Apolipoprotein B Research Articles

High-selenium exposure is associated with modulation of serum lipid metabolism.

At present, there is no consensus on the relationship between selenium (Se) exposure and human serum lipid metabolism. The etiological role of high-Se exposure in lipid markers, dyslipidemia, and nonalcoholic fatty liver (NAFLD) remains unclear. We used serum untargeted metabolomics analysis to evaluate whether high-Se exposure is cross-sectionally associated with lipid metabolism in adults from high-Se exposure area (n = 112) and control area (n = 101) in Hubei Province, China. An untargeted liquid chromatography/mass spectrometry (LC/MS)-based metabolomic analysis identified 144 differential pathways and yielded 204 differentially abundant metabolites, including 32 lipid metabolites associated with lipids profiles. To further explore the correlation between Se exposure and serum lipid metabolism, we measured serum levels of lipid profiles among all the people, including serum cholesterol (CHOL), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and apolipoprotein B (APOB). The average serum Se level of the high-Se exposure group was 537.18 μg/L, significantly higher than 72.98 μg/L in the control group (p < 0.0001). The measurement levels of serum TG, LDL-C, HDL-C, and APOB in the high-Se exposure group were 1.03 (0.76, 1.34) mmol/L, 2.25 ± 0.48 mmol/L, 1.12 ± 0.24 mmol/L, and 0.77 ± 0.15 g/L, respectively, while the control group were 1.13 (0.84, 1.80) mmol/L, 2.56 ± 0.61 mmol/L, 1.02 ± 0.22 mmol/L, and 0.83 ± 0.16 g/L, respectively (all p values <0.05). Correlation analysis showed a significant negative correlation between serum Se and CHOL (r = -0.201, p < 0.01), serum Se is also associated with metabolomics markers, the negative correlation includes glyceric acid and ect., the positive correlation includes phosphorylcholine and ect. Our study suggests that high-Se exposure is negatively associated with serum lipid profiles and decreases the risk of high-TC and HDL-C dyslipidemia.

Prevalence, genetic variants, and clinical implications of hypocholesterolemia in children.

In contrast to extensively studied hypercholesterolemia, knowledge of hypocholesterolemia is limited. This study aims to assess the prevalence, clinical characteristics, and genetics of children and adolescents with hypocholesterolemia. This national prospective cross-sectional cohort study was part of Slovenia's universal opt-out cholesterol screening program. The first part assessed hypocholesterolemia prevalence among 3538 children aged 5 years, randomly selected at the mandatory check-up. The second part included analysis of demographic and clinical data and genetic testing of 71 individuals with suspected hypocholesterolemia (total cholesterol [TC]<3.0mmol/L [116.0mg/dL]) referred to the Lipid Clinic of University Children's Hospital Ljubljana. The prevalence of hypocholesterolemia among 3538 children was 2.66% (95% CI: 2.13-3.19%). Among the 71 genetically tested individuals with suspected hypocholesterolemia, those with pathogenic variants had lower TC (2.58±0.44mmol/L vs. 2.85±0.42mmol/L [99.77±17.02mg/dL vs. 110.20±16.24mg/dL]; p=0.037) and low-density lipoprotein cholesterol (1.00±0.40mmol/L vs. 1.33±0.40mmol/L [38.67±15.47mg/dL vs. 51.43±15.47mg/dL]; p=0.014) compared to those without such variants. Genetic testing identified pathogenic alterations in 15 subjects, including 4 novel loss-of-function variants in the APOB gene. All but one subject were asymptomatic. This study provides new clinical and genetic insights into hypocholesterolemia. Asymptomatic patients with hypocholesterolemia may not require further evaluation, but additional research is needed to understand hypocholesterolemia better.

XbaI polymorphism in the APOB gene and its association with increased cholesterol in children and adolescents: Ouro Preto study

Atherosclerotic vascular changes can begin during childhood, providing risk for cardiovascular disease (CVD) in adulthood. Identifiable risk factors such as dyslipidemia accelerate this process for some children. The apolipoprotein B (APOB) gene could help explain the inter-individual variability in lipid levels among young individuals and identify groups that require greater attention to prevent CVD. A cross-sectional study was conducted with school-aged children and adolescents in Ouro Preto, Minas Gerais. The study evaluated cardiovascular risk factors’ variables and XbaI polymorphism in the APOB gene for associations with increased total cholesterol (TC). The prevalence of increased TC was notably high, reaching 68.9% in the study population. Carriers of the variant T allele were 1.45 times more likely to develop increased TC in a dominant model (1.09–1.94, p = 0.011). After adjustments, excess weight and a family history of dyslipidemia interacted significantly with XbaI polymorphism in increased TC, resulting in Odds Ratio of 1.74 (1.11–2.71, p = 0.015) and 2.04 (1.14–3.67, p = 0.016), respectively. The results suggest that XbaI polymorphism in the APOB gene may affect the lipid profile of Brazilian children and adolescents and could contribute to the CVD in adulthood.

Familial hypercholesterolemia in Chinese children and adolescents: a multicenter study

BackgroundFamilial hypercholesterolemia (FH) is an inherited disorder mainly marked by increased low-density lipoprotein cholesterol (LDL-C) concentrations and a heightened risk of early-onset arteriosclerotic cardiovascular disease (ASCVD). This study seeks to characterize the genetic spectrum and genotype‒phenotype correlations of FH in Chinese pediatric individuals.MethodsData were gathered from individuals diagnosed with FH either clinically or genetically at multiple hospitals across mainland China from January 2016 to June 2024.ResultsIn total, 140 children and adolescents (mean age of 6.00 years) with clinically and genetically diagnosed FH were enrolled in the study, with 87 distinct variants identified in the LDLR, APOB and PCSK9 genes. Among the variants, 11 variants were newly identified worldwide, with 9 classified as “pathogenic” or “likely pathogenic”, and 2 classified as “variants of uncertain significance”. Additionally, the 5 most common variants in the study were c.1448G > A (p.W483*), c.1879G > A (p.A627T), c.1216C > A (p.R406R), and c.1747C > T (p.H583Y) in the LDLR gene, as well as c.10579C > T (p.R3527W) in the APOB gene, accounting for 49.29% (69/140) of all patients. These variants are primarily observed in the Asian or Chinese population and are distinct from those present in Caucasian groups. In this cohort, 105 patients were diagnosed with heterozygous FH (HeFH), while 35 were diagnosed with homozygous FH (HoFH). Finally, only 28.57% of the patients (40/140) were using lipid-lowering medications with 33.33% of HoFH patients initiating treatment after the age of 8. Additionally, only 3 compound heterozygous patients (2.14%) underwent liver transplantation because of significantly high lipid levels.ConclusionThis study reveals the variable genotypes and phenotypes of children with FH in China and illustrates that the genotypes in the Chinese population differ from those in Caucasians, providing a valuable dataset for the clinical genetic screening of FH in China. Furthermore, the older age at diagnosis and treatment highlights the underdiagnosis and undertreatment of Chinese FH pediatric patients, suggesting that early identification should be improved through lipid or genetic screening, and that more timely and regular pharmacological treatments should be implemented.

Newly Initiated Statin Treatment Is Associated with Decreased Plasma Coenzyme Q10 Level After Acute ST-Elevation Myocardial Infarction.

Coenzyme Q10 (CoQ10) plays a crucial role in facilitating electron transport during oxidative phosphorylation, thus contributing to cellular energy production. Statin treatment causes a decrease in CoQ10 levels in muscle tissue as well as in serum, which may contribute to the musculoskeletal side effects. Therefore, we aimed to assess the effect of newly initiated statin treatment on serum CoQ10 levels after acute ST-elevation myocardial infarction (STEMI) and the correlation of CoQ10 levels with key biomarkers of subclinical or clinically overt myopathy. In this study, we enrolled 67 non-diabetic, statin-naïve early-onset STEMI patients with preserved renal function. Plasma CoQ10 level was determined by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC/MS-MS), while the myopathy marker serum fatty acid-binding protein 3 (FABP3) level was measured with enzyme-linked immunosorbent assay (ELISA) at hospital admission and after 3 months of statin treatment. The treatment significantly decreased the plasma CoQ10 (by 43%) and FABP3 levels (by 79%) as well as total cholesterol, low-density lipoprotein cholesterol (LDL-C), apolipoprotein B100 (ApoB100), and oxidized LDL (oxLDL) levels. The change in CoQ10 level showed significant positive correlations with the changes in total cholesterol, LDL-C, ApoB100, and oxLDL levels, while it did not correlate with the change in FABP3 level. Our results prove the CoQ10-reducing effect of statin treatment and demonstrate its lipid-lowering efficacy but contradict the role of CoQ10 reduction in statin-induced myopathy.

Vaccination as a Promising Approach in Cardiovascular Risk Mitigation: Are We Ready to Embrace a Vaccine Strategy?

Cardiovascular disease (CVD) remains a leading global health concern, with atherosclerosis being its principal cause. Standard CVD treatments primarily focus on mitigating cardiovascular (CV) risk factors through lifestyle changes and cholesterol-lowering therapies. As atherosclerosis is marked by chronic arterial inflammation, the innate and adaptive immune systems play vital roles in its progression, either exacerbating or alleviating disease development. This intricate interplay positions the immune system as a compelling therapeutic target. Consequently, immunomodulatory strategies have gained increasing attention, though none have yet reached widespread clinical adoption. Safety concerns, particularly the suppression of host immune defenses, remain a significant barrier to the clinical application of anti-inflammatory therapies. Recent decades have revealed the significant role of adaptive immune responses to plaque-associated autoantigens in atherogenesis, opening new perspectives for targeted immunological interventions. Preclinical models indicate that vaccines targeting specific atherosclerosis-related autoantigens can slow disease progression while preserving systemic immune function. In this context, numerous experimental studies have advanced the understanding of vaccine development by exploring diverse targeting pathways. Key strategies include passive immunization using naturally occurring immunoglobulin G (IgG) antibodies and active immunization targeting low-density lipoprotein cholesterol (LDL-C) and apolipoproteins, such as apolipoprotein B100 (ApoB100) and apolipoprotein CIII (ApoCIII). Other approaches involve vaccine formulations aimed at proteins that regulate lipoprotein metabolism, including proprotein convertase subtilisin/kexin type 9 (PCSK9), cholesteryl ester transfer protein (CETP), and angiopoietin-like protein 3 (ANGPTL3). Furthermore, the literature highlights the potential for developing non-lipid-related vaccines, with key targets including heat shock proteins (HSPs), interleukins (ILs), angiotensin III (Ang III), and a disintegrin and metalloproteinase with thrombospondin motifs 7 (ADAMTS-7). However, translating these promising findings into safe and effective clinical therapies presents substantial challenges. This review provides a critical evaluation of current anti-atherosclerotic vaccination strategies, examines their proposed mechanisms of action, and discusses key challenges that need to be overcome to enable clinical translation.

Proteomics for Biomarker Discovery in Gynecological Cancers: A Systematic Review.

The present study aims to summarize the current biomarker landscape in gynecological cancers (GCs) and incorporate bioinformatics analysis to highlight specific biological processes. The literature was retrieved from PubMed, Web of Science, Embase, Scopus, Ovid Medline, and Cochrane Library. The final search was conducted on December 7, 2022. Prospective registration was completed with the PROSPERO with registration number CRD42023477145. This systematic review covered proteomic research on biomarkers for cervical, endometrial, and ovarian cancers. The PANTHER classification system was used to classify the shortlisted candidate biomarkers (CBs), and the STRING database was utilized to visualize protein-protein interaction networks. A total of 23 articles were included in this systematic review. Consistently regulated CBs in the GCs include collagen alpha-2(I) chain, collagen alpha-1(III) chain, collagen alpha-2(V) chain, calreticulin, protein disulfide-isomerase A3, heat shock protein family A (Hsp70) member 5, prolyl 4-hydroxylase, beta polypeptide, fibrinogen alpha chain, fibrinogen gamma chain, apolipoprotein B-100, apolipoprotein C-IV, and apolipoprotein M. In conclusion, collagens, fibrinogens, chaperones, and apolipoproteins were revealed to be replicated in GCs and to be regulated consistently. These CBs contribute to GC etiology and physiology by participating in collagen fibril organization, blood coagulation, protein folding in endoplasmic reticulum, and lipid transporter activity.

Non-HDL Cholesterol May Be Preferred over Apolipoprotein B-100 for Risk Assessment when Evaluated by Receiver Operator Characteristic Curve Analysis.

Most studies found that apolipoprotein B (apo B)-100 is a superior marker for coronary risk to non-high-density lipoprotein (HDL) cholesterol (C). Usually, studies use multivariant analysis with single-point odds/risk ratios. In multivariant analysis, when variables are highly correlated they are difficult to interpret. Effects cannot be well discriminated. Brief review and examination of diagnostic sensitivity and specificity by receiver operator characteristic (ROC) curves at decision levels so that discrimination can be well compared. Since apo B has additional expense, clinical value should be compared in an appropriate format. Apo B and cholesterols were measured in 382 angiographically defined patients. Non-HDLC and apo B were stronger markers than low-density lipoprotein (LDL)C, when examined by logistic regression, but as a result of strong collinearity, non-HDLC appeared weaker than LDLC in the presence of apo B, based on P values. This was true when analyzed with and without nonlipid risk factors. On ROC analysis, apo B and non-HDLC showed stronger C statistics than LDLC and total C. When analyzed alone apo B showed about 6.1% greater sensitivity than non-HDLC. After adjustment for nonlipid risk factors, the C statistics for apo B and non-HDLC were 0.74 and 0.73, and there was little difference in diagnostic specificity. Risk is calculated from an algorithm that includes nonlipid risk factors similar to those examined here along with cholesterols. When assessed by the 10-year screening algorithm, these data support the view that non-HDLC would be less expensive than apo B with similar clinical efficacy.

The structure of apolipoprotein B100 from human low-density lipoprotein.

Low-density lipoprotein (LDL) plays a central role in lipid and cholesterol metabolism and is a key agent in the development and progression of atherosclerosis, the leading cause of mortality worldwide1,2. Apolipoprotein B100 (apoB100), one of the largest proteins in the genome, is the primary structural and functional component of LDL, yet its size and complex lipid associations have posed major challenges for structural studies3. Here we present the first structure of apoB100 resolved to sub-nanometer resolution in most regions using an integrative approach of cryo-electron microscopy, AlphaFold24, and molecular dynamics-based refinement5. The structure consists of a large globular N-terminal domain and a ~61 nm long continuous amphipathic β-sheet that wraps around the LDL particle like a belt. Distributed quasi-symmetrically across the two sides of the "β-belt" are 9 strategically located inter-strand inserts that extend across the lipid surface to provide additional structural support through a network of long-range interactions. We further compare our structure to a comprehensive list of >200 intramolecular crosslinks and find close agreement between the two. These results suggest a mechanism for how the various domains of apoB100 act in concert to maintain LDL shape and cohesion across a range of particle sizes. More generally, they advance our fundamental understanding of LDL synthesis, form and function and will help accelerate the design of potential new therapeutics.

Structure of apolipoprotein B100 bound to the low-density lipoprotein receptor.

Apolipoprotein B100 (apoB100) is a structural component of low-density lipoprotein (LDL) and a ligand for the LDL receptor (LDLR)1. Mutations in apoB100 or in LDLR cause familial hypercholesterolaemia, an autosomal dominant disease that is characterized by a marked increase in LDL cholesterol (LDL-C) and a higher risk of cardiovascular disease2. The structure of apoB100 on LDL and its interaction with LDLR are poorly understood. Here we present the cryo-electron microscopy structures of apoB100on LDL bound to the LDLRand a nanobody complex, which can form a C2-symmetric, higher-order complex. Using local refinement, we determined high-resolution structures of the interfaces between apoB100 and LDLR. One binding interface is formed between several small-ligand-binding modules of LDLR and a series of basic patches that are scattered along a β-belt formed by apoB100, encircling LDL. The other binding interface is formed between the β-propeller domain of LDLR and the N-terminal domain of apoB100. Our results reveal how both interfaces are involved in LDL dimer formation, and how LDLR cycles between LDL- and self-bound conformations. In addition, known mutations in either apoB100 or LDLR, associated with high levels of LDL-C,are located at the LDL-LDLR interface.

Enhanced levels of IL-6 and PAI-1 and decreased levels of MMP-3 in cytomegalovirus seropositive patients with prior myocardial infarction

BackgroundEfforts to understand atherosclerosis, a major cause of ischemic heart disease, have linked several lifestyle factors to increased risk for developing cardiovascular disease. Some studies suggest that cytomegalovirus (CMV), a widely prevalent herpesvirus, is reactivated in atherosclerotic plaques and associated with higher cardiovascular mortality risk. We aimed to explore whether CMV seropositivity and CMV-IgG antibody levels correlate with relevant biomarkers in a cohort of patients with myocardial infarction (MI) and matched controls. Methods and resultsWe analyzed a dataset from 324 survivors of MI treated in Stockholm between 1996 and 2001. Blood samples collected three months after MI were used to measure protective Apo B100 autoantibodies, metabolic, and inflammatory biomarkers. CMV serology was performed on stored serum samples. Correlation analyses were conducted between biomarkers and CMV serostatus in 324 patients and age- and sex-matched controls. While CMV seroprevalence was equal, the CMV-IgG levels were higher in controls. Among various factors examined, CMV seropositive MI patients had elevated levels of plasminogen activator inhibitor-1 (PAI-1) and interleukin-6, along with lower levels of MMP-3, than CMV seronegative MI patients. CMV-IgG levels correlated positively with PAI-1 levels in patients. Although CMV seropositivity was associated with increased proinsulin levels, there was no correlation with diabetes diagnosis. ConclusionsOur findings suggest an enhanced inflammatory and prothrombotic state in CMV seropositive patients after MI. Notably, patients had lower levels of CMV IgG than controls.

Lipoprotein (a) Screening, and What's Next?

Abstract Lipoprotein (a) (Lp(a)) is a low-density lipoprotein (LDL) in which the apolipoprotein B100 molecule is bound to a highly polymorphic, plasminogen-like apolipoprotein (a). The concentration of Lp(a) is predominantly genetically determined, governed by the expression of the LPA gene, and remains relatively stable from early adulthood. The physiological function of Lp(a) is unknown. Lp(a) is recognized as a main carrier of oxidized phospholipids and has strong atherogenic, prothrombotic, and pro-inflammatory properties. Elevated levels of Lp(a) above 75 nmol/l (30 mg/dl) are an independent risk factor for cardiovascular diseases. According to the recommendations of the Polish Lipid Association, Lp(a) should be measured once in a lifetime. This study presents an analysis of the results of Lp(a) screening among adult patients from southeastern Poland (Nowy Sącz county) and evaluates further diagnostic and therapeutic measures for patients with elevated Lp(a) levels. Among the 231 patients screened, 48 individuals (20.8%) had increased Lp(a) levels above 75 nmol/l. Patients with elevated Lp(a) concentration were invited to participate in a survey, in which 31 individuals ultimately took part. Of these, 4 individuals were classified into the moderate risk group, 25 into the high-risk group, and 2 into the very high-risk group for cardiovascular diseases. The pharmacotherapy directly targeting the reduction of Lp(a) is currently undergoing clinical trials. Its effectiveness in decreasing Lp(a) concentrations, along with demonstrating desirable and positive effects on cardiovascular disease outcomes, requires further investigation due to the lack of conclusive evidence. Diet and physical activity have limited impact on Lp(a) concentration. It is recommended to expand preventive programs, including for younger populations, which could help identify patients with genetically determined cardiovascular risk. Additionally, there is a necessity to educate the medical personnel and patients about the significance of Lp(a) and the implementation of guidelines for managing patients with elevated Lp(a) levels.

Oxidized Phospholipids and Calcific Aortic Valvular Disease

BackgroundOxidized phospholipids (OxPLs) are carried by apolipoprotein B-100–containing lipoproteins (OxPL-apoB) including lipoprotein(a) (Lp[a]). Both OxPL-apoB and Lp(a) have been associated with calcific aortic valve disease (CAVD). ObjectivesThis study aimed to evaluate the associations between OxPL-apoB, Lp(a) and the prevalence, incidence, and progression of CAVD. MethodsOxPL-apoB and Lp(a) were evaluated in MESA (Multi-Ethnic Study of Atherosclerosis) and a participant-level meta-analysis of 4 randomized trials of participants with established aortic stenosis (AS). In MESA, the association of OxPL-apoB and Lp(a) with aortic valve calcium (AVC) at baseline and 9.5 years was evaluated using multivariable ordinal regression models. In the meta-analysis, the association between OxPL-apoB and Lp(a) with AS progression (annualized change in peak aortic valve jet velocity) was evaluated using multivariable linear regression models. ResultsIn MESA, both OxPL-apoB and Lp(a) were associated with prevalent AVC (OR per SD: 1.19 [95% CI: 1.07-1.32] and 1.13 [95% CI: 1.01-1.27], respectively) with a significant interaction between the two (P < 0.01). Both OxPL-apoB and Lp(a) were associated with incident AVC at 9.5 years when evaluated individually (interaction P < 0.01). The OxPL-apoB∗Lp(a) interaction demonstrated higher odds of prevalent and incident AVC for OxPL-apoB with increasing Lp(a) levels. In the meta-analysis, when analyzed separately, both OxPL-apoB and Lp(a) were associated with faster increase in peak aortic valve jet velocity, but when evaluated together, only OxPL-apoB remained significant (ß: 0.07; 95% CI: 0.01-0.12). ConclusionsOxPL-apoB is a predictor of the presence, incidence, and progression of AVC and established AS, particularly in the setting of elevated Lp(a) levels, and may represent a novel therapeutic target for CAVD.

Inducible global knockout of surfeit locus protein 4 in adult mice results in hypolipidemia, intestinal lipid accumulation, liver injury, and increased mortality

Surfeit locus protein 4 (SURF4) acts as a cargo receptor to mediate endoplasmic reticulum export of various cargos. We have shown that SURF4 is essential for secretion of hepatic very low-density lipoprotein and intestinal chylomicron. Knockdown of hepatic Surf4 also significantly reduces the development of atherosclerosis and liver fibrosis without causing overt liver damage. However, constitutive global Surf4 knockout results in embryonic lethality. To further understand the physiological role of SURF4, we generated tamoxifen-inducible global Surf4 knockout mice. We found that conditional knockout of Surf4 in adult mice (Surf4ig-ko) significantly reduced mouse body weight. Male and female Surf4ig-ko mice died approximately 30 and 50 days after tamoxifen administration, respectively. Triglyceride secretion and serum levels of total cholesterol, triglycerides, free fatty acids, apolipoprotein B-100, and apolipoprotein B-48 were significantly reduced in Surf4ig-ko mice compared with Surf4flox mice. Proteomics analysis of mouse serum samples revealed 308 proteins with significantly altered expression in Surf4ig-ko mice that have unique functions and are involved in various biological processes. In addition, Surf4ig-ko mice exhibited lipid accumulation in the intestine but not in the liver. However, in Surf4ig-ko mice, liver weight was significantly reduced, and serum transaminase activity was significantly increased, indicating liver damage. Therefore, SURF4 is essential for survival in adult mice, suggesting that the therapeutic use of SURF4 requires precise tissue/cell type-specific targeting.

Variants in LPA are associated with Familial Hypercholesterolaemia: whole genome sequencing analysis in the 100,000 Genomes Project.

Familial Hypercholesterolaemia (FH) is an inherited disease of high LDL-cholesterol (LDL-C) caused by defects in LDLR, APOB, APOE and PCSK9 genes. A pathogenic variant cannot be found in ∼60% of clinical FH patients. Using whole genome sequencing (WGS) we examined genetic determinants of FH. WGS data generated by the 100,000 Genomes Project (100KGP) included 536 FH patients diagnosed using the FH Simon Broome criteria. Rare variants in known FH genes were analysed. Genome-wide association study (GWAS) between 443 FH variant-negative unrelated FH cases and 77,275 control participants of the 100KGP was run using high coverage WGS data. Polygenic risk scores for LDL-C (LDL PRS) and lipoprotein(a) (Lp(a) PRS) were computed. An FH-causing variant was found in 17.4% of FH cases. GWAS identified the LPA gene locus being significantly associated (p<1x10-8). FH variant-negative participants had higher LDL and Lp(a) PRSs in comparison to the controls (p<1.0×10-16 and p<4.09×10-6, respectively). Similar associations were found in the monogenic FH with both LDL and Lp(a) PRSs being higher than in controls (p<4.03×10-4 and p<3.01x10-3, respectively). High LDL PRS was observed in 36.4% of FH variant-negative cases, whereas high Lp(a) PRS in 18.5%, with 7.0% having both high LDL and Lp(a) PRSs. This genome-wide analysis of monogenic and polygenic FH causes confirms a complex and heterogenous architecture of hypercholesterolaemia, with the LPA gene playing a significant role. Both Lp(a) and LDL-C should be measured for precision FH diagnosis. Specific therapies to lower Lp(a) should be targeted to those who will benefit most.

Adipocytes reprogram the proteome of breast cancer cells in organotypic three-dimensional cell cultures.

While epidemiological evidence has long linked obesity with an increased risk of breast cancer, the intricate interactions between adipocytes and cancer cells within the tumor microenvironment remain largely uncharted territory. The use of organotypic three-dimensional (3D) cell cultures that more accurately mimic the spatial architecture of tumors represents an innovative approach to this complex issue. In the present study, we investigated the effects of adipocytes on the proteome of Hs578t breast cancer cells cultured in a 3D microenvironment. Using different treatments, we rigorously optimized the experimental conditions to induce the optimal differentiation of 3T3-L1 fibroblasts into mature adipocytes. Then, we grow the Hs578t cells in a simulated microenvironment using an on-top model for organotypic 3D cultures. Our data showed that cancer cells formed 3D stellate-like architectures when grown over an extracellular matrix proteins-enriched scaffold for 48h. Proteomic profiling using LC-MS/MS mass spectrometry of Hs578t cells grown in 3D conditions with or without the adipocyte-enriched culture discovered 916 unique proteins. Of these, 605 showed no significant changes in abundance, whereas 87 proteins were significantly upregulated and 224 downregulated after interaction with fat cells (p < 0.05, FC > 2.0). Bioinformatic analysis of upregulated proteins indicated that the most enriched GO terms and molecular functions were related to lipids transport, cell differentiation, hypoxia response, and cell junctions. In addition, several modulated proteins have been previously associated with breast cancer progression. Interestingly, lipid transport proteins, including PITPNM2, ATP2C1, ABCA12, HDLBP, and APOB, showed perturbations in their expression, which were also associated with low overall survival in breast cancer patients. Functional studies showed that the knockdown of apolipoprotein B (APOB) expression in Hs578t cells reduced the size of 3D cellular structures. Moreover, APOB-knocked cells cocultured with adipocytes for 48h exhibited a significant decrease of intracellular lipids, whereas an increase in the adipocytes was found. Our results indicate that the 3D microenvironment and the adipocytes crosstalk reprogram the proteome of breast cancer cells. These data help us understand the environmental effects in gene expression and contribute to discovering novel tumor proteins with potential intervention in breast cancer therapy.

CDR identification, epitope mapping and binding affinity determination of novel monoclonal antibodies generated against human apolipoprotein B-100

In-house generated mAbs to apolipoprotein B-100 (apoB-100) clones hLDL-E8, hLDL-2D8 and hLDL-F5 were extensively studied to determine their complementarity-determining regions (CDRs), binding epitopes and affinity. RT-PCR revealed that all mAbs consisted of kappa light chains and gamma heavy chains. DNA sequencing and bioinformatic analysis showed that the variable gene and protein sequences of their CDRs shared over 50 % identity with the existing databases. The 3D structures of the mAb variable fragments (Fv) with a QSQE score above 0.7 were constructed using the SWISS-MODEL platform. The structural accuracy was confirmed by Ramachandran plots, with 99 % of amino acid residues falling within acceptable regions. Thrombolytic cleavage of apoB-100 and Western blot analysis demonstrated that hLDL-E8 and hLDL-F5 specifically bind to the T3 fragment (aa 1297–3249), whereas hLDL-2D8 binds to the T4 fragment (aa 1–1297). These findings were supported with epitope-binding assays using inhibition ELISA, which indicated that hLDL-E8 binds at different epitopes from hLDL-2D8 and has some overlap with hLDL-F5. Lastly, the binding affinity of the mAbs was examined by indirect ELISA. The average affinity constants (Kaff) for mAbs hLDL-2D8, hLDL-E8 and hLDL-F5 are 1.51 ± 0.69 × 109 Mol−1, 7.25 ± 3.56 × 108 Mol−1 and 4.39 ± 2.63 × 106 Mol−1, respectively. Additionally, the behavior of the antibodies in the dose-response curve revealed that hLDL-F5 may recognize two epitopes of apoB-100 or have very low binding affinity. In contrast, hLDL-2D8 and hLDL-E8 each recognize a single epitope. These findings provide information that will be useful when selecting mAbs for both laboratory and clinical research purposes.

Clinical reality and challenges with familial hypercholesterolemia patients' management. 2024 results from the Regional Center for Rare Diseases (RCRD) Registry in Poland

BackgroundDespite advancements in early diagnosis and effective medications in last decade, most heterozygous familial hypercholesterolemia (heFH) patients still fail to achieve their low-density lipoprotein cholesterol (LDL-C) goals and remain at residual cardiovascular disease risk. We present recent data from the regional FH registry in Poland, highlighting the challenges and real-life clinical management of FH patients. MethodsThe registry is held at the Regional Centre for Rare Diseases, founded in 2016, at the 2nd largest, supraregional hospital in Poland, where >80 different rare diseases in patients from all over Poland are diagnosed and treated, including phenotypically or genetically diagnosed FH patients. Our analysis focused on both children and adult FH patients, excluding those treated with inclisiran due to a small sample size (n = 5). ResultsWe studied 173 consecutive heFH patients, median age for adult population was 40 years (range: 27–57), of whom 56.14 % were women. Among the population, 82.1 % were adults (n = 142), and 31 were children (17.92 %; median age 9 (8–13), females 58.16 %). Children exhibited lower total cholesterol and triglyceride levels compared to adults, with no significant differences in LDL-C and high-density lipoprotein cholesterol (HDL-C) levels. Molecular diagnosis in the whole population revealed that 76.6 % had an LDL receptor (LDLR) mutation, while 23.4 % had an apolipoprotein B (APOB) mutation. Risk assessment categorized patients into high (70.7 %), very high (22.1 %), and extremely high (7.1 %) risk groups. Triple therapy achieved treatment goals in 61.76 % of adults and 70.97 % of children. At baseline, 36.62 % of adult patients were not using statins. High-intensity statin therapy combined with ezetimibe was initiated for the remaining patients. Only 3.33 % of patients avoided statins due to complete intolerance. Ezetimibe was used in 57.27 % of patients (mostly in combination therapy), and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors were prescribed for 28.17 % FH patients. In adults receiving statin and ezetimibe therapy, median achieved LDL-C was 141 mg/dl (107–184). For triple therapy, median achieved LDL-C was 52.5 mg/dL (32–86.5). Overall median achieved LDL-C in the study population was 99.5 mg/dl (57.5–145.4). PCSK9 inhibitors reduced LDL-C by 165.6 mg/dl. Combination therapy did not significantly alter baseline lipoprotein(a) (Lp(a)) levels (p = 0.134), and PCSK9 inhibitors led to a mean Lp(a) reduction of 18.66 mg/dl (45 % reduction; p = 0.013). Multivariable regression analysis identified key factors for achieving LDL-C targets in FH patients: DLCN total score, DLCN category, ezetimibe use, and PCSK9 inhibitors. ConclusionsIn Poland, FH patients are often diagnosed too late (usually over 40 years of age), and many still do not reach their LDL-C goals. Combination LLT double or triple therapy significantly increases the likelihood of achieving LDL-C targets – even up to fivefold. Therefore, unrestricted access to PCSK9 inhibitors for all FH patients is crucial, without the current limitations imposed by drug reimbursement programs like B101.

Extracellular vesicles and citrullination signatures are novel biomarkers in sturgeon (Acipenser gueldenstaedtii) during chronic stress due to seasonal temperature challenge

Acipenser gueldenstaedtii is one of the most cultured sturgeon species worldwide and of considerable economic value for caviar production. There are though considerable challenges around chronic stress responses due to increased summer temperatures, impacting sturgeons’ immune responses and their susceptibility to opportunistic infections. The identification of molecular and cellular pathways involved in stress responses may contribute to identifying novel biomarkers reflective of fish health status, crucial for successful sturgeon aquaculture. Protein citrullination is a calcium-catalysed post-translational modification caused by peptidylarginine deiminases (PADs), altering target protein function and affecting protein interactions in physiological and pathobiological processes. PADs can also modulate extracellular vesicle (EVs) profiles, which play critical roles in cellular communication, via transport of their cargoes (proteins, including post-translationally modified proteins, genetic material and micro-RNAs).This study identified differences in EV signatures, and citrullinated proteins in sera from winter and summer farmed sturegeons. EVs were significantly elevated in sera of the summer chronically stressed group. The citrullinated proteins and associated gene ontology (GO) pathways in sera and serum-EVs of chronically heat stressed A. gueldenstaedtii, showed some changes, with specific citrullinated serum protein targets including alpa-2-macroglobulin, alpha globin, calcium-dependent secretion activator, ceruloplasmin, chemokine XC receptor, complement C3 isoforms, complement C9, plectin, selenoprotein and vitellogenin. In serum-EVs, citrullinated protein cargoes identified only in the chronically stressed summer group included alpha-1-antiproteinase, apolipoprotein B-100, microtubule actin crosslinking factor and histone H3. Biological gene ontology (GO) pathways related to citrullinated serum proteins in the chronically stressed group were associated with innate and adaptive immune responses, stress responses and metabolic processes. In serum-EVs of the heat-stressed group the citrullinome associated with various metabolic GO pathways.In addition to modified citrullinated protein content, Serum-EVs from the stressed summer group showed significantly increased levels of the inflammatory associated miR-155 and the hypoxia-associated miR-210, but significantly reduced levels of the growth-associated miR-206.Our findings highlight roles for protein citrullination and EV signatures in response to chronic heat stress in A. gueldenstaedtii, indicating a trade-off in immunity versus growth and may be of value for sturgeon aquaculture.

FITM2 deficiency results in ER lipid accumulation, ER stress, and reduced apolipoprotein B lipidation and VLDL triglyceride secretion in vitro and in mouse liver

ObjectiveTriglycerides (TGs) associate with apolipoprotein B100 (apoB100) to form very low density lipoproteins (VLDLs) in the liver. The repertoire of factors that facilitate this association is incompletely understood. FITM2, an integral endoplasmic reticulum (ER) protein, was originally discovered as a factor participating in cytosolic lipid droplet (LD) biogenesis in tissues that do not form VLDL. We hypothesized that in the liver, in addition to promoting cytosolic LD formation, FITM2 would also transfer TG from its site of synthesis in the ER membrane to nascent VLDL particles within the ER lumen. MethodsExperiments were conducted using a rat hepatic cell line (McArdle-RH7777, or McA cells), an established model of mammalian lipoprotein metabolism, and mice. FITM2 expression was reduced using siRNA in cells and by liver specific cre-recombinase mediated deletion of the Fitm2 gene in mice. Effects of FITM2 deficiency on VLDL assembly and secretion in vitro and in vivo were measured by multiple methods, including density gradient ultracentrifugation, chromatography, mass spectrometry, stimulated Raman scattering (SRS) microscopy, sub-cellular fractionation, immunoprecipitation, immunofluorescence, and electron microscopy. Main findings1) FITM2-deficient hepatic cells in vitro and in vivo secrete TG-depleted VLDL particles, but the number of particles is unchanged compared to controls; 2) FITM2 deficiency in mice on a high fat diet (HFD) results in decreased plasma TG levels. The number of apoB100-containing lipoproteins remains similar, but shift from VLDL to low density lipoprotein (LDL) density; 3) Both in vitro and in vivo, when TG synthesis is stimulated and FITM2 is deficient, TG accumulates in the ER, and despite its availability this pool is unable to fully lipidate apoB100 particles; 4) FITM2 deficiency disrupts ER morphology and results in ER stress. ConclusionThe results suggest that FITM2 contributes to VLDL lipidation, especially when newly synthesized hepatic TG is in abundance. In addition to its fundamental importance in VLDL assembly, the results also suggest that under dysmetabolic conditions, FITM2 may be an important factor in the partitioning of TG between cytosolic LDs and VLDL particles.