previously reported higher surfaceome abundance of SCRB1. In total, 165 addition-al receptors were found on HAEC upon VEGF-A treatment representing SCRB1 co-regulated re-ceptors potentially involved in HDL function. Using the HATRIC-LRC technology on human endothelial cells, we specifically aimed for the identification of other bona fide (co-)receptors of HDL beyond SCRB1. HATRIC-LRC enabled, next to SCRB1, the identification of the receptor ty-rosine-protein kinase Mer (MERTK). Through RNA interference, we revealed its contribution to endothelial HDL binding and uptake. Furthermore, subsequent proximity ligation assays (PLAs) demonstrated the spatial vicinity of MERTK and SCRB1 on the endothelial cell surface. The data shown provide direct evidence for a complex and dynamic HDL receptome and that receptor nanoscale organization may influence binding and uptake of HDL.
2020-11-02 | MSV000086397 | MassIVE
Project description:High-density lipoprotein (HDL) is a mixture of complex particles mediating reverse cholesterol transport (RCT) and several cytoprotective activities. Despite its relevance for human health, many aspects of HDL-mediated lipid trafficking and cellular signaling remain elusive at the molecular level. During HDL's journey throughout the body, its functions are mediated through interactions with cell surface receptors on different cell types. To characterize and better understand the functional interplay between HDL particles and tissue, we analyzed the surfaceome-residing receptor neighborhoods with which HDL potentially interacts. We applied a combination of chemoproteomic technologies including automated cell surface capturing (auto-CSC) and HATRIC-based ligand-receptor capturing (HATRIC-LRC) on four different cellular model systems mimicking tissues relevant for RCT. The surfaceome analysis of EA.hy926, HEPG2, foam cells, and human aortic endothelial cells (HAECs) revealed the main currently known HDL receptor scavenger receptor B1 (SCRB1), as well as 155 shared cell surface receptors representing potential HDL interaction candidates. Since vascular endothelial growth factor A (VEGF-A) was recently found as a regulatory factor of transendothelial transport of HDL, we next analyzed the VEGF-modulated surfaceome of HAEC using the auto-CSC technology. VEGF-A treatment led to the remodeling of the surfaceome of HAEC cells, including the previously reported higher surfaceome abundance of SCRB1. In total, 165 additional receptors were found on HAEC upon VEGF-A treatment representing SCRB1 co-regulated receptors potentially involved in HDL function. Using the HATRIC-LRC technology on human endothelial cells, we specifically aimed for the identification of other bona fide (co-)receptors of HDL beyond SCRB1. HATRIC-LRC enabled, next to SCRB1, the identification of the receptor tyrosine-protein kinase Mer (MERTK). Through RNA interference, we revealed its contribution to endothelial HDL binding and uptake. Furthermore, subsequent proximity ligation assays (PLAs) demonstrated the spatial vicinity of MERTK and SCRB1 on the endothelial cell surface. The data shown provide direct evidence for a complex and dynamic HDL receptome and that receptor nanoscale organization may influence binding and uptake of HDL.
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Project description:The LDL receptor (LDLR) and scavenger receptor class B type I (SR-BI) play physiological roles in LDL and HDL metabolism in vivo. In this study, we explored HDL metabolism in LDLR-deficient mice in comparison with WT littermates. Murine HDL was radiolabeled in the protein ((125)I) and in the cholesteryl ester (CE) moiety ([(3)H]). The metabolism of (125)I-/[(3)H]HDL was investigated in plasma and in tissues of mice and in murine hepatocytes. In WT mice, liver and adrenals selectively take up HDL-associated CE ([(3)H]). In contrast, in LDLR(-/-) mice, selective HDL CE uptake is significantly reduced in liver and adrenals. In hepatocytes isolated from LDLR(-/-) mice, selective HDL CE uptake is substantially diminished compared with WT liver cells. Hepatic and adrenal protein expression of lipoprotein receptors SR-BI, cluster of differentiation 36 (CD36), and LDL receptor-related protein 1 (LRP1) was analyzed by immunoblots. The respective protein levels were identical both in hepatic and adrenal membranes prepared from WT or from LDLR(-/-) mice. In summary, an LDLR deficiency substantially decreases selective HDL CE uptake by liver and adrenals. This decrease is independent from regulation of receptor proteins like SR-BI, CD36, and LRP1. Thus, LDLR expression has a substantial impact on both HDL and LDL metabolism in mice.
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Project description:This submission contains a result of a study of transcriptiomic profiles of blood of COVID-19 patients.
2022-01-10 | GSE185863 | GEO
Project description:Dyslipidemia treatment is of major importance in reducing the risk of atherosclerotic cardiovascular disease (ASCVD), which is still the most common cause of death worldwide. During the last decade, a novel lipid-lowering drug category has emerged, i.e., proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors. Apart from the two available anti-PCSK9 monoclonal antibodies (alirocumab and evolocumab), other nucleic acid-based therapies that inhibit or "silence" the expression of PCSK9 are being developed. Among them, inclisiran is the first-in-class small interfering RNA (siRNA) against PCSK9 that has been approved by both the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of hypercholesterolemia. Importantly, inclisiran therapy may improve low-density lipoprotein cholesterol (LDL-C) target achievement by offering a prolonged and significant LDL-C-lowering effect with the administration of only two doses per year. The present narrative review discusses the ORION/VICTORION clinical trial program that has been designed to investigate the impact of inclisiran on atherogenic lipoproteins and major adverse cardiac events in different patient populations. The results of the completed clinical trials are presented, focusing on the effects of inclisiran on LDL-C and lipoprotein (a) (Lp(a)) levels as well as on other lipid parameters such as apolipoprotein B and non-high-density lipoprotein cholesterol (non-HDL-C). Ongoing clinical trials with inclisiran are also discussed.
| S-EPMC10143414 | biostudies-literature