The aim of optimalization the insemination doses production is to provide the highest fertilization ability of spermatozoa during the demanding proces of processing fresh semen and its subsequent cryopreservation. Temperature changes causes spermatozoa damage during the cooling and freezing. Spermatozoa is exposed to cold shock and many others limiting factors, which leads to cell death and therefore to decline of fertilization ability of thawed insemination doses. For increasing spermatozoa resistance, exactly the plasma membrane resistance against cold shock was fraction of egg yolk LDL cholesterol (low density lipoprotein) at various concentrations into the comercially produced diluents added. It is believed that LDL acts possitively to plasma membrane and helps to maintain the fertilization ability of spermatozoa after thawing. Following step in the proces of insemination doses production is slow cooling of diluted semen and equilibration, when the straws are store at cooling box for 30 minutes to 240 hours. This period is necessary to penetrate of certain diluent components into the spermatazoa also maintain the balance between their intracellular and extracellular concentration. Also important is subsequent freezing temperature gradient of insemination doses. The most suitable freezing method is based on computer controlled temperature decline in freezing chamber which allows the precise control of ice crystals formation that could tear and kill the cell. During 2012 to 2016 was repeatedly collected semen from the group of breeding bulls (n = 27, Holstein and Czech Fleckvieh breed) at AI centre. Semen which fulfill the standard entrance conditions in first step was evenly into several parts divided. For dilution the three types of comercially diluents AndroMed, Bioxcell and Triladyl with and without LDL addition were used. Into the diluents AndroMed and Bioxcell the concentration of LDL 4 %, 6 % and 8% into the dilent Triladyl 6 %, 8 % and 10 % was added. Diluted semen was filled into the glass capillares with volume 0,1 ml and temperature +4 °C. Subsequently the sample was placed to cold bath (0°C) for 10 minutes. Then the volume of capillare with physiological solution (37 °C) was mixed and for next 120 minutes was incubate. The effect of cold shock to proportion of live spermatozoa was evaluated by using Eosin and Nigrosine staining technique during heat test of spermatozoa survivability after spermatozoa heating and after 120 minutes of incubation. The more suitable semen diluents which provide the higher spermatozoa resistance against cold shock were AndroMed and Bioxcell. Together the possitive effect of LDL addition into the diluents to lower decrease of proportion of live spermatozoa during heat test was found (P<0.05). The most suitable LDL concentration which had a favorable influence at spermatozoa resistance against cold shock was 6 % in diluent Bioxcell. Values of the proportion of live sperm were higher at the beginning of the heat test (+1.31% to + 3.2%) and after 120 minute incubation (+5.82% to +8.41%) compared to other diluents with and without addition of LDL. In the next step the process of equilibration was optimized, is an important part of insemination doses production. The effect of the length of equilibration for subsequent fertilization ability of spermatozoa was evaluated using spermatozoa motility based of CASA and proportion of live spermatozoa after thawing and during heat survival test lasting 120 minutes (37 ° C). Suitable semen was diluted by comercially used diluent AndroMed based on soya lecithin, filled into the straws (0.25 ml), cooled and equilibrated in cooling box for 30, 120 and 240 minutes and freezed in programmable freezing box applying four types of freezing curves differing in temperature rate decline. There was used standard and by producer recommended 3. phase freezing curve, then 2. phase freezing curve, and 3. phase freezing curve with slower as well as rapid decline of temperature rate in freezing chamber, compared with standard freezing curve. The highest spermatozoa motility was found using 240 minutes of equilibration by +2.72% and +4.58% compared to other lengths of equilibration (P <0.05 to 0.01). The highest proportion of live spermatozoa was found using 120 minutes of equilibration (+6.87 % and +8.68 %). The highest average spermatozoa motility during heat test after thawing was achieved by using 2. phase freezing curve (from +2.97% to +10.37%, P <0.05), also in the proportion of live spermatozoa (from + 4.37% to +8.82%, P <0.01). When evaluating interaction between the length of equilibration and freezing curve (standard 3. phase and 2 . phase freezing curve), the highest average spermatozoa motility and proportion of live spermatozoa using 240 minutes of equilibration by both freezing curves was reached, there was no statistically significant differences. As well as, in all evaluated parts of this study the individual differences between ejaculate of bulls and within semen from one bull (P <0.05) as secondary effect were found. To maintain good fertilization ability of semen during cryopreservation is necessary to increase the spermatozoa resistance against cold shock using addition of correct concentration of LDL into the commercially used diluents AndroMed and Bioxcell. Subsequently the fertilization ability of insemination dose is influenced by cooling, the length of equilibration and freezing. The length of equilibration 120 minutes and more as well as gentle way of freezing according to freezing curve, which ensures a gradual decrease of temperature in freezing chamber provided the higher average spermatozoa motility and proportion of live spermatozoa.

Purpose: To assess whether the participation in food assistance programs (Community Kitchens and Glass of Milk) was associated with lipid profile patterns in the Peruvian population. We conducted a secondary data analysis using data from the National Survey of Nutritional, Biochemical, Socioeconomic, and Cultural Indicators related to Chronic Degenerative Diseases. The sample included individuals aged ≥20 years, selected from five geographic strata in Peru. From each stratum a random sample of clusters was chosen. Different Poisson regression models with robust variance were built to determine the association between food assistance programs and participant lipid profile (total cholesterol (TC), HDL-cholesterol (HDL-c), LDL-cholesterol and triglycerides (TG)). Data from 4028 participants was analyzed, 123 (3.1%) reported being beneficiaries of the Community Kitchens program and 827 (20.5%) were beneficiaries of the Glass of Milk program. An association between being a beneficiary of Community Kitchens and increased LDL-c (Prevalence ratio (PR)= 2.33; 95% CI: 1.18–4.59) was found. Being a beneficiary of the Glass of Milk program increased the probability of having low HDL-c levels (PR= 1.08; 95% CI: 1.02–1.14), but reduced the probability of hypertriglyceridemia (PR= 0.70; 95% CI: 0.56–0.88). Being a beneficiary of the Community Kitchen program was associated with increased LDL-c levels; while, being a beneficiary of the Glass of Milk increased the probability of low HDL-c, but reduced the probability of developing hypertriglyceridemia. / Revisión por pares

Elevated plasma cholesterol is a risk factor for cardiovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) hinders the uptake of low-density lipoprotein cholesterol (LDL-c) by mediating degradation of LDL receptors (LDLRs) in the liver. Gain-of-function (GOF) mutations in PCSK9 cause familial hypercholesterolemia (FH). In normolipidemic human plasma, 30-40% of PCSK9 is bound to LDL particles, and this association with LDL inhibits PCSK9’s ability to mediate LDLR degradation in cultured cells. To further investigate the physiological relevance of this interaction, we analyzed natural GOF mutations in PCSK9 and assessed their effects in vitro on LDL binding, LDLR binding and LDLR degradation. Our results indicate that several GOF mutations severely inhibit LDL binding compared to wild type (WT) PCSK9, and only modestly affect LDLR affinity and LDLR degradation. These findings shed light on the potential physiological relevance of the PCSK9-LDL interaction, which may have an inhibitory effect on PCSK9 activity in vivo.

Purpose: Fasting has been promoted as a method of preventing disease and aging for thousands of years. With heart disease being a leading cause of death in the U.S., researchers have explored the effects of fasting on variables that influence cardiovascular disease (CVD), like LDL cholesterol. Therefore, the purpose of this study was to assess the effects of weekly water-only fasting on LDL cholesterol (LDL-C) in men and women with metabolic risk factors for CVD. Methods: This study was a randomized control trial in adult men and women. Participants were randomized to fasting (treatment) or normal diet (control). The fasting protocol consisted of four weeks of two 24-hour water-only fasts, followed by 22 weeks of once-weekly water-only 24-hour fasts. Measurements such as height, weight, waist circumference and LDL-C were assessed at baseline, 4 weeks, 13 weeks, and 26 weeks. Results: Intermittent fasting (n = 50) and control (n = 53) participants were 49.3 ± 12.0 and 47.0 ± 9.8 years, respectively, predominantly females (66.0% and 67.9%), overweight (103 ± 24 and 100 ± 21 kg), and with mild LDL-C elevation (124 ± 19 and 128 ± 20 mg/dL). Change in weight was −1.70 ± 4.69 (kg) in the fasting group and 0.20 ± 3.45 (kg) in the control group and not different between conditions (p = 0.06). There was no condition-by-period interaction for LDL-C (p = 0.06). Similarly, the change in LDL-C from baseline to follow-up was not different between conditions (t = −0.538, p = 0.59; Cohens D = 0.12) Conclusions: A once-per-week intermittent fasting regimen did not reduce weight or LDL-C. Further research of such fasting regimens is needed to evaluate their potential impact on cardiometabolic health.

AIMS: Macrophages (MPs) and vascular smooth muscle cells (VSMCs) closely interact within the growing atherosclerotic plaque. An in vitro co-culture model was established to study how MPs modulate VSMC behaviour. METHODS AND RESULTS: MPs were exposed to fluorescence-labelled-acetylated LDL (FL-acLDL) prior to co-culture with VSMCs. Fluorescence microscopy visualized first transport of FL-acLDL within 6 h after co-culture implementation. When MPs had been fed with FL-acLDL in complex with fluorescence-labelled cholesterol (FL-Chol), these complexes were also transferred during co-culture and resulted in cholesterol positive lipid droplet formation in VSMCs. When infected with a virus coding for a fusion protein of Rab5a and fluorescent protein reporter (FP) to mark early endosomes, no co-localization between Rab5a-FP and the transported FL-acLDL within VSMCs was detected implying a mechanism independent of phagocytosis. Next, expression of lysosome-associated membrane glycoprotein 1 (LAMP1)-FP, marking all lysosomes in VSMCs, revealed that the FL-acLDL was located in non-acidic lysosomes. MPs infected with virus encoding for LAMP1-FP prior to co-culture demonstrated that intact fluorescence-marked lysosomes were transported into the VSMC, instead. Xenogenic cell composition (rat VSMC, human MP) and subsequent quantitative RT-PCR with rat-specific primers rendered induction of genes typical for MPs and down-regulation of the cholesterol sensitive HMG-CoA reductase. CONCLUSION: Our results demonstrate that acLDL/cholesterol-loaded lysosomes are transported from MPs into VSMCs in vitro. Lysosomal transfer results in a phenotypic alteration of the VSMC towards a foam cell-like cell. This way VSMCs may lose their plaque stabilizing properties and rather contribute to plaque destabilization and rupture.

A series of investigations were undertaken to determine whether physical or emotional stress, catecholamines or naloxone (B-endorphon blocker) would increase serum total cholesterol and LDL and HDL levels. Physical stress given daily over a period of time caused a steady increase in serum total cholesterol and LDL without a significantly altering high density lipoproteins (HDL) or serum triglycerides. Daily injections of epinerphrine in oil caused an increase in both serum total cholesterol and LDL levels while daily injections of norepinephrine did not. Reversal of the treatments caused a reversed response in both groups of rats. Similar increases in both total cholesterol and LDL levels occurred in graduate students during preparation for their comprehensive written or oral thesis/dissertation defense. Injection of eigher dichloroisoproterenol (M.W. = 248) or naloxone (M.W. = 346) in rats prior to stress inhibited the increase in total cholesterol and LDL levels, although naloxone at the dosage given was more effective, possibly due to its larger molecular weight. When naloxone plus epinephrine was injected into non-stressed rats, there was a significant increase in total cholesterol and LDL levels, but the increase was not as great as that of groups injected with epinephrine only. Stressed, adrenalectomized rats exhibited higher cholesterol and LDL levels than the normal reported range for rats of their age and weight, but their levels did not differ from those of stressed, sham-operated rats indicating that the adrenals per se are not needed for stress-induced elevation of blood LDL levels.

Made available in DSpace on 2015-12-06T22:54:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2009 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Objetivos: Lipoproteínas oxidadas e anticorpos (Abs) anti-LDL oxidada (antiLDLox) foram detectados no plasma em resposta à elevação da pressão arterial, sugerindo a participação do sistema imune adaptativo. Postulamos que o tratamento da hipertensão possa modificar a resposta imune diminuindo o estímulo oxidativo. Analisamos os títulos de Abs anti-LDLox após adequado controle da hipertensão arterial. Métodos: Títulos de Abs anti-LDLox foram examinados em pacientes com hipertensão arterial no estágio 1 (n=94), sem manifestação clínica prévia de aterosclerose ou fatores de risco clássicos para a doença coronariana e isentos de medicações anti-hipertensivas. Pacientes foram distribuídos aleatoriamente para terapia com perindopril, hidroclorotiazida ou indapamida por 12 semanas, com adição de perindopril, se necessário para adequado controle pressórico. Títulos de Abs anti-LDLox foram mensurados por ELISA. Resultados: O tratamento anti-hipertensivo reduziu as pressões arteriais casuais e as obtidas pela MAPA (p<0,0005), diminuiu os níveis plasmáticos de substâncias reativas ao ácido tiobarbitúrico (p<0,05) e aumentou os títulos dos Ab anti-LDLox (p<0,005) e a dilatação mediada pelo fluxo (p<0,0005), independentemente do tratamento instituído. Conclusões: Aumento nos títulos de Abs anti-LDLox após terapia anti-hipertensiva ao lado de melhor função endotelial e diminuição do estresse oxidativo sugerem que a despeito de maior estímulo oxidativo estar presente na hipertensão, os Abs anti-LDLox podem ser potenciais biomarcadores protetores para monitorizar o tratamento da hipertensão. / Objectives: Oxidized lipoproteins and autoantibodies anti-oxidized LDL (antioxLDL) have been detected in human plasma in response to blood pressure elevation. Thus, treatment of hypertension, by decreasing the stimuli for oxidation, may have an effect on the immune response. We aimed to assess early changes on anti-LDLox Abs after blood pressure control. Methods: Titers of human antioxLDL Abs were measured in subjects with recently diagnosed hypertension on stage 1 (n=94), in primary prevention of coronary disease, with no other risk factors, and naïve of anti-hypertensive medication at entry. Subjects were randomly assigned to receive perindopril, hydrochlorothiazide or indapamide during 12 weeks, with addition of perindopril if necessary to achieve blood pressure control. Abs anti-oxLDL were measured by ELISA. Results: Twelve-week antihypertensive treatment reduced both office-based and ambulatory blood pressure monitoring measurements (p<0.0005), decreased plasma TBARS (p<0.05), increased titers of anti-oxLDL Abs (p<0.005), and improved endothelial dependent vasodilation (p<0.0005), independently of treatment assignment. Conclusions: Increase in titers of Abs anti-oxLDL with improvement in endothelial function and reduction in oxidative stress suggests that albeit hypertension increases the oxidation stimuli, Abs anti-oxLDL may be protective and potential markers in the follow-up of hypertension treatment. / FAPESP: 2004/00324-1 / BV UNIFESP: Teses e dissertações

The aim of this thesis is to assess the influence of the polyunsaturated fatty acids, especially DHA and EPA in the diet on the animal organism in accordance to the levels of total HDL and LDL cholesterol and triglycerides in plasma. As experimental animals were used rats and they were divided into 3 groups of 10 pieces: control group with the addition of 6 % Carthamus tinctorius (n-6 flares), the group with the addition of 6 % fish oil (n-3: EPA, DHA fish) and the group with 6 % addition of oil from Schizochytrium (n-3: DHA, DHA). After 40 days the animals were putted to death according to relevant regulations. Blood lipids were obtained from blood samples by spectrophotometry. Value of polyunsaturated fatty acids of n-3 and n-6 was set by gas chromatography in the liver, muscle and adipose tissue. In accordance to the increase of content n-3 PUFA in group with fish oil and with oil from Schizochytrium, total and LDL cholesterol was reduced in comparison of samples in control group. In comparison to the control group were TAG reduced only in the group with addition of oil.

Coronary artery disease is the leading cause of death worldwide and results from progression of atherosclerosis, which is triggered in part by elevated plasma concentrations of LDL cholesterol. Genome-wide association studies have identified many loci that are associated with circulating lipid levels and bioinformatics tools have been implemented to prioritize positional candidate genes. This project aims to better understand the genetics underlying the regulation of plasma LDL levels and their effect of atherosclerosis using a zebrafish (Danio rerio) model system. A multiplex line with the genes abcg5, abcg8, myrf, col4a3bpa, col4a3bpb, st3gal3, ywhaqa and ywhaqb targeted by CRISPR/Cas9 technique was established using zebrafish with fluorescently labeled macrophages (Tg[mpeg1:mCherry]) and neutrophils (Tg[mpo:EGFP]). Monodansylpentane cadaverase was used to visualize lipids droplets, together with macrophages and neutrophils, in 384 overfed larvae, allowing the visualization and quantification of vascular atherogenic traits at 10 days post-fertilization. Euthanized larvae were homogenized for the quantification of triglycerides, total cholesterol, LDL, HDL, glucose and protein levels. DNA was extracted and larvae were paired-end sequenced for the CRISPR-targeted sites. Linear regression analysis to compare the wild-type larvae against homozygous mutants and additive models for orthologous genes were performed. The lower accumulation of lipids and the lower co-localization of macrophages and neutrophils in the vasculature suggested that the larvae with mutations in the gene abcg5, abcg8, col4a3bpb, and ywhaqb resulted in larvae more protected against atherosclerotic phenotype. The study suggested that loss of function of the targeted genes was associated with atherogenic traits, helping to understand the pathophysiology of atherosclerosis.

Abstract Estrogen replacement therapy (ERT) has been associated with favorable effects on risk factors for atherosclerosis. In observational studies ERT was also suggested to reduce the risk of cardiovascular disease in postmenopausal women, but the cardioprotective role of estrogen has been challenged after negative results in randomized trials. However, the mechanisms of estrogen action in atherosclerosis development are only partially known. In order to investigate the regulation of plasma low-density lipoprotein (LDL) cholesterol in postmenopausal women and the effects of ERT on cholesterol and glucose metabolism and blood pressure, 79 hysterectomized, non-diabetic postmenopausal women were randomized in a double-blind, double-dummy study to receive either peroral estradiol valerate (2 mg/day) or transdermal 17β-estradiol gel (1 mg estradiol/day) for six months. At baseline the level of LDL cholesterol was related to body mass index, the fractional catabolic rate (FCR) and the production of LDL apolipoprotein B (apo B), but not to cholesterol absorption efficiency. Both peroral and transdermal ERT decreased plasma total and LDL cholesterol, while high-density lipoprotein cholesterol and triglycerides increased only in the peroral group. The LDL-lowering response was related to changes in estrogen levels, which presumably enhance LDL receptor activity shown as an increase in FCR for LDL apo B. In contrast, the determined genetic factors, apo E phenotype, EcoRI and XbaI polymorphisms of the apo B gene and polymorphism of 7α-hydroxylase gene, were not significant in regulation of LDL cholesterol, neither did they modify the response of ERT in these postmenopausal women. Similar outcomes were observed with both peroral and transdermal ERT as regards glucose metabolism and blood pressure. The overall effect of ERT on glucose tolerance was found to be neutral. Blood pressure decreased among non-hypertensive subjects on both estrogens, which could be related, at least in part, to the alterations in vasoactive peptides. The data of the present study suggest an overall favorable effect of both peroral and transdermal estrogen on common cardiovascular risk factors. However, the clinical significance of these findings in the prevention of cardiovascular diseases needs to be proven in long-term, randomized trials.