Algorithm for detection and screening of familial hypercholesterolemia in Lithuanian population.

BACKGROUND: Familial hypercholesterolemia (FH) is one of the most common autosomal dominant diseases. FH causes a lifelong increase in low-density lipoprotein cholesterol (LDL-C) levels, which in turn leads to atherosclerotic cardiovascular disease. The incidence of FH is widely underestimated and undertreated, despite the availability and effectiveness of lipid-lowering therapy. Patients with FH have an increased cardiovascular risk; therefore, early diagnosis and treatment are vital. To address the burden of FH, several countries have implemented national FH screening programmes. The currently used method for FH detection in Lithuania is mainly based on opportunistic testing with subsequent cascade screening of index cases' first-degree relatives. METHODS: A total of 428 patients were included in this study. Patients with suspected FH are referred to a lipidology center for thorough evaluation. Patients who met the criteria for probable or definite FH according to the Dutch Lipid Clinic Network (DLCN) scoring system and/or had LDL-C > = 6.5 mmol/l were subjected to genetic testing. Laboratory and instrumental tests, vascular marker data of early atherosclerosis, and consultations by other specialists, such as radiologists and ophthalmologists, were also recorded. RESULTS: A total of 127/428 (30%) patients were genetically tested. FH-related mutations were found in 38.6% (n = 49/127) of the patients. Coronary artery disease (CAD) was diagnosed in 13% (n = 57/428) of the included patients, whereas premature CAD was found in 47/428 (11%) patients. CAD was diagnosed in 19% (n = 9/49) of patients with FH-related mutations, and this diagnosis was premature for all of them. CONCLUSIONS: Most patients in this study were classified as probable or possible FH without difference of age and sex. The median age of FH diagnosis was 47 years with significantly older females than males, which refers to the strong interface of this study with the LitHir programme. CAD and premature CAD were more common among patients with probable and definite FH, as well as those with an FH-causing mutation. The algorithm described in this study is the first attempt in Lithuania to implement a specific tool which allows to maximise FH detection rates, establish an accurate diagnosis of FH, excluding secondary causes of dyslipidaemia, and to select patients for cascade screening initiation more precisely.

Gallic Acid Can Promote Low-Density Lipoprotein Uptake in HepG2 Cells via Increasing Low-Density Lipoprotein Receptor Accumulation.

Gallic acid (GA) is a type of polyphenolic compound that can be found in a range of fruits, vegetables, and tea. Although it has been confirmed it improves non-alcoholic fatty liver disease (NAFLD), it is still unknown whether GA can improve the occurrence of NAFLD by increasing the low-density lipoprotein receptor (LDLR) accumulation and alleviating cholesterol metabolism disorders. Therefore, the present study explored the effect of GA on LDLR and its mechanism of action. The findings indicated that the increase in LDLR accumulation in HepG2 cells induced by GA was associated with the stimulation of the epidermal growth factor receptor-extracellular regulated protein kinase (EGFR-ERK1/2) signaling pathway. When the pathway was inhibited by EGFR mab cetuximab, it was observed that the activation of the EGFR-ERK1/2 signaling pathway induced by GA was also blocked. At the same time, the accumulation of LDLR protein and the uptake of LDL were also suppressed. Additionally, GA can also promote the accumulation of forkhead box O3 (FOXO3) and suppress the accumulation of hepatocyte nuclear factor-1α (HNF1α), leading to the inhibition of proprotein convertase subtilisin/kexin 9 (PCSK9) mRNA expression and protein accumulation. This ultimately results in increased LDLR protein accumulation and enhanced uptake of LDL in cells. In summary, the present study revealed the potential mechanism of GA's role in ameliorating NAFLD, with a view of providing a theoretical basis for the dietary supplementation of GA.

[The influence of knocking down the expression of low-density lipoprotein receptor associated proteins on the vascular abnormalities in hepatocellular carcinoma and its mechanisms].

Objectives: To investigate the effect of the expression of low-density lipoprotein receptor associated protein (LDLR) on the vascular abnormalities in hepatocellular carcinoma (HCC) and its mechanisms. Methods: Based on the information of Oncomine Cancer GeneChip database, we analyzed the correlation between the expression level of LDLR and the expression level of carcinoembryonic antigen (CEA) and CD31 in hepatocellular carcinoma tissues. Lentiviral transfection of short hairpin RNA target genes was used to construct LDLR-knockdown MHCC-97H and HLE hepatocellular carcinoma cells. The differential genes and their expression level changes in LDLR-knockdown hepatocellular carcinoma cells were detected by transcriptome sequencing, real-time fluorescence quantitative polymerase chain reaction, and protein immunoblotting. The gene-related signaling pathways that involve LDLR were clarified by enrichment analysis. The effect of LDLR on CEA was assessed by the detection of CEA content in conditioned medium of hepatocellular carcinoma cells. Angiogenesis assay was used to detect the effect of LDLR on the angiogenic capacity of human umbilical vein endothelial cells, as well as the role of CEA in the regulation of angiogenesis by LDLR. Immunohistochemical staining was used to detect the expression levels of LDLR in 176 hepatocellular carcinoma tissues, and CEA and CD31 in 146 hepatocellular carcinoma tissues, and analyze the correlations between the expression levels of LDLR, CEA, and CD31 in the tissues, serum CEA, and alanine transaminase (ALT). Results: Oncomine database analysis showed that the expressions of LDLR and CEA in the tissues of hepatocellular carcinoma patients with portal vein metastasis were negatively correlated (r=-0.64, P=0.001), whereas the expressions of CEA and CD31 in these tissues were positively correlated ( r=0.46, P=0.010). The transcriptome sequencing results showed that there were a total of 1 032 differentially expressed genes in the LDLR-knockdown group and the control group of MHCC-97H cells, of which 517 genes were up-regulated and 515 genes were down-regulated. The transcript expression level of CEACAM5 was significantly up-regulated in the cells of the LDLR-knockdown group. The Gene Ontology (GO) function enrichment analysis showed that the differential genes were most obviously enriched in the angiogenesis function. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis showed that the relevant pathways involved mainly included the cellular adhesion patch, the extracellular matrix receptor interactions, and the interactions with the extracellular matrix receptors. The CEA content in the conditioned medium of the LDLR-knockdown group was 43.75±8.43, which was higher than that of the control group (1.15±0.14, P＜0.001). The results of angiogenesis experiments showed that at 5 h, the number of main junctions, the number of main segments, and the total area of the lattice formed by HUVEC cells cultured with the conditioned medium of MHCC-97H cells in the LDLR-knockdown group were 295.3±26.4, 552.5±63.8, and 2 239 781.0±13 8211.9 square pixels, which were higher than those of the control group (113.3±23.5, 194.8±36.5, and 660 621.0±280 328.3 square pixels, respectively, all P＜0.01).The number of vascular major junctions, the number of major segments, and the total area of the lattice formed by HUVEC cells cultured in conditioned medium with HLE cells in the LDLR-knockdown group were 245.3±42.4, 257.5±20.4, and 2 535 754.5±249 094.2 square pixels, respectively, which were all higher than those of the control group (113.3±23.5, 114.3±12.2, and 1 565 456.5±219 259.7 square pixels, respectively, all P＜0.01). In the conditioned medium for the control group of MHCC-97H cells,the number of main junctions, the number of main segments, and the total area of the lattice formed by the addition of CEA to cultured HUVEC cells were 178.9±12.0, 286.9±12.3, and 1 966 990.0±126 249.5 spixels, which were higher than those in the control group (119.7±22.1, 202.7±33.7, and 1 421 191.0±189 837.8 square pixels, respectively). The expression of LDLR in hepatocellular carcinoma tissues was not correlated with the expression of CEA, but was negatively correlated with the expression of CD31 (r=-0.167, P=0.044), the level of serum CEA (r=-0.061, P=0.032), and the level of serum ALT(r=-0.147,P=0.05). The expression of CEA in hepatocellular carcinoma tissues was positively correlated with the expression of CD31 (r=0.192, P=0.020). The level of serum CEA was positively correlated with the level of serum ALT (r=0.164, P=0.029). Conclusion: Knocking down LDLR can promote vascular abnormalities in HCC by releasing CEA.

Analysis of 3760 hematologic malignancies reveals rare transcriptomic aberrations of driver genes.

BACKGROUND: Rare oncogenic driver events, particularly affecting the expression or splicing of driver genes, are suspected to substantially contribute to the large heterogeneity of hematologic malignancies. However, their identification remains challenging. METHODS: To address this issue, we generated the largest dataset to date of matched whole genome sequencing and total RNA sequencing of hematologic malignancies from 3760 patients spanning 24 disease entities. Taking advantage of our dataset size, we focused on discovering rare regulatory aberrations. Therefore, we called expression and splicing outliers using an extension of the workflow DROP (Detection of RNA Outliers Pipeline) and AbSplice, a variant effect predictor that identifies genetic variants causing aberrant splicing. We next trained a machine learning model integrating these results to prioritize new candidate disease-specific driver genes. RESULTS: We found a median of seven expression outlier genes, two splicing outlier genes, and two rare splice-affecting variants per sample. Each category showed significant enrichment for already well-characterized driver genes, with odds ratios exceeding three among genes called in more than five samples. On held-out data, our integrative modeling significantly outperformed modeling based solely on genomic data and revealed promising novel candidate driver genes. Remarkably, we found a truncated form of the low density lipoprotein receptor LRP1B transcript to be aberrantly overexpressed in about half of hairy cell leukemia variant (HCL-V) samples and, to a lesser extent, in closely related B-cell neoplasms. This observation, which was confirmed in an independent cohort, suggests LRP1B as a novel marker for a HCL-V subclass and a yet unreported functional role of LRP1B within these rare entities. CONCLUSIONS: Altogether, our census of expression and splicing outliers for 24 hematologic malignancy entities and the companion computational workflow constitute unique resources to deepen our understanding of rare oncogenic events in hematologic cancers.

Clinical relevance of somatic mutations in Chinese lung adenocarcinoma and their prognostic implications for survival.

BACKGROUND: To comprehensively elucidate the genomic and mutational features of lung cancer cases, and lung adenocarcinoma (LUAD), it is imperative to conduct ongoing investigations into the genomic landscape. In this study, we aim to analyze the somatic mutation profile and assessed the significance of these informative genes utilizing a retrospective LUAD cohort. METHODS: A total of 247 Chinese samples were analyzed to exhibit the tumor somatic genomic alterations in patients with LUAD. The Cox regression analysis was employed to identify prognosis-related genes and establish a predictive model for stratifying patients with LUAD. RESULTS: In the Dianjiang People's Hospital (DPH) cohort, the top five frequent mutated genes were (Epidermal growth factor receptor) EGFR (68%), TP53 (30%), RBM10 (13%), LRP1B (9%), and KRAS (9%). Of which, EGFR is a mostly altered driver gene, and most mutation sites are located in tyrosine kinase regions. Oncogene pathway alteration and mutation signature analysis demonstrated the RTK-RAS pathway alteration, and smoking was the main carcinogenic factor of the DPH cohort. Furthermore, we identified 34 driver genes in the DPH cohort, including EGFR (68%), TP53 (30.4%), RBM10 (12.6%), KRAS (8.5%), LRP1B (8.5%), and so on, and 45 Clinical Characteristic-Related Genes (CCRGs) were found to closely related to the clinical high-risk factors. We developed a Multiple Parameter Gene Mutation (MPGM) risk model by integrating critical genes and oncogenic pathway alterations in LUAD patients from the DPH cohort. Based on publicly available LUAD datasets, we identified five genes, including BRCA2, Anaplastic lymphoma kinase (ALK), BRAF, EGFR, and Platelet-Derived Growth Factor Receptor Alpha (PDGFRA), according to the multivariable Cox regression analysis. The MPGM-low group showed significantly better overall survival (OS) compared to the MPGM-high group (p < 0.0001, area under the curve (AUC) = 0.754). The robust performance was validated in 55 LUAD patients from the DPH cohort and another LUAD dataset. Immune characteristics analysis revealed a higher proportion of primarily DCs and mononuclear cells in the MPGM-low risk group, while the MPGM-high risk group showed lower immune cells and higher tumor cell infiltration. CONCLUSION: This study provides a comprehensive genomic landscape of Chinese LUAD patients and develops an MPGM risk model for LUAD prognosis stratification. Further follow-up will be performed for the patients in the DPH cohort consistently to explore the resistance and prognosis genetic features.

Prenatal Choline Supplementation Improves Glucose Tolerance and Reduces Liver Fat Accumulation in Mouse Offspring Exposed to Ethanol during the Prenatal and Postnatal Periods.

Prenatal alcohol exposure (AE) affects cognitive development. However, it is unclear whether prenatal AE influences the metabolic health of offspring and whether postnatal AE exacerbates metabolic deterioration resulting from prenatal AE. Choline is a semi-essential nutrient that has been demonstrated to mitigate the cognitive impairment of prenatal AE. This study investigated how maternal choline supplementation (CS) may modify the metabolic health of offspring with prenatal and postnatal AE (AE/AE). C57BL/6J female mice were fed either a Lieber-DeCarli diet with 1.4% ethanol between embryonic day (E) 9.5 and E17.5 or a control diet. Choline was supplemented with 4 × concentrations versus the control throughout pregnancy. At postnatal week 7, offspring mice were exposed to 1.4% ethanol for females and 3.9% ethanol for males for 4 weeks. AE/AE increased hepatic triglyceride accumulation in male offspring only, which was normalized by prenatal CS. Prenatal CS also improved glucose tolerance compared to AE/AE animals. AE/AE suppressed hepatic gene expression of peroxisome proliferator activated receptor alpha (Ppara) and low-density lipoprotein receptor (Ldlr), which regulate fatty acid catabolism and cholesterol reuptake, respectively, in male offspring. However, these changes were not rectified by prenatal CS. In conclusion, AE/AE led to an increased risk of steatosis and was partially prevented by prenatal CS in male mice.

LDL receptor-related protein 5 selectively transports unesterified polyunsaturated fatty acids to intracellular compartments.

Polyunsaturated fatty acids (PUFAs), which cannot be synthesized by animals and must be supplied from the diet, have been strongly associated with human health. However, the mechanisms for their accretion remain poorly understood. Here, we show that LDL receptor-related protein 5 (LRP5), but not its homolog LRP6, selectively transports unesterified PUFAs into a number of cell types. The LDLa ligand-binding repeats of LRP5 directly bind to PUFAs and are required and sufficient for PUFA transport. In contrast to the known PUFA transporters Mfsd2a, CD36 and FATP2, LRP5 transports unesterified PUFAs via internalization to intracellular compartments including lysosomes, and n-3 PUFAs depend on this transport mechanism to inhibit mTORC1. This LRP5-mediated PUFA transport mechanism suppresses extracellular trap formation in neutrophils and protects mice from myocardial injury during ischemia-reperfusion. Thus, this study reveals a biologically important mechanism for unesterified PUFA transport to intracellular compartments.

Targeting PCSK9 to upregulate MHC-II on the surface of tumor cells in tumor immunotherapy.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9), the last member of the proprotein convertase family, functions as a classic regulator of low-density lipoprotein (LDL) by interacting with low-density lipoprotein receptor (LDLR). Recent studies have shown that PCSK9 can affect the occurrence and development of tumors and can be used as a novel therapeutic target. However, a comprehensive pan-cancer analysis of PCSK9 has yet to be conducted. METHODS: The potential oncogenic effects of PCSK9 in 33 types of tumors were explored based on the datasets of The Cancer Genome Atlas (TCGA) dataset. In addition, the immune regulatory role of PCSK9 inhibition was evaluated via in vitro cell coculture and the tumor-bearing mouse model. Finally, the antitumor efficacy of targeted PCSK9 combined with OVA-II vaccines was verified. RESULTS: Our results indicated that PCSK9 was highly expressed in most tumor types and was significantly correlated with late disease stage and poor prognosis. Additionally, PCSK9 may regulate the tumor immune matrix score, immune cell infiltration, immune checkpoint expression, and major histocompatibility complex expression. Notably, we first found that dendritic cell (DC) infiltration and major histocompatibility complex-II (MHC-II) expression could be upregulated by PCSK9 inhibition and improve CD8+ T cell activation in the tumor immune microenvironment, thereby achieving potent tumor control. Combining PCSK9 inhibitors could enhance the efficacies of OVA-II tumor vaccine monotherapy. CONCLUSIONS: Conclusively, our pan-cancer analysis provided a more comprehensive understanding of the oncogenic and immunoregulatory roles of PCSK9 and demonstrated that targeting PCSK9 could increase the efficacy of long peptide vaccines by upregulating DC infiltration and MHC-II expression on the surface of tumor cells. This study reveals the critical oncogenic and immunoregulatory roles of PCSK9 in various tumors and shows the promise of PCSK9 as a potent immunotherapy target.

LDL Receptor-Related Protein 1B Polymorphisms Associated with Increased Risk of Lymph Node Metastasis in Oral Cancer Group with Diabetes Mellitus.

Oral cancer ranks fourth among malignancies among Taiwanese men and is the eighth most common cancer among men worldwide in terms of general diagnosis. The purpose of the current study was to investigate how low-density lipoprotein receptor-related protein 1B (LDL receptor related protein 1B; LRP1B) gene polymorphisms affect oral squamous cell carcinoma (OSCC) risk and progression in individuals with diabetes mellitus (DM). Three LRP1B single-nucleotide polymorphisms (SNPs), including rs10496915, rs431809, and rs6742944, were evaluated in 311 OSCC cases and 300 controls. Between the case and control groups, we found no evidence of a significant correlation between the risk of OSCC and any of the three specific SNPs. Nevertheless, in evaluating the clinicopathological criteria, individuals with DM who possess a minimum of one minor allele of rs10496915 (AC + CC; p = 0.046) were significantly associated with tumor size compared with those with homozygous major alleles (AA). Similarly, compared to genotypes homologous for the main allele (GG), rs6742944 genotypes (GA + AA; p = 0.010) were more likely to develop lymph node metastases. The tongue and the rs6742944 genotypes (GA + AA) exhibited higher rates of advanced clinical stages (p = 0.024) and lymph node metastases (p = 0.007) when compared to homozygous alleles (GG). LRP1B genetic polymorphisms appear to be prognostic and diagnostic markers for OSCC and DM, as well as contributing to genetic profiling research for personalized medicine.

Leveraging Bidirectional Nature of Allostery To Inhibit Protein-Protein Interactions (PPIs): A Case Study of PCSK9-LDLR Interaction.

The protein PCSK9 (proprotein convertase subtilisin/Kexin type 9) negatively regulates the recycling of LDLR (low-density lipoprotein receptor), leading to an elevated plasma level of LDL. Inhibition of PCSK9-LDLR interaction has emerged as a promising therapeutic strategy to manage hypercholesterolemia. However, the large interaction surface area between PCSK9 and LDLR makes it challenging to identify a small molecule competitive inhibitor. An alternative strategy would be to identify distal cryptic sites as targets for allosteric inhibitors that can remotely modulate PCSK9-LDLR interaction. Using several microseconds long molecular dynamics (MD) simulations, we demonstrate that on binding with LDLR, there is a significant conformational change (population shift) in a distal loop (residues 211-222) region of PCSK9. Consistent with the bidirectional nature of allostery, we establish a clear correlation between the loop conformation and the binding affinity with LDLR. Using a thermodynamic argument, we establish that the loop conformations predominantly present in the apo state of PCSK9 would have lower LDLR binding affinity, and they would be potential targets for designing allosteric inhibitors. We elucidate the molecular origin of the allosteric coupling between this loop and the LDLR binding interface in terms of the population shift in a set of salt bridges and hydrogen bonds. Overall, our work provides a general strategy toward identifying allosteric hotspots: compare the conformational ensemble of the receptor between the apo and bound states of the protein and identify distal conformational changes, if any. The inhibitors should be designed to bind and stabilize the apo-specific conformations.

Joint genotypic and phenotypic outcome modeling improves base editing variant effect quantification.

CRISPR base editing screens enable analysis of disease-associated variants at scale; however, variable efficiency and precision confounds the assessment of variant-induced phenotypes. Here, we provide an integrated experimental and computational pipeline that improves estimation of variant effects in base editing screens. We use a reporter construct to measure guide RNA (gRNA) editing outcomes alongside their phenotypic consequences and introduce base editor screen analysis with activity normalization (BEAN), a Bayesian network that uses per-guide editing outcomes provided by the reporter and target site chromatin accessibility to estimate variant impacts. BEAN outperforms existing tools in variant effect quantification. We use BEAN to pinpoint common regulatory variants that alter low-density lipoprotein (LDL) uptake, implicating previously unreported genes. Additionally, through saturation base editing of LDLR, we accurately quantify missense variant pathogenicity that is consistent with measurements in UK Biobank patients and identify underlying structural mechanisms. This work provides a widely applicable approach to improve the power of base editing screens for disease-associated variant characterization.

Recombinant human proteoglycan 4 lowers inflammation and atherosclerosis susceptibility in female low-density lipoprotein receptor knockout mice.

Recombinant human proteoglycan 4 (rhPRG4) is a macromolecular mucin-like glycoprotein that is classically studied as a lubricant within eyes and joints. Given that endogenously produced PRG4 is present within atherosclerotic lesions and genetic PRG4 deficiency increases atherosclerosis susceptibility in mice, in the current study we investigated the anti-atherogenic potential of chronic rhPRG4 treatment. Female low-density lipoprotein receptor knockout mice were fed an atherogenic Western-type diet for 6 weeks and injected three times per week intraperitoneally with 0.5 mg rhPRG4 or PBS as control. Treatment with rhPRG4 was associated with a small decrease in plasma-free cholesterol levels, without a change in cholesteryl ester levels. A marked increase in the number of peritoneal foam cells was detected in response to the peritoneal rhPRG4 administration, which could be attributed to elevated peritoneal leukocyte MSR1 expression levels. However, rhPRG4-treated mice exhibited significantly smaller aortic root lesions of 278 ± 21 × 103 µm2 compared with 339 ± 15 × 103 µm2 in the aortic root of control mice. The overall decreased atherosclerosis susceptibility coincided with a shift in the monocyte and macrophage polarization states towards the patrolling and anti-inflammatory M2-like phenotypes, respectively. Furthermore, rhPRG4 treatment significantly reduced macrophage gene expression levels as well as plasma protein levels of the pro-inflammatory/pro-atherogenic cytokine TNF-alpha. In conclusion, we have shown that peritoneal administration and subsequent systemic exposure to rhPRG4 beneficially impacts the inflammatory state and reduces atherosclerosis susceptibility in mice. Our findings highlight that PRG4 is not only a lubricant but also acts as an anti-inflammatory agent. KEY POINTS: Endogenously produced proteoglycan 4 is found in atherosclerotic lesions and its genetic deficiency in mice is associated with enhanced atherosclerosis susceptibility. In this study we investigated the anti-atherogenic potential of chronic treatment with recombinant human PRG4 in hypercholesterolaemic female low-density lipoprotein receptor knockout mice. We show that recombinant human PRG4 stimulates macrophage foam cell formation, but also dampens the pro-inflammatory state of monocyte/macrophages, eventually leading to a significant reduction in plasma TNF-alpha levels and a lowered atherosclerosis susceptibility. Our findings highlight that peritoneal recombinant human PRG4 treatment can execute effects both locally and systemically and suggest that it will be of interest to study whether rhPRG4 treatment is also able to inhibit the progression and/or induce regression of previously established atherosclerotic lesions.

A high-cholesterol diet promotes the intravasation of breast tumor cells through an LDL-LDLR axis.

Most metastases in breast cancer occur via the dissemination of tumor cells through the bloodstream. How tumor cells enter the blood (intravasation) is, however, a poorly understood mechanism at the cellular and molecular levels. Particularly uncharacterized is how intravasation is affected by systemic nutrients. High levels of systemic LDL-cholesterol have been shown to contribute to breast cancer progression and metastasis in various models, but the cellular and molecular mechanisms involved are still undisclosed. Here we show that a high- cholesterol diet promotes intravasation in two mouse models of breast cancer and that this could be reverted by blocking LDL binding to LDLR in tumor cells. Moreover, we show that LDL promotes vascular invasion in vitro and the intercalation of tumor cells with endothelial cells, a phenotypic change resembling vascular mimicry (VM). At the molecular level, LDL increases the expression of SERPINE2, previously shown to be required for both VM and intravasation. Overall, our manuscript unravels novel mechanisms by which systemic hypercholesterolemia may affect the onset of metastatic breast cancer by favouring phenotypic changes in breast cancer cells and increasing intravasation.

The immune response-related genomic alterations in patients with malignant melanoma.

Immune checkpoint inhibitors (ICIs) significantly improve the survival outcomes of patients with advanced melanoma. However, response varies among from patient to patient and predictive biomarkers are urgently needed. We integrated mutational profiles from next-generation sequencing (NGS) data and clinicopathologic characteristics of melanoma patients to investigate whether tumor genomic profiling contribute to clinical benefit of ICIs treatment. The majority of genes identified with high mutation frequency have all been reported as well-known immunotherapy-related genes. Thirty-five patients (43.2%) had at least 1 BRAF/RAS/NF1 mutation. The other 46 (56.8%) melanomas without BRAF/RAS/NF1 mutation were classified as Triple-WT. We identified mutational signature 6 (known as associated with defective DNA mismatch repair) among cases in this cohort. Compared to patients with PD-L1 expression (TPS < 1%), patients with PD-L1 expression (TPS ≥ 1%) had significantly higher median progression-free survival (mPFS), but no significantly higher durable clinical benefit (DCB) rate. In contrast, FAT1, ATM, BRCA2, LRP1B, and PBRM1 mutations only occurred frequently in patients with DCB, irrespective of PD-L1 expression status. Our study explored molecular signatures of melanoma patients who respond to ICIs treatment and identified a series of mutated genes that might serve as predictive biomarker for ICIs responses in melanoma.

Dihydrostilbenes and flavonoids from whole plants of Jacobaea vulgaris.

The isolation of previously undescribed 12 compounds from the MeOH extract of Jacobaea vulgaris whole plants is disclosed, comprising 11 dihydrostilbenes (1-11) and one flavanone (12), and eight known compounds (six flavonoids, one dihydrostilbene, and one caffeoylquinic acid). Structural elucidation employed spectroscopic methods, including 1D and 2D NMR spectroscopy, HRESIMS, and ECD calculations. Evaluation of the compounds' effects on PCSK9 and LDLR mRNA expression revealed that compounds 1 and 3 downregulated PCSK9 mRNA while increasing LDLR mRNA expression, suggesting potential cholesterol-lowering properties.

MaiJiTong granule attenuates atherosclerosis by reducing ferroptosis via activating STAT6-mediated inhibition of DMT1 and SOCS1/p53 pathways in LDLR-/- mice.

BACKGROUND AND PURPOSE: Atherosclerosis is the primary pathological basis of cardiovascular disease. Ferroptosis is a regulated form of cell death, a process of lipid peroxidation driven by iron, which can initiate and promote atherosclerosis. STAT6 is a signal transducer that shows a potential role in regulating ferroptosis, but, the exact role in ferroptosis during atherogenesis remains unclear. The Traditional Chinese Medicine Maijitong granule (MJT) is used for treating cardiovascular disease and shows a potential inhibitory effect on ferroptosis. However, the antiatherogenic effect and the underlying mechanism remain unclear. In this study, we determined the role of STAT6 in ferroptosis during atherogenesis, investigated the antiatherogenic effect of MJT, and determined whether its antiatherogenic effect was dependent on the inhibition of ferroptosis. METHODS: 8-week-old male LDLR-/- mice were fed a high-fat diet (HFD) at 1st and 10th week, respectively, to assess the preventive and therapeutic effects of MJT on atherosclerosis and ferroptosis. Simultaneously, the anti-ferroptotic effects and mechanism of MJT were determined by evaluating the expression of genes responsible for lipid peroxidation and iron metabolism. Subsequently, we reanalyzed microarray data in the GSE28117 obtained from cells after STAT6 knockdown or overexpression and analyzed the correlation between STAT6 and ferroptosis. Finally, the STAT6-/- mice were fed HFD and injected with AAV-PCSK9 to validate the role of STAT6 in ferroptosis during atherogenesis and revealed the antiatherogenic and anti-ferroptotic effect of MJT. RESULTS: MJT attenuated atherosclerosis by reducing plaque lesion area and enhancing plaque stability in both preventive and therapeutic groups. MJT reduced inflammation via suppressing inflammatory cytokines and inhibited foam cell formation by lowering the LDL level and promoting ABCA1/G1-mediated lipid efflux. MJT ameliorated the ferroptosis by reducing lipid peroxidation and iron dysregulation during atherogenesis. Mechanistically, STAT6 negatively regulated ferroptosis by transcriptionally suppressing SOCS1/p53 and DMT1 pathways. MJT suppressed the DMT1 and SOCS1/p53 via stimulating STAT6 phosphorylation. In addition, STAT6 knockout exacerbated atherosclerosis and ferroptosis, which abolished the antiatherogenic and anti-ferroptotic effects of MJT. CONCLUSION: STAT6 acts as a negative regulator of ferroptosis and atherosclerosis via transcriptionally suppressing DMT1 and SOCS1 expression and MJT attenuates atherosclerosis and ferroptosis by activating the STAT6-mediated inhibition of DMT1 and SOCS1/p53 pathways, which indicated that STAT6 acts a novel promising therapeutic target to ameliorate atherosclerosis by inhibiting ferroptosis and MJT can serve as a new therapy for atherosclerosis treatment.

PCSK9 stimulates Syk, PKCδ, and NF-κB, leading to atherosclerosis progression independently of LDL receptor.

Proprotein convertase subtilisin/kexin type-9 (PCSK9) binds to and degrades low-density lipoprotein (LDL) receptor, leading to increase of LDL cholesterol in blood. Its blockers have emerged as promising therapeutics for cardiovascular diseases. Here we show that PCSK9 itself directly induces inflammation and aggravates atherosclerosis independently of the LDL receptor. PCSK9 exacerbates atherosclerosis in LDL receptor knockout mice. Adenylyl cyclase-associated protein 1 (CAP1) is the main binding partner of PCSK9 and indispensable for the inflammatory action of PCSK9, including induction of cytokines, Toll like receptor 4, and scavenger receptors, enhancing the uptake of oxidized LDL. We find spleen tyrosine kinase (Syk) and protein kinase C delta (PKCδ) to be the key mediators of inflammation after PCSK9-CAP1 binding. In human peripheral blood mononuclear cells, serum PCSK9 levels are positively correlated with Syk, PKCδ, and p65 phosphorylation. The CAP1-fragment crystallizable region (CAP1-Fc) mitigates PCSK9-mediated inflammatory signal transduction more than the PCSK9 blocking antibody evolocumab does.

High Prevalence of Familial Hypercholesterolemia Due to the Founder Effect of the LDLR c.2271del Variant in Communities of Oaxaca, Mexico.

INTRODUCTION: In Mexico, familial hypercholesterolemia (FH) is underdiagnosed, but population screening in small communities where at least one homozygous patient has already been detected results in a useful and inexpensive approach to reduce this problem. Considering that we previously reported nine homozygous cases from the state of Oaxaca, we decided to perform a population screening to identify patients with FH and to describe both their biochemical and genetic characteristics. METHODS: LDL cholesterol (LDLc) was quantified in 2,093 individuals from 11 communities in Oaxaca; either adults with LDLc levels ≥170 mg/dL or children with LDLc ≥130 mg/dL were classified as suggestive of FH and therefore included in the genetic study. LDLR and APOB (547bp fragment of exon 26) genes were screened by sequencing and MLPA analysis. RESULTS: Two hundred and five individuals had suggestive FH, with a mean LDLc of 223 ± 54 mg/dL (range: 131-383 mg/dL). Two pathogenic variants in the LDLR gene were detected in 149 individuals: c.-139_-130del (n = 1) and c.2271del (n = 148). All patients had a heterozygous genotype. With the cascade screening of their relatives (n = 177), 15 heterozygous individuals for the c.2271del variant were identified, presenting a mean LDLc of 133 ± 35 mg/dL (range: 60-168 mg/dL). CONCLUSIONS: The FH frequency in this study was 7.8% (164/2093), the highest reported worldwide. A founder effect combined with inbreeding could be responsible for the high percentage of patients with the LDLR c.2271del variant (99.4%), which allowed us to detect both significant biochemical heterogeneity and incomplete penetrance; hence, we assumed the presence of phenotype-modifying variants.

In vivo chronic exposure to inorganic mercury worsens hypercholesterolemia, oxidative stress and atherosclerosis in the LDL receptor knockout mice.

Heavy metal exposure leads to multiple system dysfunctions. The mechanisms are likely multifactorial and involve inflammation and oxidative stress. The aim of this study was to evaluate markers and risk factors for atherosclerosis in the LDL receptor knockout mouse model chronically exposed to inorganic mercury (Hg) in the drinking water. Results revealed that Hg exposed mice present increased plasma levels of cholesterol, without alterations in glucose. As a major source and target of oxidants, we evaluated mitochondrial function. We found that liver mitochondria from Hg treated mice show worse respiratory control, lower oxidative phosphorylation efficiency and increased H2O2 release. In addition, Hg induced mitochondrial membrane permeability transition. Erythrocytes from Hg treated mice showed a 50% reduction in their ability to take up oxygen, lower levels of reduced glutathione (GSH) and of antioxidant enzymes (SOD, catalase and GPx). The Hg treatment disturbed immune system cells counting and function. While lymphocytes were reduced, monocytes, eosinophils and neutrophils were increased. Peritoneal macrophages from Hg treated mice showed increased phagocytic activity. Hg exposed mice tissues present metal impregnation and parenchymal architecture alterations. In agreement, increased systemic markers of liver and kidney dysfunction were observed. Plasma, liver and kidney oxidative damage indicators (MDA and carbonyl) were increased while GSH and thiol groups were diminished by Hg exposure. Importantly, atherosclerotic lesion size in the aorta root of Hg exposed mice were larger than in controls. In conclusion, in vivo chronic exposure to Hg worsens the hypercholesterolemia, impairs mitochondrial bioenergetics and redox function, alters immune cells profile and function, causes several tissues oxidative damage and accelerates atherosclerosis development.

Deficiency of lncRNA MERRICAL abrogates macrophage chemotaxis and diabetes-associated atherosclerosis.

Diabetes-associated atherosclerosis involves excessive immune cell recruitment and plaque formation. However, the mechanisms remain poorly understood. Transcriptomic analysis of the aortic intima in Ldlr-/- mice on a high-fat, high-sucrose-containing (HFSC) diet identifies a macrophage-enriched nuclear long noncoding RNA (lncRNA), MERRICAL (macrophage-enriched lncRNA regulates inflammation, chemotaxis, and atherosclerosis). MERRICAL expression increases by 249% in intimal lesions during progression. lncRNA-mRNA pair genomic mapping reveals that MERRICAL positively correlates with the chemokines Ccl3 and Ccl4. MERRICAL-deficient macrophages exhibit lower Ccl3 and Ccl4 expression, chemotaxis, and inflammatory responses. Mechanistically, MERRICAL guides the WDR5-MLL1 complex to activate CCL3 and CCL4 transcription via H3K4me3 modification. MERRICAL deficiency in HFSC diet-fed Ldlr-/- mice reduces lesion formation by 74% in the aortic sinus and 86% in the descending aorta by inhibiting leukocyte recruitment into the aortic wall and pro-inflammatory responses. These findings unveil a regulatory mechanism whereby a macrophage-enriched lncRNA potently inhibits chemotactic responses, alleviating lesion progression in diabetes.