GENETIC LCAT DEFICIENCY AND ATHEROSCLEROSIS: STUDIES ON ENDOTHELIAL CELLS AND MONOCYTES

Introduzione La lecitin:cholesterol aciltransferasi (LCAT) \ue8 l\u2019unico enzima nell\u2019uomo in grado di esterificare il colesterolo nel plasma. Il deficit genetico di LCAT influenza il metabolismo delle lipoproteine ad alta densit\ue0 (HDL), portando a concentrazioni plasmatiche ridotte di queste lipoproteine. Si ritiene che LCAT sia un\u2019importante driving force per il trasporto inverso del colesterolo (RCT) nei macrofagi e che pertanto giochi un ruolo importante nell\u2019ateroprotezione. Nonostante i bassi livelli plasmatici di HDL-C, il grado di aterosclerosi preclinica non \ue8 risulta aumentato nel deficit di LCAT che addirittura sembra essere protettivo. Abbiamo quindi ipotizzato che questa discrepanza potesse essere spiegata da un aumento della funzionalit\ue0 delle particelle HDL che sono pi\uf9 efficienti nel proteggere contro la disfunzione endoteliale. Metodi Le HDL sono state isolate da soggetti portatori di deficit di LCAT e testate in vitro per la loro capacit\ue0 di stimolare la produzione di NO e di inibire l\u2019espressione di molecole di adesione in cellule endoteliali. La misura della vasodilatazione flusso-mediata (FMD) \ue8 stata utilizzata come indice in vivo di disfunzione endoteliale. HDL ricostituite, contenenti solo apoA-I (LpA-I) o apoA-I e apoA-II (LpA-I:A-II) sono state utilizzate per dimostrare come quali modifiche strutturali siano responsabili dell\u2019aumentata funzionalit\ue0. Analisi ex vivo del fenotipo e della funzionalit\ue0 di monociti isolati da portatori di deficit di LCAT sono stati condotti tramite citometria a flusso, stimolazione e saggi di transmigrazione endoteliale. Portatori di altri deficit genetici di HDL sono stati utilizzati come confronto. Risultati Il deficit di LCAT genetico \ue8 caratterizzato da una deplezione selettiva di LpA-I: A-II e da alte concentrazioni di pre\u3b2-HDL immature. Le HDL isolate dai portatori si sono dimostrate pi\uf9 efficaci nel promuovere l'attivazione di eNOS in vitro mediante fosforilazione e, di conseguenza, la produzione di NO nelle cellule endoteliali, rispetto ai controlli, con un effetto gene-dose. Inoltre, le HDL dei portatori hanno una maggiore capacit\ue0 di inibire l'espressione di VCAM-1. L'effetto \ue8 probabilmente dipendente dalla deplezione selettiva in lipoproteine LpA-I:A-II. Inoltre, i monociti dei portatori presentavano un fenotipo meno infiammatorio rispetto ai controlli, come evidenziato dalla ridotta espressione dell\u2019integrina CD11c, dalla ridotta capacit\ue0 di trasmigrazione e dalla minore produzione di citochine dopo stimolazione. Al contrario, nessuna variazione \ue8 stata individuata nell\u2019immunofenotipo di monociti isolati in soggetti con altri deficit genetici di HDL. A conferma in vivo di quanto individuato precedentemente non \ue8 stata trovata alcuna differenza nella FMD tra portatori di deficit genetico di LCAT e controlli. Conclusioni Nonostante le ridotte concentrazioni plasmatiche di HDL-C, il deficit di LCAT non \ue8 associato a una maggiore disfunzione endoteliale dovuta all'aumentata efficacia delle particelle HDL nella stimolazione della produzione di NO endoteliale e nell'inibizione dell'espressione delle molecole di adesione, nonch\ue9 al ridotto potenziale pro-infiammatorio dei monociti dei portatori.Background Lecithin:cholesterol acyltransferase (LCAT) is the unique enzyme in human able to esterify free cholesterol in plasma. Genetic lack of LCAT affects high-density lipoprotein (HDL) metabolism, leading to very low plasma concentrations of these lipoproteins. LCAT was believed to be an important driving force behind macrophage reverse cholesterol transport (RCT) and therefore playing a role in atheroprotection. Despite the low plasma HDL-C, LCAT deficiency does not remarkably increase preclinical atherosclerosis or could even be protective. We hypothesized that this discrepancy can be explained by an increased functionality of HDL particles, that are more effective in protecting against endothelium dysfunction. Methods HDLs were isolated from LCAT-deficient carriers and tested in vitro for their capacity to promote NO production and to inhibit vascular cell adhesion molecules in endothelial cells. Measurement of flow-mediated vasodilation (FMD) was used as marker of endothelial dysfunction in vivo. Reconstituted HDL containing only apoA-I (LpA-I) or apoA-I and apoA-II (LpA-I:A-II) were used to prove the structural responsible for increased HDL functionality. Ex vivo analysis of monocytes in carriers of LCAT deficiency, to assessed phenotype and function, was performed with fluorescence-activated cell sorter analysis, inflammatory stimulation and transendothelial migration assays. Comparison with other genetic hypoalphalipoproteinemia was conducted for the monocyte study. Results Genetic LCAT deficiency is characterized by selective depletion of LpA-I:A-II and high presence of immature pre\u3b2-HDL. HDLs isolated from carriers were more effective in promoting in vitro eNOS activation by phosphorylation and consequently NO production in endothelial cells, compared to controls, with a gene-dose dependent effect. Moreover, HDLs from carriers have an increased capability in inhibiting VCAM-1 expression. The effect was likely dependent on the selective depletion in LpA-I:A-II particles. In addition carriers\u2019 monocyte displayed a less inflammatory phenotype compared to controls, as evidenced by a reduced expression of integrin CD11c, a decreased capacity to transmigrate and a lower production of cytokines upon stimulation. Conversely, no changes in monocyte immunophenotype was found in other genetic HDL deficiency. As in vivo confirmation, no difference in FMD was found between carriers of LCAT deficiency and controls. Conclusions Despite the low plasma HDL-C concentration, LCAT deficiency is not associated with increased endothelial dysfunction due to the enhance efficacy of HDL particles in stimulating endothelial NO production and in inhibiting adhesion molecules expression, as well to the reduce pro-inflammatory potential of carriers\u2019 monocytes

Treatment with fibrates is associated with higher LAL activity in dyslipidemic patients

Lysosomal acid lipase (LAL) is responsible for the hydrolysis of cholesteryl esters (CE) and triglycerides (TG) within the lysosomes; generated cholesterol and free fatty acids (FFA) are released in the cytosol where they can regulate their own synthesis and metabolism. When LAL is not active, as in case of genetic mutations, CE and TG accumulate in the lysosomal compartment, while the lack of release of cholesterol and FFA in the cytosol leads to an upregulation of their synthesis. Thus, LAL plays a central role in the intracellular homeostasis of lipids. Since there are no indications about the effect of different lipid-lowering agents on LAL activity, aim of the study was to address the relationship between LAL activity and the type of lipid-lowering therapy in a cohort of dyslipidemic patients. LAL activity was measured on dried blood spot from 120 patients with hypercholesterolemia or mixed dyslipidemia and was negatively correlated to LDL-cholesterol levels. Among enrolled patients, ninety-one were taking one or more lipid-lowering drugs, as statins, fibrates, ezetimibe and omega-3 polyunsaturated fatty acids. When patients were stratified according to the type of lipid-lowering treatment, i.e. untreated, taking statins or taking fibrates, LAL activity was significantly higher in those with fibrates, even after adjustment for sex, age, BMI, lipid parameters, liver function, metabolic syndrome, diabetes and statin use. In a subset of patients tested after 3 months of treatment with micronized fenofibrate, LAL activity raised by 21%; the increase was negatively correlated with baseline LAL activity. Thus, the use of fibrates is independently associated with higher LAL activity in dyslipidemic patients, suggesting that the positive effects of PPAR-\u3b1 activation on cellular and systemic lipid homeostasis can also include an improved LAL activity

Effect of soy on metabolic syndrome and cardiovascular risk factors : a randomized controlled trial

Background: Cardiovascular diseases are currently the commonest cause of death worldwide. Different strategies for their primary prevention have been planned, taking into account the main known risk factors, which include an atherogenic lipid profile and visceral fat excess. Methods: The study was designed as a randomized, parallel, single-center study with a nutritional intervention duration of 12 weeks. Whole soy foods corresponding to 30 g/day soy protein were given in substitution of animal foods containing the same protein amount. Results: Soy nutritional intervention resulted in a reduction in the number of MetS features in 13/26 subjects. Moreover, in the soy group we observed a significant improvement of median percentage changes for body weight ( 121.5 %) and BMI ( 121.5 %), as well as for atherogenic lipid markers, namely TC ( 124.85 %), LDL-C ( 125.25 %), non-HDL-C ( 127.14 %) and apoB ( 1214.8 %). Since the majority of the studied variables were strongly correlated, three factors were identified which explained the majority (52 %) of the total variance in the whole data set. Among them, factor 1, which loaded lipid and adipose variables, explained the 22 % of total variance, showing a statistically significant difference between treatment arms (p = 0.002). Conclusions: The inclusion of whole soy foods (corresponding to 30 g/day protein) in a lipid-lowering diet significantly improved a relevant set of biomarkers associated with cardiovascular risk

A proteomic approach to identify novel disease biomarkers in LCAT deficiency

Genetic LCAT deficiency is a rare recessive autosomal disease due to loss-of-function mutations in the gene coding for the enzyme lecithin:cholesterol acyltransferase (LCAT). Homozygous carriers are characterized by corneal opacity, haemolytic anaemia and renal disease, which represent the first cause of morbidity and mortality in these subjects. Diagnostic and prognostic markers capable of early detecting declining kidney function in these subjects are not available, and the specific serum or urine proteomic signature of LCAT deficient carriers has never been assessed. Taking advantage of a proteomic approach, we performed 2-DE analysis of carriers' plasma and identified proteins present at different concentration in samples from homozygous carriers. Our data confirm the well-known alterations in the concentration of circulating apolipoproteins, with a statistically significant decrease of both apoA-I and apoA-II and a statistically significant increase of apoC-III. Furthermore, we observed increased level of alpha-1-antitrypsin, zinc-alpha-2-glycoprotein and retinol-binding protein 4, and reduced level of clusterin and haptoglobin. Interestingly, only beta but not alpha subunit of haptoglobin is significant reduced in homozygous subjects. Despite the limited sample size, our findings set the basis for assessing the identified protein in a larger population and for correlating their levels with clinical markers of renal function and anaemia. Significance: This investigation defines the effects of LCAT deficiency on the level of the major plasma proteins in homozygous and heterozygous carriers. Increase for some proteins, with different function, together with a drop for haptoglobin, and specifically for haptoglobin beta chains, are reported for the first time as part of a coherent signature. We are glad to have the opportunity to report our findings on this subject, which is one of the main interests for our research group, when Journal of Proteomics celebrates its 10th anniversary. With its various sections devoted to different areas of research, this journal is a privileged forum for publishing proteomic investigations without restrictions either in sample type or in technical approach. It is as well a privileged forum for reviewing literature data on various topics related to proteomics investigation, as colleagues in our research group have done over the years; by the way, a good share of the reviewed papers were as well reports published in Journal of Proteomics itself. The journal also offers opportunities for focused surveys through thematic issues devoted to a variety of subjects, timely selected for their current relevance in research; it was an honour for colleagues in our group to recently act as editors of one of those. Out of this diverse experience, we express our appreciation for the endeavour of Journal of Proteomics in its first 10 years of life \u2013 and wish identical and possibly greater success for the time to come

Silent polymorphisms in the RYR1 gene do not\ud modify the phenotype of the p.4898 I>T\ud pathogenic mutation in central core disease:\ud a case report

Background: Central core disease is a congenital myopathy, characterized by presence of central core-like areas in\ud muscle fibers. Patients have mild or moderate weakness, hypotonia and motor developmental delay. The disease is\ud caused by mutations in the human ryanodine receptor gene (RYR1), which encodes a calcium-release channel.\ud Since the RYR1 gene is huge, containing 106 exons, mutation screening has been limited to three ‘hot spots’, with\ud particular attention to the C-terminal region. Recent next- generation sequencing methods are now identifying\ud multiple numbers of variants in patients, in which interpretation and phenotype prevision is difficult.\ud Case presentation: In a Brazilian Caucasian family, clinical, histopathological and molecular analysis identified a\ud new case of central core disease in a 48-year female. Sanger sequencing of the C-terminal region of the RYR1\ud gene identified two different missense mutations: c.14256 A > C polymorphism in exon 98 and c.14693 T > C in\ud exon 102, which have already been described as pathogenic. Trans-position of the 2 mutations was confirmed\ud because patient’s daughter, mother and sister carried only the exon 98’s mutation, a synonymous variant that was\ud subsequently found in the frequency of 013–0,05 of alleles. Further next generation sequencing study of the whole\ud RYR1 gene in the patient revealed the presence of additional 5 common silent polymorphisms in homozygosis and\ud 8 polymorphisms in heterozygosis.\ud Conclusions: Considering that patient’s relatives showed no pathologic phenotype, and the phenotype presented\ud by the patient is within the range observed in other central core disease patients with the same mutation, it was\ud concluded that the c.14256 A > C polymorphism alone is not responsible for disease, and the associated additional\ud silent polymorphisms are not acting as modifiers of the primary pathogenic mutation in the affected patient. The\ud case described above illustrates the present reality where new methods for wide genome screening are becoming\ud more accessible and able to identify a great variety of mutations and polymorphisms of unknown function in\ud patients and their families.Fundação de Amparo a Pesquisa do Estado de São Paulo - Centro de Pesquisa, Inovação e Difusão (FAPESP-CEPID)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-INCT)Associação Brasileira de Distrofia Muscular (ABDIM)CAPES-COFECU

A PROBLEMÁTICA DO MONITORAMENTO DAS INFECÇÕES DE SÍTIO CIRÚRGICO E A NECESSIDADE DE PADRONIZAÇÃO DE CRITÉRIOS PARA SEU DIAGNÓSTICO E NOTIFICAÇÃO.

Las infecciones hospitalarias (IH) ocurren como un problema de salud pública mundial, siendo la Infección del Sitio Quirúrgico la tercera topografia más común, de 14% a 16% de todas las IH. Cuando un paciente muere por una causa asociada a la IH, 77% están relacionadas con la ISC; 93% de ellas con infecciones serias que invaden órganos o espacios accedidos durante un procedimiento quirúrgico. Directamente, las repercusiones de las ISC aparecen en los costos hospitalarios, pues aumentan la permanencia hospitalaria entre 7 a 10 días, amén de las readmisiones; pero, de manera indirecta, igualmente o más importante, están los costos indirectos que causan impactos emocionales desastrosos en los pacientes y en la familia. Los criterios más utilizados para diagnóstico de ISC son los Centers for Diseases Control (CDC); otros criterios desarrollados por especialistas ingleses como el National Prevalence Survey Study (NPS) también son utilizados. El objetivo de este estudio fue buscar una respuesta, en la literatura, para la práctica del controlador de infección hospitalaria en lo que respecta a la existencia de un “patrón-oro” para el diagnóstico de ISC, con el fin de soportar los resultados obtenidos y las consecuentes acciones. Una revisión sistemática ha mostrado que comparándose las definiciones del CDC y NPS de 93 heridas operatorias, 24% han quedado sin diagnóstico al utilizarse criterios del CDC y 19% cuando se utilizaron criterios del NPS. La conclusión de este estudio es que no hay un “patrón-oro” para el diagnóstico de ISC, pues el juicio es subjetivo, y sujeto a variaciones de acuerdo con el observador. Es necesario que cada servicio de salud junto al grupo Comissão de Controle de Infeccção Hospitalar -CCIH (Comisión de Control de Infección Hospitalaria) asuma y reglamente, por medio de la mejor evidencia científica, cuáles son los mejores criterios para diagnóstico y notificación de ISC, cuál es el mejor método de trabajo para vigilancia después del alta, teniendo en cuenta la factibilidad y las necesidades locales.As infecções hospitalares (IH) surgem como um problema de saúde pública mundial sendo a Infecção de Sítio Cirúrgico (ISC) a terceira topografia mais comum, de 14% a 16% de todas as IH. Quando um paciente morre por causa associada à IH, 77% estão relacionadas a ISC; 93% deles com infecções sérias que invadem órgãos ou espaços acessados durante o procedimento cirúrgico. Diretamente, as repercussões das ISC aparecem nos custos hospitalares pois aumentam a permanência hospitalar entre 7 a 10 dias, além das readmissões; mas de forma indireta e tão ou mais importante, estão os custos indiretos que provocam impactos emocionais desastrosos nos pacientes e familiares. Os critérios mais utilizados para diagnóstico de ISC são os do Centers for Diseases Control (CDC), outros critérios desenvolvidos por especialistas ingleses como o National Prevalence Survey Study (NPS) também são utilizados. O objetivo deste estudo foi buscar resposta, na literatura, para a pratica do controlador de infecção hospitalar no que tange a existência de um padrão ouro para o diagnostico de ISC, a fim de respaldar os resultados obtidos e as conseqüentes ações. Uma revisão sistemática mostrou que se comparando as definições do CDC e NPS de 93 feridas operatórias, 24% ficaram sem diagnóstico quando usados critérios do CDC e 19% quando usados critérios do NPS. A conclusão deste estudo é que não há padrão ouro, no diagnóstico de ISC, pois o julgamento é subjetivo e sujeito a variações de acordo com o observador. É preciso que cada serviço de saúde, junto ao grupo da Comissão de Controle de Infecção Hospitalar (CCIH) assuma e normatize, por meio da melhor evidência científica, quais os melhores critérios para diagnóstico e notificação de ISC, qual o melhor método de trabalho para vigilância no pós-alta; levando em conta a factibilidade e as necessidades locais

Individuals with familial hypercholesterolemia and cardiovascular events have higher circulating Lp(a) levels

BACKGROUND: Cardiovascular disease (CVD) is a major cause of mortality and morbidity. Increased low-density lipoprotein cholesterol (LDL-C) level is its major risk factor. Familial hypercholesterolemia (FH) is a genetic disorder characterized by elevated LDL-C since birth and subsequent premature CVD. There is a heterogeneity in the CVD onset in patients with FH. This is potentially due to the presence of other independent risk factors. Lipoprotein(a) [Lp(a)] is an LDL-like particle and represents a strong risk factor for CVD. OBJECTIVE: Our objective was to understand the contribution of Lp(a) in the susceptibility to CVD in individuals with genetic diagnosis of FH. METHODS: We measured Lp(a) levels in 2 independent and well-characterized genetic-FH cohorts: the FH-Gothenburg cohort (n = 190) and the FH-CEGP Milan cohort (n = 160). The genetic diagnosis was performed by targeted next-generation sequencing (FH-Gothenburg and part of the FH-CEGP Milan cohort), or by Sanger sequencing. RESULTS: We show that among individuals with genetic diagnosis of FH, those with previous CVD had higher Lp(a) levels. In addition, analyzing the response to the lipid-lowering therapies, we have also shown that statins had the same LDL-C-lowering effect irrespective of the type of FH-causative mutation. However, when we examined the lipid-lowering effect of proprotein convertase subtilisin/kexin type 9 inhibition by antibodies, we observed a trend in a better reduction of the LDL-C level in carriers of nonsense mutations. CONCLUSION: In conclusion, our results suggest that Lp(a) contributes to CVD onset in individuals with genetic diagnosis of FH. Our finding supports the importance to identify an efficacious therapy to lower Lp(a) in patients with FH to prevent CVD onset or recurrence

Influence of body variables in the development of metabolic syndrome : A long term follow-up study

Objectives. The body variable associated with the diagnosis of Metabolic Syndrome (MetS) is an elevated waist circumference (WC), although a number of other variables have been suggested. Among these, an elevated waist-to-height ratio (WHtR), ie a value higher than 0.5, that may identify abnormality, independently from height. An elevated WHtR provided the best correlation with MetS in a prior study in a large Italian population. In order to assess the validity of this conclusion, a long-term follow-up study re-examined this population, also in order to detect possible associations with cardiovascular (CV) risk. Methods and Results. 1,071 subjects with a complete follow-up of over 6 years were evaluated with a comparative assessment of the three anthropometric variables, namely WHtR, WC and body mass index (BMI). WHtR 65 0.5 had the highest sensitivity for the identification of MetS, both in males and females (94.1% and 86.7% respectively). WHtR was of reduced specificity, occurring, yet less frequently (17.7% in males and 30% in females), in patients without MetS. By contrast, enlarged WC occurred with a lower frequency in male patients who developed MetS (30.2%) whereas in females, frequency was higher than in males (69.3%). Finally, a BMI 65 25 kg/m2 had intermediate sensitivity and specificity regardless of gender. WC showed the highest odds ratio (2.62, 95%CI: 1.18-5.78) for the prediction of CV occurrence. Conclusion. The present study confirms WHtR as an excellent screening tool in identifying MetS carriers, but, different from reports in other countries, it shows a lower specificity in our population

Analysis of HDL-microRNA panel in heterozygous familial hypercholesterolemia subjects with LDL receptor null or defective mutation

In the last years increasing attention has been given to the connection between genotype/phenotype and cardiovascular events in subjects with familial hypercholesterolemia (FH). MicroRNAs (miRs) bound to high-density lipoprotein (HDL) may contribute to better discriminate the cardiovascular risk of FH subjects. Our aim was to evaluate the HDL-miR panel in heterozygous FH (HeFH) patients with an LDLR null or defective mutation and its association with pulse wave velocity (PWV). We evaluated lipid panel, HDL-miR panel and PWV in 32 LDLR null mutation (LDLR-null group) and 35 LDLR defective variant (LDLR-defective group) HeFH patients. HDL-miR-486 and HDL-miR-92a levels were more expressed in the LDLR-null group than the LDLR-defective group. When we further stratified the study population into three groups according to both the LDLR genotype and history of ASCVD (LDLR-null/not-ASCVD, LDLR-defective/not-ASCVD and LDLR/ASCVD groups), both the LDLR/ASCVD and the LDLR-null/not-ASCVD groups had a higher expression of HDL-miR-486 and HDL-miR-92a than the LDLR-defective/not-ASCVD group. Finally, HDL-miR-486 and HDL-miR-92a were independently associated with PWV. In conclusion, the LDLR-null group exhibited HDL-miR-486 and HDL-miR-92a levels more expressed than the LDLR-defective group. Further studies are needed to evaluate these HDL-miRs as predictive biomarkers of cardiovascular events in FH

Effects of PCSK9 inhibitors on HDL cholesterol efflux and serum cholesterol loading capacity in familial hypercholesterolemia subjects: a multi-lipid-center real-world evaluation

Proprotein convertase subtilisin/kexin type 9 (PCSK9), beyond regulating LDL cholesterol (LDL-c) plasma levels, exerts several pleiotropic effects by modulating lipid metabolism in extrahepatic cells such as macrophages. Macrophage cholesterol homeostasis depends on serum lipoprotein functions, including the HDL capacity to promote cell cholesterol efflux (CEC) and the serum capacity to promote cell cholesterol loading (CLC). The aim of this observational study was to investigate the effect of PCSK9 inhibitors (PCSK9-i) treatment on HDL-CEC and serum CLC in patients with familial hypercholesterolemia (FH). 31 genetically confirmed FH patients were recruited. Blood was collected and serum isolated at baseline and after 6 months of PCSK9-i treatment. HDL-CEC was evaluated through the main pathways with a radioisotopic cell-based assay. Serum CLC was assessed fluorimetrically in human THP-1 monocyte-derived macrophages. After treatment with PCSK9-i, total cholesterol and LDL-c significantly decreased (−41.6%, p < 0.0001 and −56.7%, p < 0.0001, respectively). Total HDL-CEC was not different between patients before and after treatment. Conversely, despite no changes in HDL-c levels between the groups, ABCG1 HDL-CEC significantly increased after treatment (+22.2%, p < 0.0001) as well as HDL-CEC by aqueous diffusion (+7.8%, p = 0.0008). Only a trend towards reduction of ABCA1 HDL-CEC was observed after treatment. PCSK9-i significantly decreased serum CLC (−6.6%, p = 0.0272). This effect was only partly related to the reduction of LDL-c levels. In conclusion, PCSK9-i treatment significantly increased HDL-CEC through ABCG1 and aqueous diffusion pathways and reduced the serum CLC in FH patients. The favorable effect of PCSK9-i on functional lipid profile could contribute to the cardiovascular benefit of these drugs in FH patients