Lipophorin receptors regulate mushroom body development and complex behaviors in Drosophila.

BACKGROUND: Drosophila melanogaster lipophorin receptors (LpRs), LpR1 and LpR2, are members of the LDLR family known to mediate lipid uptake in a range of organisms from Drosophila to humans. The vertebrate orthologs of LpRs, ApoER2 and VLDL-R, function as receptors of a glycoprotein involved in development of the central nervous system, Reelin, which is not present in flies. ApoER2 and VLDL-R are associated with the development and function of the hippocampus and cerebral cortex, important association areas in the mammalian brain, as well as with neurodevelopmental and neurodegenerative disorders linked to those regions. It is currently unknown whether LpRs play similar roles in the Drosophila brain. RESULTS: We report that LpR-deficient flies exhibit impaired olfactory memory and sleep patterns, which seem to reflect anatomical defects found in a critical brain association area, the mushroom bodies (MB). Moreover, cultured MB neurons respond to mammalian Reelin by increasing the complexity of their neurite arborization. This effect depends on LpRs and Dab, the Drosophila ortholog of the Reelin signaling adaptor protein Dab1. In vitro, two of the long isoforms of LpRs allow the internalization of Reelin, suggesting that Drosophila LpRs interact with human Reelin to induce downstream cellular events. CONCLUSIONS: These findings demonstrate that LpRs contribute to MB development and function, supporting the existence of a LpR-dependent signaling in Drosophila, and advance our understanding of the molecular factors functioning in neural systems to generate complex behaviors in this model. Our results further emphasize the importance of Drosophila as a model to investigate the alterations in specific genes contributing to neural disorders.

Relationship between Discordance of Low-Density Lipoprotein and Non-High-Density Lipoprotein Cholesterol and Risk Stratification in Acute Myocardial Infarction

Abstract Background: Sizeable proportion of patients have discordant low-density lipoprotein cholesterol (LDL-C) and non-high density lipoprotein cholesterol (NHDL-C). It has been shown that discordance of LDL-C and NHDL-C either underestimates or overestimates coronary risk. Objectıve: We assessed whether this discordance has an impact on GRACE and TIMI risk scores in patients with acute myocardial infarction (AMI). Methods: We retrospectively evaluated the data of 198 consecutive patients with AMI. Fasting serum lipid profiles were recorded, GRACE and TIMI scores were calculated. Patients were divided into 3 groups according to LDL-C and NHDL-C percentiles: Discordant group: LDL-C<NHDL-C (n=38), concordant group: LDL-C=NHDL-C (n=112) and discordant group LDL-C>NHDL-C (n=48). GRACE and TIMI scores, mortality and cardiovascular events (heart failure, non-fatal myocardial infarction and angina) at sixth month were compared between these three groups. Differences between these groups were analyzed with One-way ANOVA or Kruskal-Wallis rank test, and with chi-square for percentages. Also, post hoc LSD or Conover-Iman's non-parametric multiple comparison test were used. A p value <0.05 was accepted as statistically significant. Results: TIMI risk score didn't differ between discordant or concordant groups. Mean GRACE (death) and GRACE (death and MI) scores were higher in group with LDL-C<NHDL-C than with LDL-C=NHDL-C and LDL-C>NHDL-C (p=0.029 and 0.008, respectively). Cardiovascular events and mortality at sixth month were not different among groups (p=0.473 and p=0.176, respectively). Conclusion: GRACE score was higher in discordant group with LDL-C<NHDL-C, but there is no difference regarding TIMI scores between discordant and concordant groups in AMI patients.

Oxidized low-density lipoprotein stimulates dendritic cells maturation via LOX-1-mediated MAPK/NF-κB pathway

Dendritic cells (DCs) play a crucial role as central orchestrators of immune system response in atherosclerosis initiation and progression. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is involved in the immune maturation of DCs, but the underlying mechanisms remain unclear. We isolated mouse bone marrow progenitors and stimulated them with granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4 to induce immature DCs. We then treated DCs with oxidized low-density lipoprotein (oxLDL) to induce maturation. LOX-1 siRNA was used to investigate the modulation of LOX-1 on the development of DCs and the underlying signal pathways. CD11c-positive DCs were successfully derived from mouse bone marrow progenitors. OxLDL promoted the expressions of DCs maturation markers and pro-inflammatory cytokines. OxLDL also upregulated LOX-1 expression and activated MAPK/NF-κB pathways. LOX-1 siRNA could attenuate the expression of MAPK/NF-κB pathways and inflammatory cytokines. In conclusion, oxLDL induced the maturation of DCs via LOX-1-mediated MAPK/NF-κB pathway, which contributed to the initiation and progression of atherosclerosis.

Cholesterol metabolism in mice models of genetic hypercholesterolemia.

Monogenic familial hypercholesterolemia is characterized by impaired cellular uptake of apolipoprotein B containing lipoproteins. However, its consequences on whole-body cholesterol metabolism are unclear. We investigated cholesterol metabolism in wild-type mice (control) and in knockout (KO) mice for the low-density lipoprotein receptor (LDLR-KO) and for apolipoprotein E (apoE-KO) containing the genetic basis of the C57BL/6J mice, under a cholesterol-free diet. Cholesterol and "non-cholesterol" sterols (cholestanol, desmosterol, and lathosterol) were measured in plasma, tissues, as well as in feces as cholesterol and its bacterial modified products (neutral sterols) using gas chromatography/mass spectrometry, and bile acids were measured by an enzymatic method. Compared to controls, LDLR-KO mice have elevated plasma and whole-body cholesterol concentrations, but total fecal sterols are not modified, characterizing unaltered body cholesterol synthesis together with impaired body cholesterol excretion. ApoE-KO mice presented the highest concentrations of plasma cholesterol, whole-body cholesterol, cholestanol, total fecal sterols, and cholestanol, compatible with high cholesterol synthesis rate; the latter seems attributed to elevated body desmosterol (Bloch cholesterol synthesis pathway). Nonetheless, whole-body lathosterol (Kandutsch-Russel cholesterol synthesis pathway) decreased in both KO models, likely explaining the diminished fecal bile acids. We have demonstrated for the first time quantitative changes of cholesterol metabolism in experimental mouse models that explain differences between LDLR-KO and apoE-KO mice. These findings contribute to elucidate the metabolism of cholesterol in human hypercholesterolemia of genetic origin.

Characterization of LDLR rs5925 and PCSK9 rs505151 genetic variants frequencies in healthy subjects from northern Chile: Influence on plasma lipid levels.

BACKGROUND: Identification and characterization of genetic variants and their effects on human health may allow to establish relationships between genetic background and susceptibility to developing cardiovascular diseases. LDLR and PCSK9 polymorphisms have been associated with higher lipid levels and risk of cardiovascular diseases. Thus, the main aim of this study was to evaluate genotype distribution and relative allelic frequency of LDLR rs5925 (1959C > T) and PCSK9 rs505151 (23968 A > G) genetic variants and their effects on lipid levels of healthy subjects from northern Chile. METHODS: A total of 178 healthy individuals were recruited for this study. The genotyping of rs5925 (LDLR) and rs505151 (PCSK9) polymorphisms was performed by PCR-RFLP and qPCR, respectively. In addition, glucose and lipid levels were determined and associated with the genetic data. RESULTS: Genotype distribution for LDLR rs5925 polymorphism was as follows: CC = 19%; CT = 53%; and TT = 28% (HWE: χ2  = 0.80; P = .37), and for PCSK9 rs505151 genetic variant was as follows: AA = 93%; AG = 7%; and GG = 0% (HWE: χ2  = 0.22; P = .64). The frequency of T (rs5925) and G (rs505151) mutated alleles was 0.55 and 0.03, respectively. Data showed that individuals carrying LDLR mutated allele (T) presented lower values of total cholesterol, triglycerides, and LDL-cholesterol when compared to CC homozygous genotype (P < .05). Subgroup analysis revealed that women carrying the PCSK9 mutated allele (G) exhibited higher values of total cholesterol, triglycerides, HDL-C, and LDL-C when compared to male group carrying the same genotype (P < .05). CONCLUSIONS: The effect of LDLR rs5925 and PCSK9 rs505151 gene polymorphisms on lipid levels is associated with gender among healthy subjects from northern Chile.

Phytosterol containing diet increases plasma and whole body concentration of phytosterols in apoE-KO but not in LDLR-KO mice.

Phytosterol metabolism is unknown in the hypercholesterolemia of genetic origin. We investigated the metabolism of phytosterols in a cholesterol-free, phytosterol-containing standard diet in hypercholesterolemic mice knockouts for low density lipoprotein receptor (LDLR) and apolipoprotein E (apoE) mice compared to wild-type mice (controls). Phytosterols were measured in mice tissues by GCMS. ApoE-KO mice absorbed less phytosterols than LDLR-KO and the latter absorbed less phytosterols than control mice, because the intestinal campesterol content was low in both KO mice, and sitosterol was low in the intestine in apoE-KO mice as compared to LDLR-KO mice. Although the diet contained nine times more sitosterol than campesterol, the concentration of sitosterol was lower than that of campesterol in plasma in LDLR-KO, and in the liver in controls and in LDLR-KO, but only in apoE-KO. On the other hand, in the intestine sitosterol was higher than campesterol in controls, and in LDLR-KO but with a tendency only in apoE-KO. Because of the high dietary supply of sitosterol, sitosterol was better taken up by the intestine than campesterol, but the amount of sitosterol was lower than that of campesterol in the liver, while in the whole body the amounts of these phytosterols do not differ from each other. Therefore, via intestinal lymph less sitosterol than campesterol was transferred to the body. However, as compared to controls, in apoE-KO mice, but not in LDLR-KO mice, the increase in campesterol and sitosterol in plasma and in the whole body indicating that apoE-KO mice have a marked defect in the elimination of both phytosterols from the body.

Mutational analysis of the LDLR gene in a cohort of Colombian families with familial hypercholesterolemia.

BACKGROUND AND AIMS: Familial hypercholesterolemia (FH) is characterized by elevated serum cholesterol levels due to high low-density lipoprotein (LDL) cholesterol levels. FH is an autosomal dominant genetic disorder and one of the most common dominant hereditary diseases in the world. However, the frequency of mutations in Colombia is unknown. The purpose of this preliminary study was to identify mutations in the LDL receptor (LDLR) gene in a Colombian population with FH. METHODS: The study included 24 families with clinical diagnosis of sure/probable FH. The 18 exons of the LDLR were sequenced by Sanger method. RESULTS: Among 18 variants identified, 3 were known pathogenic mutations and were identified in nine individuals in five unrelated families. Five affected individuals were heterozygous for one mutation each. They were the p.W4X in two, the p.D139G in two and the p.G396D in one. Two affected individuals were homozygous for p.G396D. The variant c.1187-1G > T, which has uncertain significance in FH pathogenesis, was present in all the individuals with the p.D139G mutation. CONCLUSIONS: In total, 18 variants were identified, of which 14 correspond to known nonpathogenic variants. Three pathogenic variants were identified in the LDLR. No pathological mutations were identified in the LDLR in 79% of the study population.

mRNA levels of low-density lipoprotein receptors are overexpressed in the foci of deep bowel endometriosis.

STUDY QUESTION: Is mRNA expression of LDL receptors altered in deep bowel endometriotic foci? SUMMARY ANSWER: mRNA expression of LDL receptors is up-regulated in deep bowel endometriotic foci of patients with endometriosis. WHAT IS KNOWN ALREADY: Several studies have demonstrated the overexpression of low-density lipoprotein receptors in various tumour cell lines and endometriosis has similar aspects to cancer, mainly concerning the pathogenesis of both diseases. This is the first study we know of to investigate lipoprotein receptors expression in deep endometriosis with bowel involvement. STUDY DESIGN, SIZE, DURATION: During 2014-2015, an exploratory case-control study was conducted with 39 patients, including 20 women with a histological diagnosis of deep endometriosis compromising the bowel and 19 women without endometriosis who underwent laparoscopic tubal ligation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Peripheral blood samples were collected on the day of surgery for lipid profile analysis, and samples of endometrial tissue and of bowel endometriotic lesions were also collected. The tissue samples were sent for histopathological analysis and for LDL-R and LRP-1 gene expression screening using quantitative real-time PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Patients with deep endometriosis had lower LDL-cholesterol than patients without the disease (119 ± 23 versus 156 ± 35; P = 0.001). Gene expression analysis of LDL receptors revealed that LDL-R was more highly expressed in endometriotic lesions when compared to the endometrium of the same patient but not more than in the endometrium of women without endometriosis (0.027 ± 0.022 versus 0.012 ± 0.009 versus 0.019 ± 0.01, respectively; P < 0.001). LRP-1 was more highly expressed in endometriotic lesions, both when compared with the endometrium of the same patient and when compared with the endometrium of patients without the disease (0.307 ± 0.207 versus 0.089 ± 0.076 and versus 0.126 ± 0.072, respectively; P < 0.001). The study also showed that LDL-R gene expression in the endometrium of women with endometriosis was higher during the secretory phase of the menstrual cycle (P = 0.001). LRP-1 gene expression was increased during the secretory phase in the endometrium of women without the disease (P = 0.008). LIMITATIONS, REASONS FOR CAUTION: In the endometriotic lesions, the presence of fibrosis is substantial, restricting access to the stromal and glandular components of the lesion. Despite that, we found that LDL receptor mRNA was overexpressed. Future studies may perform laser microdissection to isolate the area of interest in the target tissue, excluding fibrosis contamination. WIDER IMPLICATIONS OF THE FINDINGS: This study supports the feasibility of LDL-R targeted therapy in the treatment of deep endometriosis. STUDY FUNDING/COMPETING INTERESTS: This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP #2011/17245-0). The authors have no conflicts of interest to declare.

Is there an association between hypercholesterolemia and depression? Behavioral evidence from the LDLr(-/-) mouse experimental model.

Although epidemiological studies have reported an association between hypercholesterolemia and mood disorders, there is a lack of data regarding depressive-like behavior in animal models of hypercholesterolemia. To address these questions, we assessed depressive-like behavior and hippocampal and cortical monoaminergic metabolism in three-month-old, low-density lipoprotein receptor knockout (LDLr(-/-)) and C57BL/6 wild-type mice. The LDLr(-/-) mice exhibited depressive-like behavior in the sucrose preference test, splash test, and tail suspension test. Increased monoamine oxidase (MAO) A and B activity was evidenced in the hippocampus of LDLr(-/-) mice. Furthermore, to address whether or not cholesterol modulates MAO activity, we exposed SH-SY5Y human neuroblastoma cells to human isolated low-density lipoprotein (LDL). Notably, LDL increased the activity of MAO-A and stimulated the reactive species generation in vitro. These findings indicate that depressive-like behavior in hypercholesterolemic mice is accompanied by alterations in the monoaminergic metabolism, providing new evidence about the association between hypercholesterolemia and depression.

Familial hypercholesterolemia in Brazil: cascade screening program, clinical and genetic aspects.

BACKGROUND: There is little knowledge about familial hypercholesterolemia in Brazil. This study presents the first results of genetic cascade screening performed in the city of Sao Paulo. MATERIAL AND METHODS: Two-hundred and forty-eight suspected index cases were initially included. DNA was extracted from peripheral blood and the complete coding sequence of low-density lipoprotein receptor, exon 7 of proprotein convertase subtilisin/kexin type 9 gene and part of exon 26 of apolipoprotein B genes were sequenced. Multiplex Ligation-dependent Probe Amplification was performed on cases where a causal mutation was not identified through sequencing. After the identification of a causal mutation screening in first-degree relatives was pursued. RESULTS: From 248 index cases, a mutation was found in 125 individuals (50.4%). 394 relatives were included in the cascade screening program and a mutation was identified in 59.4%. Seventy different causal mutations in the low-density lipoprotein receptor gene (97.2%) and 2 in the apolipoprotein B gene (2.8%) were found. No mutations were encountered in the proprotein convertase subtilisin/kexin type 9 gene. Mutations in exons 14 and 4 were the most prevalent and, 10 cases of true homozygotes (8 index cases and 2 relatives) and 1 compound heterozygote were identified. The most frequent mutation found was of Lebanese origin, the p.(Cys681*) mutation in exon 14 (8.5%). CONCLUSION: Genetic familial hypercholesterolemia cascade screening is feasible in Brazil and leads to identification of a mutation in approximately half of the index cases with higher rates of success in their relatives.

Polimorfismos genéticos associados ao metabolismo lipídico envolvidos na fisiopatologia do acidente vascular encefálico isquêmico / Genetic polymorphisms associated with lipid metabolism involved in the pathophysiology of ischemic stroke

O acidente vascular encefálico (AVE) é uma desordem complexa, multifatorial e poligênica decorrente da interação entre componentes genéticos do indivíduo e fatores ambientais. Estudos prévios têm estabelecido a hipertensão arterial, tabagismo, diabetes mellitus, dislipidemia, elevado índice de massa corpórea, distúrbios da coagulação e aumento da idade como fatores de risco preditores de AVE. A dislipidemia tem sido associada à fisiopatologia do AVE isquêmico e polimorfismos genéticos que ocorrem na via metabólica dos lipídeos têm sido propostos como fatores genéticos associados ao AVE isquêmico. O componente genético na causa da dislipidemia tem sido intensamente investigado nos últimos anos. Entre os vários polimorfismos genéticos,os que ocorrem nos genes Apo E, Apo B, LDLR, Apo A-I, Apo C-III, lipase hepática, proteína transferidorade ésteres de colesterol (CETP), lipase lipoprotéica (LPL) e proteína convertase subtilisina/kexina tipo 9(PCSK9) têm sido objeto de estudos na população em geral. Os dados sobre o perfil lipídico e o estudo dos polimorfismos dos genes que codificam proteínas estruturais e enzimas relacionadas com o metabolismo dos lipídeos podem revelar a prevalência das dislipidemias em uma população, possibilitando uma intervenção direcionada para o controle e prevenção das doenças ateroscleróticas como o AVE isquêmico.

The stroke is a complex, multifactorial, and polygenic disorder that results from the interaction betweenthe individual genetic components and environmental factors. Previous studies have established hypertension, smoking, diabetes mellitus, dyslipidemia, elevated body mass index, disturbances of coagulation and increasing age as predictors of stroke risk factors. Dyslipidemia has been associated with pathophysiology of ischemic stroke and genetic polymorphisms that occur in the metabolic pathway, such as lipids metabolism, has been one of the hereditary factors related to ischemic stroke. The genetic component in the cause of dyslipidemia has been intensively investigated in recent years. Among the several genetic polymorphisms, those that occur in the Apo E, Apo B, low-density lipoprotein receptor (LDLR), Apo A-I, Apo C-III, hepatic lipase, cholesteryl ester transfer protein (CETP), lipoprotein lipase(LPL) and proprotein convertase subtilisin/kexin type 9 (PCSK9). have been the object of many studiesin the population worldwide. Data on lipid profile and study of polymorphisms of genes encoding structural proteins and enzymes related to lipid metabolism may reveal the prevalence of dyslipidemia ina population, enabling a targeted intervention for the control and prevention of atherosclerotic diseasessuch as ischemic stroke.

Impact of genetic variants of IL-6, IL6R, LRP5, ESR1 and SP7 genes on bone mineral density in postmenopausal Mexican-Mestizo women with obesity.

BACKGROUND: Since obesity and osteoporosis present a high genetic predisposition and polymorphisms of IL-6, IL6R, LRP5, ESR1 and SP7 may influence the risk of both diseases, the aim of this study was to analyze the possible association of polymorphisms in these genes, as well as their haplotypes, with BMD variations in postmenopausal Mexican-Mestizo women with grade 2 or grade 3 obesity. METHODS: One hundred eighty unrelated postmenopausal women with grade 2 or grade 3 obesity were included. BMD was measured in total hip and lumbar spine by dual-energy X-ray absorptiometry. DNA was obtained from blood leukocytes. Rs1800795 of IL-6, rs2228145 of IL6R, rs3736228 of LRP5, rs9340799 (XbaI) and rs2234693 (PvuII), of ESR1, rs10876432 and rs2016266, of SP7 (and their haplotypes), were studied by real-time PCR allelic discrimination. Deviations from Hardy-Weinberg equilibrium were tested. Pairwise linkage disequilibrium between single nucleotide polymorphisms was calculated by direct correlation r(2), and haplotype analysis was conducted. RESULTS: Using WHO criteria, 54.5% had grade 2 obesity, and 45.5% had grade 3 obesity. Regarding DXA results, 11.1% women had osteoporosis, 41.7% had osteopenia, and 47.2% had normal BMD. Genotype and haplotype analysis showed no significant differences with BMD variations at the lumbar spine, total hip or femoral neck. CONCLUSIONS: We did not find a significant association between the polymorphisms analyzed or their haplotypes and BMD variations in postmenopausal women with obesity. The higher BMD observed in women with obesity could be the result of an adaptive response to the higher loading of the skeleton.

Respuesta del receptor de lipoproteína de muy baja densidad a estresores inflamatorios / Very low density lipoprotein receptor response to inflammatory stressors

Se estudió la regulación del receptor de lipoproteína de muy baja densidad (VLDLR) en dos tipos de células que intervienen en la respuesta inflamatoria: hepatocitos (HepG2) y células linfocitarias de bazo de ratón (CMB) por agentes implicados en la inflamación: VLDL o Gemfibrozil, lipopolisacárido bacteriano (LPS) para HepG2 o Concanavalina A (ConA) para CMB. Se determinaron por citometría de flujo las subpoblaciones de CMB y de HepG2 en distintas fases del ciclo celular (G1 y G2/M) con ioduro de propidio y las VLDLR+ con VLDL fluorescente. Entre 10 a 60 por ciento de células expresaron VLDLR dependiendo de las condiciones experimentales. El enfrentamiento con LPS o ConA produjo un aumento de células VLDLR+. El tratamiento con Gemfibrozil disminuyó el número de hepatocitos en reposo VLDLR+ pero incrementó significativamente (más de dos veces) los hepatocitos VLDLR+ en fase G2/M. En los cultivos de CMB Gemfibrozil aumentó por igual el porcentaje de células VLDLR+ quiescentes y en G2/M. El comportamiento de estos tipos celulares con VLDL fue distinto: CMB no mostró cambios en las subpoblaciones VLDLR+, los hepatocitos mostraron disminución de VLDLR+ tanto en fase G1 como en fase G2/M. Se concluyó que el estudio de la regulación de VLDLR en distintas células facilitará el diseño de fármacos específicos para el tratamiento de enfermedades de etiología inflamatoria.

The regulation of the VLDL receptor (VLDLR) by molecules involved in the inflammation process (lipopolysaccharide, LPS; Concanavalin A, ConA, VLDL or Gemfibrozil) in two cellular types implied in the inflammatory response, hepatocytes (HepG2) and lymphocitary cells from mice spleen (CMB), was studied. Different subpopulations of CMB and HepG2 in different cell cycle phases (G1 and G2/M) were analyzed using flow citometry techniques with propidium iodide, and the VLDLR+ with fluorescent VLDL. In cultures of both cell types, it was observed that 10 to 60% of the cells expressed the VLDLR (VLDLR+ cells) indistinctly of the culture conditions. VLDLR+ cells belonged equally to cells in the quiescent and in the synthesis or mitosis phase of the cell cycle. Challenging them with LPS or ConA an increase in the percentage of VLDLR+ cells was produced. Gemfibrozil treatment decreased the number of resting hepatocytes VLDLR+ but increased significantly (more than twice) the number of hepatocytes VLDLR+ in phase G2/M. In hepatocytes there was almost the same proportion of VLDLR+ cells that were in the G1 or in the G2/M phase of the cell cycle. It is concluded that the study of VLDLR regulation will facilitate the design of new drugs to treat inflammatory diseases.