Angiopoietin-like 4 facilitates human aortic smooth muscle cell phenotype switch and dysfunctions through the PI3K/Akt signaling in aortic dissection.

PURPOSE: Vascular smooth muscle cell (VSMC) phenotype switch and dysfunctions have been reported to participate in aortic dissection (AD) progression. This study was aimed to investigate the role of angiopoietin-like 4 (ANGPTL4) in regulating VSMCs phenotype switch. MATERIALS AND METHODS: Key genes were analyzed in AD using public datasets, and it was found that the central differential gene ANGPTL4 was up-regulated in AD. The KEGG signaling pathway annotation was performed to validate the associated pathways, and the expression of ANGPTL4 was verified using multiple datasets and clinical samples. Furthermore, the specific functions of ANGPTL4 on platelet-derived growth factor-BB (PDGF-BB)-treated human aortic smooth muscle cell (HASMC) phenotypes were investigated. The dynamic effects of ANGPTL4 and core signaling antagonists on HASMC phenotypes were examined. RESULTS: Hub gene ANGPTL4 was significantly up-regulated in AD. ANGPTL4 was linked to the PI3K/Akt signaling, angiogenesis, and neovascularization and remodeling. ANGPTL4 overexpression further enhanced PDGF-BB effects on HASMC phenotypes, including promoted cell viability and migration, decreased contractile VSMC markers α-SMA and SM22α, elevated ECM degradation markers MMP-2 and MMP-9, and promoted phosphorylation of PI3K and Akt. ANGPTL4 knockdown partially abolished PDGF-BB-induced contractile/synthetic VSMCs imbalance and HASMC dysfunctions. Furthermore, in ANGPTL4-overexpressing HASMCs pre-treated with PDGF-BB, the PI3K/Akt signaling inhibitor LY294002 also partially eliminated the effects caused by the PDGF-BB treatment and ANGPTL4 overexpression. CONCLUSIONS: ANGPTL4 is significantly up-regulated in AD. ANGPTL4 overexpression further enhanced PDGF-BB effects on HASMC phenotype switch and dysfunctions, which might be involved in the PI3K/Akt signaling.

ANGPTL4-the Link Binding Lipid Metabolism and Inflammation.

BACKGROUND: Angiopoietin-like 4 (ANGPTL4) belongs to the family of angiopoietin- like proteins. The involvement of ANGPTL4 in various aspects of lipid metabolism and inflammation has become an important area of research. METHODS: A thorough search on PubMed related to ANGPTL4, lipid metabolism, and inflammation was performed. RESULTS: Over the past two decades, the recognition of ANGPTL4 as a potent regulator of lipid metabolism has substantially increased. As part of the senescence-associated secretory phenotype, ANGPTL4 also serves as an inflammatory mediator. Considering the advancements in ANGPTL4 research, we have highlighted that ANGPTL4 acts as a key node linking lipid metabolism and inflammation. ANGPTL4 impacts inflammation by regulating lipid metabolism. It affects critical enzymes (lipoprotein lipase, hepatic lipase, endothelial lipase, and acetyl-CoA carboxylase), regulatory factors (AMPK, cAMP, SLC7A11, GPX4, and mTOR), and receptors (LepR, CD36, and PPARγ) of lipid oxidation, synthesis, and peroxidation, thereby affecting immune cells and inflammatory pathways. CONCLUSION: Understanding the potential association and the therapeutic value of ANGPTL4 for regulating lipid metabolism and inflammation could contribute to drug discovery and therapeutic development.

Anoikis-related subtype and prognosis analyses based on bioinformatics, and an expression verification of ANGPTL4 based on experiments of lung adenocarcinoma.

Background: Lung adenocarcinoma (LUAD) is one of the most common malignant tumors with high mortality. Anoikis resistance is an important mechanism of tumor cell proliferation and migration. Our research is devoted to exploring the role of anoikis in the diagnosis, classification, and prognosis of LUAD. Methods: We downloaded the expression profile, mutation, and clinical data of LUAD from The Cancer Genome Atlas (TCGA) database. The "ConsensusClusterPlus" package was then used for the cluster analysis, and least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression analyses were used to establish the prognostic model. We verified the reliability of the model using a Gene Expression Omnibus (GEO) data set. A gene set variation analysis (GSVA) was conducted to investigate the functional enrichment differences in the different clusters and risk groups. The CIBERSORT algorithm and a single-sample gene set enrichment analysis (ssGSEA) were used to analyze immune cell infiltration. The tumor mutation burden (TMB) and Tumor Immune Dysfunction and Exclusion (TIDE) scores were used to evaluate the patients' sensitivity to immunotherapy. Immunohistochemical staining of tissue microarrays was used to verify the correlation between ANGPTL4 expression and the clinicopathological characteristics and prognosis of LUAD patients. Results: First, we screened 135 differentially expressed anoikis-related genes (ARGs) and 23 prognosis-related ARGs from TCGA-LUAD data set. Next, 494 LUAD samples were allocated to cluster A and cluster B based on the 23 prognosis-related ARGs. The Kaplan-Meier (K-M) analysis showed the overall survival (OS) of cluster B was better than that of cluster A. The clinicopathological characteristics and functional enrichment analyses revealed significant differences between clusters A and B. The tumor microenvironment (TME) analysis showed that cluster B had more immune cell infiltration and a higher TME score than cluster A. Subsequently, a LASSO Cox regression model of LUAD was constructed with ten ARGs. The K-M analysis showed that the low-risk patients had longer OS than the high-risk patients. The receiver operating characteristic curve, nomogram, and GEO data set verification results showed that the model had high accuracy and reliability. The level of immune cell infiltration and TME score were higher in the low-risk group than the high-risk group. The high-risk group had stronger sensitivity to immune checkpoint block therapy and weaker sensitivity to chemotherapy drugs than the low-risk group. ANGPTL4 expression was correlated with stage, tumor differentiation, tumor size, lymph node metastasis, and OS. Conclusions: We discovered novel molecular subtypes and constructed a novel prognostic model of LUAD. Our findings provide important insights into subtype classification and the accurate survival prediction of LUAD. We also identified ANGPTL4 as a prognostic indicator of LUAD.

Liver-targeted Angptl4 silencing by antisense oligonucleotide treatment attenuates hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice.

BACKGROUND AND AIMS: Angiopoietin-like 3 (ANGPTL3) and 4 (ANGPTL4) inhibit lipoprotein lipase to regulate tissue fatty acid uptake from triglyceride-rich lipoproteins such as VLDL. While pharmacological inhibition of ANGPTL3 is being evaluated as lipid-lowering strategy, systemic ANGPTL4 inhibition is not pursued due to adverse effects. This study aimed to compare the therapeutic potential of liver-specific Angptl3 and Angptl4 silencing to attenuate hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice, a well-established humanized model for lipoprotein metabolism. METHODS AND RESULTS: Mice were subcutaneously injected twice-weekly with saline or liver-targeted antisense oligonucleotides against Angptl3, Angptl4, both, or a scrambled oligonucleotide. Plasma lipid levels, VLDL clearance and hepatic VLDL production were determined, and atherosclerosis development was assessed. For toxicological evaluation, cynomolgus monkeys were treated with three dosages of liver-targeted ANGPTL4-silencing oligonucleotides.Liver-targeted Angptl4 silencing reduced plasma triglycerides (-48%) and total cholesterol (-56%), explained by higher VLDL-derived fatty acid uptake by brown adipose tissue and lower VLDL production by the liver. Accordingly, Angptl4 silencing reduced atherosclerotic lesion size (-86%) and improved lesion stability. Hepatic Angptl3 silencing similarly attenuated hyperlipidemia and atherosclerosis development. While Angptl3 and Angptl4 silencing lowered plasma triglycerides in the refed and fasted state, respectively, combined Angptl3/4 silencing lowered plasma triglycerides independent of nutritional state. In cynomolgus monkeys, anti-ANGPTL4 ASO treatment was well tolerated without adverse effects. CONCLUSIONS: Liver-targeted Angptl4 silencing potently attenuates hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice, and liver-targeted ANGPTL4 silencing is well-tolerated in non-human primates. These data warrant further clinical development of liver-targeted ANGPTL4 silencing.

Unveiling the bioinformatic genes and their involved regulatory mechanisms in type 2 diabetes combined with osteoarthritis.

Background: Type 2 Diabetes Mellitus (T2D) and Osteoarthritis (OA) are both prevalent diseases that significantly impact the health of patients. Increasing evidence suggests that there is a big correlation between T2D and OA, but the molecular mechanisms remain elusive. The aims of this study are to investigate the shared biomarkers and potential molecular mechanisms in T2D combined with OA. Methods: T2D and OA-related differentially expressed genes (DEGs) were identified via bioinformatic analysis on Gene Expression Omnibus (GEO) datasets GSE26168 and GSE114007 respectively. Subsequently, extensive target prediction and network analysis were finished with Gene Ontology (GO), protein-protein interaction (PPI), and pathway enrichment with DEGs. The transcription factors (TFs) and miRNAs coupled in co-expressed DEGs involved in T2D and OA were predicted as well. The key genes expressed both in the clinical tissues of T2D and OA were detected with western blot and qRT-PCR assay. Finally, the most promising candidate compounds were predicted with the Drug-Gene Interaction Database (DGIdb) and molecular docking. Results: In this study, 209 shared DEGs between T2D and OA were identified. Functional analysis disclosed that these DEGs are predominantly related to ossification, regulation of leukocyte migration, extracellular matrix (ECM) structural constituents, PI3K/AKT, and Wnt signaling pathways. Further analysis via Protein-Protein Interaction (PPI) analysis and validation with external datasets emphasized MMP9 and ANGPTL4 as crucial genes in both T2D and OA. Our findings were validated through qRT-PCR and Western blot analyses, which indicated high expression levels of these pivotal genes in T2D, OA, and T2D combined with OA cases. Additionally, the analysis of Transcription Factors (TFs)-miRNA interactions identified 7 TFs and one miRNA that jointly regulate these important genes. The Receiver Operating characteristic (ROC) analysis demonstrated the significant diagnostic potential of MMP9 and ANGPTL4.Moreover, we identified raloxifene, ezetimibe, and S-3304 as promising agents for patients with both T2D and OA. Conclusion: This study uncovers the shared signaling pathways, biomarkers, potential therapeutics, and diagnostic models for individuals suffering from both T2D and OA. These findings not only present novel perspectives on the complex interplay between T2D and OA but also hold significant promise for improving the clinical management and prognosis of patients with this concurrent condition.

Mir22hg facilitates ferritinophagy-mediated ferroptosis in sepsis by recruiting the m6A reader YTHDC1 and enhancing Angptl4 mRNA stability.

BACKGROUND: Ferritinophagy-mediated ferroptosis plays a crucial role in fighting pathogen aggression. The long non-coding RNA Mir22hg is involved in the regulation of ferroptosis and aberrantly overexpression in lipopolysaccharide (LPS)-induced sepsis mice, but whether it regulates sepsis through ferritinophagy-mediated ferroptosis is unclear. METHODS: Mir22hg was screened by bioinformatics analysis. Ferroptosis was assessed by assaying malondialdehyde (MDA), reactive oxygen species (ROS), and Fe2+ levels, glutathione (GSH) activity, as well as ferroptosis-related proteins GPX4 and SLC3A2 by using matched kits and performing western blot. Ferritinophagy was assessed by Lyso tracker staining and FerroOrange staining, immunofluorescence analysis of Ferritin and LC-3, and western blot analysis of LC-3II/I, p62, FTH1, and NCOA4. The bind of YTH domain containing 1 (YTHDC1) to Mir22hg or angiopoietin-like-4 (Angptl4) was verified by RNA pull-down and/or immunoprecipitation (RIP) assays. RESULTS: Mir22hg silencing lightened ferroptosis and ferritinophagy in LPS-induced MLE-12 cells and sepsis mouse models, as presented by the downregulated MDA, ROS, Fe2+, NCOA4, and SLC3A2 levels, upregulated GPX4, GSH, and FTH1 levels, along with a decrease in autophagy. Mir22hg could bind to the m6A reader YTHDC1 without affecting its expression. Mechanistically, Mir22hg enhanced Angptl4 mRNA stability through recruiting the m6A reader YTHDC1. Furthermore, Angptl4 overexpression partly overturned Mir22hg inhibition-mediated effects on ferroptosis and ferritinophagy in LPS-induced MLE-12 cells. CONCLUSION: Mir22hg contributed to in ferritinophagy-mediated ferroptosis in sepsis via recruiting the m6A reader YTHDC1 and strengthening Angptl4 mRNA stability, highlighting that Mir22hg may be a potential target for sepsis treatment based on ferroptosis.

ANGPTL4 Stabilizes Bone Morphogenetic Protein 7 Through Deubiquitination and Promotes HCC Proliferation via the SMAD/MAPK Pathway.

This study aimed to determine the function of angiopoietin-related protein 4 (ANGPTL4) and bone morphogenetic protein 7 (BMP7) on hepatocellular carcinoma (HCC). Overexpressing plasmids were cotransfected into HepG2 cells to determine the interaction between ANGPTL4 and BMP7. The effect of ANGPTL4 on the stability of BMP7 is examined by detecting the expression and ubiquitination levels. In vitro and in vivo experiments of knocking down ANGPTL4 while overexpressing BMP7 were performed to investigate whether the effects of ANGPTL4 on HCC proliferation, migration, and downstream signaling pathways were dependent on BMP7. ANGPTL4 is able to interact with BMP7, and knockdown of ANGPTL4 increased BMP7 expression and ubiquitination. Overexpression of BMP7 reversed the inhibition of HCC proliferation and migration as well as the decrease in the expression levels of Smad1/5/8 and MAPK14 caused by knockdown of ANGPTL4. ANGPTL4 promotes the proliferation and migration of HCC by inhibiting the ubiquitination degradation of BMP7 and the Smad/MAPK pathway, providing a novel mechanism and a potential therapeutic target for the treatment of HCC.

Enhancing chemosensitivity of PANC1 pancreatic cancer cells to gemcitabine using ANGTPL4, Notch1 and NF-κß1 siRNAs.

Aim: siRNA can silence targeted genes with lesser toxicity than therapeutic drugs. Therefore, this study aims to investigate new approaches to treat pancreatic cancer (PC) using combinations of siRNA and gemcitabine. Methods: Three genes, ANGTPL4, Notch1 and NF-κß1, were silenced using siRNA, and their anti-proliferative effects were studied in combination with gemcitabine on pancreatic cancer cell line (PANC-1) using MTT viability assay. Results: Our results showed a significant reduction in PANC-1 cells growth upon treating cells with gemcitabine and single and combinations of siRNA sequences specific for ANGTPL4, Notch1 and NF-κß1 genes. Conclusion: Co-transfection of gemcitabine-treated PANC-1 cells with ANGPTL4, Notch1 and NF-κßsiRNAs enhances the chemosensitivity of PANC-1 cells to gemcitabine can be a promising therapeutic approach.

Pancreatic cancer (PC) is prominent with its aggressive behavior and metastatic properties, making it one of the leading causes of cancer-related deaths worldwide. PC is associated with poor prognosis and low survival rate, with 5 years survival rate of less than 9%. Moreover, only 20% of PC patients could undergo surgery, which makes investigating new therapeutic approaches to treat PC necessary. In the current study, the chemosensitivity of pancreatic cancer cells to gemcitabine has been enhanced using a single and combination of ANGTPL4, Notch1 and NF-κß1 siRNA.

Angiopoietin-2 and Angiopoietin-like Proteins with a Prospective Role in Predicting Diabetic Nephropathy.

Angiopoietins are crucial growth factors for maintaining a healthy, functional endothelium. Patients with type 2 diabetes (T2D) exhibit significant levels of angiogenic markers, particularly Angiopoietin-2, which compromises endothelial integrity and is connected to symptoms of endothelial injury and failure. This report examines the levels of circulating angiopoietins in people with T2D and diabetic nephropathy (DN) and explores its link with ANGPTL proteins. We quantified circulating ANGPTL3, ANGPTL4, ANGPTL8, Ang1, and Ang2 in the fasting plasma of 117 Kuwaiti participants, of which 50 had T2D and 67 participants had DN. The Ang2 levels increased with DN (4.34 ± 0.32 ng/mL) compared with T2D (3.42 ± 0.29 ng/mL). This increase correlated with clinical parameters including the albumin-to-creatinine ratio (ACR) (r = 0.244, p = 0.047), eGFR (r = -0.282, p = 0.021), and SBP (r = -0.28, p = 0.024). Furthermore, Ang2 correlated positively to both ANGPTL4 (r = 0.541, p < 0.001) and ANGPTL8 (r = 0.41, p = 0.001). Multiple regression analysis presented elevated ANGPTL8 and ACRs as predictors for Ang2's increase in people with DN. In people with T2D, ANGPTL4 positively predicted an Ang2 increase. The area under the curve (AUC) in receiver operating characteristic (ROC) analysis of the combination of Ang2 and ANGPTL8 was 0.77 with 80.7% specificity. In conclusion, significantly elevated Ang2 in people with DN correlated with clinical markers such as the ACR, eGFR, and SBP, ANGPTL4, and ANGPTL8 levels. Collectively, this study highlights a close association between Ang2 and ANGPTL8 in a population with DN, suggesting them as DN risk predictors.

Identification of memory mechanism in tissue-resident stem cells via ANGPTL4 beyond immune cells upon viral antigen exposure.

Although memory functions of immune cells characterized by increased resistance to subsequent infections after initial pathogen exposure are well-established, it remains unclear whether non-immune cells, especially tissue-resident stem cells, exhibit similar memory mechanisms. The present study revealed that detrimental effects of initial viral antigen exposure (human papillomavirus [HPV]) on diverse stem cell functions were significantly exacerbated upon subsequent secondary exposure both in vitro and in vivo. Importantly, endometrial stem cells exhibited robust memory functions following consecutive HPV antigen exposures, whereas fully differentiated cells such as fibroblasts and vesicular cells did not show corresponding changes in response to the same antigen exposures. Deficiency of angiopoietin-like 4 (ANGPTL4) achieved through small hairpin RNA knockdown in vitro and knockout (KO) mice in vivo highlighted the critical role of ANGPTL4 in governing memory functions associated with various stem cell processes. This regulation occurred through histone H3 methylation alterations and PI3K/Akt signaling pathways in response to successive HPV antigen exposures. Furthermore, memory functions associated with various stem cell functions that were evident in wild-type mice following consecutive exposures to HPV antigen were not observed in ANGPTL4 KO mice. In summary, our findings strongly support the presence of memory mechanism in non-immune cells, particularly tissue-resident stem cells.

Renal lipid accumulation and aging linked to tubular cells injury via ANGPTL4.

Renal tubular epithelial cells are vulnerable to stress-induced damage, including excessive lipid accumulation and aging, with ANGPTL4 potentially playing a crucial bridging role between these factors. In this study, RNA-sequencing was used to identify a marked increase in ANGPTL4 expression in kidneys of diet-induced obese and aging mice. Overexpression and knockout of ANGPTL4 in renal tubular epithelial cells (HK-2) was used to investigate the underlying mechanism. Subsequently, ANGPTL4 expression in plasma and kidney tissues of normal young controls and elderly individuals was analyzed using ELISA and immunohistochemical techniques. RNA sequencing results showed that ANGPTL4 expression was significantly upregulated in the kidney tissue of diet-induced obesity and aging mice. In vitro experiments demonstrated that overexpression of ANGPTL4 in HK-2 cells led to increased lipid deposition and senescence. Conversely, the absence of ANGPTL4 appears to alleviate the impact of free fatty acids (FFA) on aging in HK-2 cells. Additionally, aging HK-2 cells exhibited elevated ANGPTL4 expression, and stress response markers associated with cell cycle arrest. Furthermore, our clinical evidence revealed dysregulation of ANGPTL4 expression in serum and kidney tissue samples obtained from elderly individuals compared to young subjects. Our study findings indicate a potential association between ANGPTL4 and age-related metabolic disorders, as well as injury to renal tubular epithelial cells. This suggests that targeting ANGPTL4 could be a viable strategy for the clinical treatment of renal aging.

Exploring Memory Function Beyond Immune Cells: ANGPTL4-Mediated Memory Functions in Tissue Resident Stem Cells.

Adapted immune cells are known to develop memory functions that increase resistance to subsequent infections after initial pathogen exposure, however, it is unclear whether non-immune cells, like tissue-resident stem cells, have similar memory functions. Here, it is found that tissue-resident stem cells crucial for tissue regeneration show diminished adverse effects on diverse stem cell functions against successive exposure to foreign antigen (ß-glucan) to maintain tissue homeostasis and stability both in vitro and in vivo. These data suggest that endometrial stem cells may possess a robust memory function, in contrast, fully differentiated cells like fibroblasts and vesicular cells do not show these memory mechanisms upon consecutive antigen exposure. Moreover, the pivotal role of Angiopoietin-like 4 (ANGPTL4) in regulating the memory functions of endometrial stem cells is identified through specific shRNA knockdown in vitro and knockout mice in vivo experiments. ANGPTL4 is associated with the alteration of diverse stem cell functions and epigenetic modifications, notably through histone H3 methylation changes and two pathways (i.e., PI3K/Akt and FAK/ERK1/2 signaling) upon consecutive antigen exposure. These findings imply the existence of inherent self-defense mechanisms through which local stem cells can adapt and protect themselves from recurrent antigenic challenges, ultimately mitigating adverse consequences.

Adipose-derived stem cells promote glycolysis and peritoneal metastasis via TGF-ß1/SMAD3/ANGPTL4 axis in colorectal cancer.

Peritoneal metastasis, the third most common metastasis in colorectal cancer (CRC), has a poor prognosis for the rapid progression and limited therapeutic strategy. However, the molecular characteristics and pathogenesis of CRC peritoneal metastasis are poorly understood. Here, we aimed to elucidate the action and mechanism of adipose-derived stem cells (ADSCs), a prominent component of the peritoneal microenvironment, in CRC peritoneal metastasis formation. Database analysis indicated that ADSCs infiltration was increased in CRC peritoneal metastases, and high expression levels of ADSCs marker genes predicted a poor prognosis. Then we investigated the effect of ADSCs on CRC cells in vitro and in vivo. The results revealed that CRC cells co-cultured with ADSCs exhibited stronger metastatic property and anoikis resistance, and ADSCs boosted the intraperitoneal seeding of CRC cells. Furthermore, RNA sequencing was carried out to identify the key target gene, angiopoietin like 4 (ANGPTL4), which was upregulated in CRC specimens, especially in peritoneal metastases. Mechanistically, TGF-ß1 secreted by ADSCs activated SMAD3 in CRC cells, and chromatin immunoprecipitation assay showed that SMAD3 facilitated ANGPTL4 transcription by directly binding to ANGPTL4 promoter. The ANGPTL4 upregulation was essential for ADSCs to promote glycolysis and anoikis resistance in CRC. Importantly, simultaneously targeting TGF-ß signaling and ANGPTL4 efficiently reduced intraperitoneal seeding in vivo. In conclusion, this study indicates that tumor-infiltrating ADSCs promote glycolysis and anoikis resistance in CRC cells and ultimately facilitate peritoneal metastasis via the TGF-ß1/SMAD3/ANGPTL4 axis. The dual-targeting of TGF-ß signaling and ANGPTL4 may be a feasible therapeutic strategy for CRC peritoneal metastasis.

A unified model for regulating lipoprotein lipase activity.

The regulation of triglyceride (TG) tissue distribution, storage, and utilization, a fundamental process of energy homeostasis, critically depends on lipoprotein lipase (LPL). We review the intricate mechanisms by which LPL activity is regulated by angiopoietin-like proteins (ANGPTL3, 4, 8), apolipoproteins (APOA5, APOC3, APOC2), and the cAMP-responsive element-binding protein H (CREBH). ANGPTL8 functions as a molecular switch, through complex formation, activating ANGPTL3 while deactivating ANGPTL4 in their LPL inhibition. The ANGPTL3-4-8 model integrates the roles of the aforementioned proteins in TG partitioning between white adipose tissue (WAT) and oxidative tissues (heart and skeletal muscles) during the feed/fast cycle. This model offers a unified perspective on LPL regulation, providing insights into TG metabolism, metabolic diseases, and therapeutics.

Correlation between the serum FABP4, ANGPTL3, and ANGPTL4 levels and coronary artery disease.

BACKGROUND: Lipid metabolism related factors, such as angiopoietin-like protein 3 (ANGPTL3), angiopoietin-like 4 (ANGPTL4), fatty acid-binding protein 4 (FABP4) are newly discovered factors that can affect coronary artery disease (CAD). In this study, we aimed to investigate the relationship between CAD and these lipid metabolism factors. HYPOTHESIS: ANGPTL3, ANGPTL4, and FABP4 may provide a new method for the control of CAD risk factors and the prevention and treatment of CAD. METHODS: We enrolled 284 consecutive inpatients with suspected CAD and divided them into CAD and non-CAD groups based on the coronary angiography results. Serum ANGPTL3, ANGPTL4, FABP4, and tumor necrosis factor-α (TNF-α) levels were estimated using the enzyme-linked immunosorbent assay. Multivariate logistic regression was used to assess the risk factors for CAD. The receiver operating characteristic curve was used to determine the cutoff and diagnostic values. RESULTS: The serum TNF-α, FABP4, ANGPTL3, and ANGPTL4 values showed a significant difference between the CAD and non-CAD groups (p < .05). After adjusting for confounding factors, the FABP4, ANGPTL3, and ANGPTL4 levels were independently associated with CAD (p < .05). The ANGPTL3 expression level was an independent risk factor for CAD in patients with hypertension, but not in those without hypertension. The ANGPTL3 > 67.53 ng/mL, ANGPTL4 > 29.95 ng/mL, and FABP4 > 1421.25 ng/L combination had the highest diagnostic value for CAD. CONCLUSION: ANGPTL3, ANGPTL4, and FABP4 were identified as independent risk factors for CAD and have valuable clinical implications for the diagnosis and treatment of CAD.

Characterization of sexual dimorphism in ANGPTL4 levels and function.

ANGPTL4 is an attractive pharmacological target for lowering plasma triglycerides and cardiovascular risk. Since most preclinical studies on ANGPTL4 were performed in male mice, little is known about sexual dimorphism in ANGPTL4 regulation and function. Here, we aimed to study potential sexual dimorphism in ANGPTL4 mRNA and protein levels and ANGPTL4 function. Additionally, we performed exploratory studies on the function of ANGPTL4 in the liver during fasting using Angptl4-transgenic and Angptl4-/- mice. Compared to female mice, male mice showed higher hepatic and adipose ANGPTL4 mRNA and protein levels, as well as a more pronounced effect of genetic ANGPTL4 modulation on plasma lipids. By contrast, very limited sexual dimorphism in ANGPTL4 levels was observed in human liver and adipose tissue. In human and mouse adipose tissue, ANGPTL8 mRNA and/or protein levels were significantly higher in females than males. Adipose LPL protein levels were higher in female than male Angptl4-/- mice, which was abolished by ANGPTL4 (over) expression. At the human genetic level, the ANGPTL4 E40K loss-of-function variant was associated with similar plasma triglyceride reductions in women and men. Finally, ANGPTL4 ablation in fasted mice was associated with changes in hepatic gene expression consistent with PPARα activation. In conclusion, the levels of ANGPTL4 and the magnitude of the effect of ANGPTL4 on plasma lipids exhibit sexual dimorphism. Nonetheless, inactivation of ANGPTL4 should confer a similar metabolic benefit in women and men. Expression levels of ANGPTL8 in human and mouse adipose tissue are highly sexually dimorphic, showing higher levels in females than males.

ANGPTL4 regulates ovarian cancer progression by activating the ERK1/2 pathway.

BACKGROUND: Ovarian cancer (OC) has the highest mortality rate among all gynecological malignancies. A hypoxic microenvironment is a common feature of solid tumors, including ovarian cancer, and an important driving factor of tumor cell survival and chemo- and radiotherapy resistance. Previous research identified the hypoxia-associated gene angiopoietin-like 4 (ANGPTL4) as both a pro-angiogenic and pro-metastatic factor in tumors. Hence, this work aimed to further elucidate the contribution of ANGPTL4 to OC progression. METHODS: The expression of hypoxia-associated ANGPTL4 in human ovarian cancer was examined by bioinformatics analysis of TCGA and GEO datasets. The CIBERSORT tool was used to analyze the distribution of tumor-infiltrating immune cells in ovarian cancer cases in TCGA. The effect of ANGPTL4 silencing and overexpression on the proliferation and migration of OVCAR3 and A2780 OC cells was studied in vitro, using CCK-8, colony formation, and Transwell assays, and in vivo, through subcutaneous tumorigenesis assays in nude mice. GO enrichment analysis and WGCNA were performed to explore biological processes and genetic networks associated with ANGPTL4. The results obtained were corroborated in OC cells in vitro by western blotting. RESULTS: Screening of hypoxia-associated genes in OC-related TCGA and GEO datasets revealed a significant negative association between ANGPTL4 expression and patient survival. Based on CIBERSORT analysis, differential representation of 14 distinct tumor-infiltrating immune cell types was detected between low- and high-risk patient groups. Silencing of ANGPTL4 inhibited OVCAR3 and A2780 cell proliferation and migration in vitro and reduced the growth rate of xenografted OVCAR3 cells in vivo. Based on results from WGCNA and previous studies, western blot assays in cultured OC cells demonstrated that ANGPTL4 activates the Extracellular signal-related kinases 1 and 2 (ERK1/2) pathway and this results in upregulation of c-Myc, Cyclin D1, and MMP2 expression. Suggesting that the above mechanism mediates the pro-oncogenic actions of ANGPTL4T in OC, the pro-survival effects of ANGPTL4 were largely abolished upon inhibition of ERK1/2 signaling with PD98059. CONCLUSIONS: Our work suggests that the hypoxia-associated gene ANGPTL4 stimulates OC progression through activation of the ERK1/2 pathway. These findings may offer a new prospect for targeted therapies for the treatment of OC.

Single-cell transcriptomic profiling of heart reveals ANGPTL4 linking fibroblasts and angiogenesis in heart failure with preserved ejection fraction.

INTRODUCTION: Despite the high morbidity and mortality, the effective therapies for heart failure with preserved fraction (HFpEF) are limited as the poor understand of its pathophysiological basis. OBJECTIVE: This study was aimed to characterize the cellular heterogeneity and potential mechanisms of HFpEF at single-cell resolution. METHODS: An HFpEF mouse model was induced by a high-fat diet with N-nitro-L-arginine methyl ester. Cells from the hearts were subjected to single-cell sequencing. The key protein expression was measured with Immunohistochemistry and immunofluorescence staining. RESULTS: In HFpEF hearts, myocardial fibroblasts exhibited higher levels of fibrosis. Furthermore, an increased number of fibroblasts differentiated into high-metabolism and high-fibrosis phenotypes. The expression levels of genes encoding certain pro-angiogenic secreted proteins were decreased in the HFpEF group, as confirmed by bulk RNA sequencing. Additionally, the proportion of the endothelial cell (EC) lineages in the HFpEF group was significantly downregulated, with low angiogenesis and high apoptosis phenotypes observed in these EC lineages. Interestingly, the fibroblasts in the HFpEF heart might cross-link with the EC lineages via over-secretion of ANGPTL4, thus displaying an anti-angiogenic function. Immunohistochemistry and immunofluorescence staining then revealed the downregulation of vascular density and upregulation of ANGPTL4 expression in HFpEF hearts. Finally, we predicted ANGPTL4as a potential druggable target using DrugnomeAI. CONCLUSION: In conclusion, this study comprehensively characterized the angiogenesis impairment in HFpEF hearts at single-cell resolution and proposed that ANGPTL4 secretion by fibroblasts may be a potential mechanism underlying this angiogenic abnormality.

Construction of an Anoikis-Related Gene Prognostic Signature and Identification of ANGPTL4 as a Key Oncogene in Lung Adenocarcinoma.

Anoikis plays an important role in cancer invasion and metastasis. However, the role of anoikis-related genes, AnRGs, in lung adenocarcinoma (LUAD) is not clear. First, anoikis-related genes (AnRGs) were obtained from the Genecard database. Second, the prognostic risk model of AnRGs was established by univariate Cox analysis, the Least Absolute Shrinkage and Selection Operator (LASSO) analysis, and multivariate Cox analysis. Finally, in vitro cell experiments were carried out to determine the expression and function of the key gene AnRGs. Three AnRGs (angiopoietin-like 4, ANGPTL4; Cyclin-Dependent Kinase Inhibitor 3, CDKN3; Solute Carrier Organic Anion Transporter Family Member 1B3, SLCO1B3) were screened for the construction of risk prediction model. Additionally, ANGPTL4 was significantly highly expressed in tumor cells, and the knockdown of ANGPTL4 expression on tumor cells could inhibit tumor cell migration and apoptosis. Constructing a risk model based on anoikis-related genes can effectively differentiate the prognosis of LUAD. ANGPTL4 can be used as a potential new target for LUAD treatment.

Knockdown of ANGPTL4 inhibits adipogenesis of preadipocyte via autophagy.

It has been demonstrated that angiopoietin-like protein 4 (ANGPTL4) plays an important regulatory role in lipid metabolism and backfat deposition appears to vary in different pig breeds. However, the correlation between ANGPTL4 and backfat deposition have not been well characterized and the role of ANGPTL4 in regulating adipogenesis remains unclear. Therefore, this study aimed to investigate correlation between ANGPTL4 and backfat deposition and to explore the effects of ANGPTL4 on preadipocyte differentiation and the underlying mechanism. Our results showed that the backfat thickness and the ANGPTL4 gene expression of Laiwu pigs were significantly higher than those in DLY pigs and the ANGPTL4 gene expression was positively correlated with backfat thickness both in DLY pigs and Laiwu pigs. Moreover, an increase in ANGPTL4 expression and activation of autophagy were observed during the differentiation of stromal vascular fraction cells. In addition, knockdown of ANGPTL4 inhibited the differentiation of 3T3-L1 cells with decreased expression of LC3-II and ATG5 and increased expression of SQSTM1, suggesting the involvement of autophagy in ANGPTL4-mediated adipogenesis. In conclusion, these results suggested that ANGPTL4 is positively correlated with backfat deposition in pigs and knockdown of ANGPTL4 inhibits adipogenesis of preadipocyte via autophagy, providing new insights into the regulation of fat deposition and to improve the carcass quality and meat quality of porcine.