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Neovascular age-related macular degeneration (nvAMD) is the leading cause of severe vision loss in the elderly in the developed world. While the introduction of therapies targeting vascular endothelial growth factor (VEGF) has provided the first opportunity to significantly improve vision in patients with nvAMD, many patients respond inadequately to current anti-VEGF therapies. It was recently demonstrated that expression of a second angiogenic mediator, angiopoietin-like 4 (ANGPTL4), synergizes with VEGF to promote choroidal neovascularization (CNV) in mice and correlates with reduced response to anti-VEGF therapy in patients with nvAMD. Here, we report that expression of ANGPTL4 in patients with nvAMD increases following treatment with anti-VEGF therapy and that this increase is dependent on accumulation of hypoxia-inducible factor (HIF)-1α in response to inhibition of VEGF/KDR signaling in the retinal pigment epithelium (RPE). We therefore explored HIF-1 inhibition with 32-134D, a recently developed pharmacologic HIF-inhibitor, for the treatment of nvAMD. 32-134D prevented the expression of both VEGF and ANGPTL4 and was at least as effective as aflibercept in treating CNV in mice. Moreover, by preventing the increase in HIF-1α accumulation in the RPE in response to anti-VEGF therapy, combining 32-134D with aflibercept was more effective than either drug alone for the treatment of CNV. Collectively, these results help explain why many patients with nvAMD respond inadequately to anti-VEGF therapy and suggest that the HIF inhibitor 32-134D will be an effective drug-alone or in combination with current anti-VEGF therapies-for the treatment of patients with this blinding disease.

Dengue virus infection can cause severe complications due to vascular leakage. Angiopoietin-like protein 4 (ANGPTL4) regulates vascular permeability, but its role in dengue pathogenesis is unclear. This study investigated the association between plasma ANGPTL4 levels and dengue severity in Singapore adults. Plasma samples from 48 dengue patients (24 severe and 24 non-severe) during acute and convalescent phases were selected from the prospective COhort study on progression of DENgue severity in Singapore adults (CODEN) cohort. The CODEN was conducted at the National Centre for Infectious Diseases, Tan Tock Seng Hospital, from June 2016 to January 2020. ANGPTL4 levels were measured and compared to 152 healthy controls. Logistic regression assessed the relationship between plasma ANGPTL4 concentrations and disease severity. There were no statistically significant differences in ANGPTL4 levels between severe and non-severe dengue patients during acute (677.4 vs. 909.1 pg/mL, p = 0.4) or convalescent phases (793.7 vs. 565.6 pg/mL, p = 0.96). Plasma ANGPTL4 levels were significantly elevated during acute dengue (4634.3 pg/mL) versus healthy controls (907.4 pg/mL), declining during convalescence. Compared to the lowest tertile, the adjusted odds ratios for severe dengue were 0.36 (95%CI: 0.08–1.65, p = 0.190) for medium tertile and 0.57 (95%CI: 0.13–2.49, p = 0.456) for high tertile. Among patients with high ANGPTL4 levels (>5000 pg/mL), 36.4% developed severe complications, including significant plasma leakage. Plasma ANGPTL4 levels were significantly higher in dengue patients than controls, suggesting its potential as a biomarker, which warrants future detailed investigations. Larger prospective studies with serial sampling, including pediatric populations, may clarify the role of ANGPTL4 in severe dengue. [ABSTRACT FROM AUTHOR]

Abstract Aims/Introduction The association between serum angiopoietin‐like 4 (ANGPTL4) levels and the severity of diabetic kidney disease (DKD) in patients with type 2 diabetes mellitus remains unclear. Methods A total of 1,115 type 2 diabetes mellitus patients were analyzed in this cross‐sectional study. DKD index included DKD stages defined by estimated glomerular filtration rate, the albuminuria grades and DKD risk management grades. Serum levels of ANGPTL4 and other biomarkers were detected. Multivariable‐adjusted linear and logistic analyses were used to study the association between ANGPTL4 and DKD. The protein levels of ANGPTL4 were assessed in the kidney. Renal tubular cells were stimulated with glucose to study ANGPTL4 expression. Results Compared with the participants in the third or fourth quantile of ANGPTL4, those in the first or second quantile of ANGPTL4 were younger, with lower glycated hemoglobin, triglycerides and urinary albumin creatinine ratio (all P

Inflammation, fibrosis and metabolic stress critically promote heart failure with preserved ejection fraction (HFpEF). Exposure to high-fat diet and nitric oxide synthase inhibitor N[w]-nitro-l-arginine methyl ester (L-NAME) recapitulate features of HFpEF in mice. To identify disease-specific traits during adverse remodeling, we profiled interstitial cells in early murine HFpEF using single-cell RNAseq (scRNAseq). Diastolic dysfunction and perivascular fibrosis were accompanied by an activation of cardiac fibroblast and macrophage subsets. Integration of fibroblasts from HFpEF with two murine models for heart failure with reduced ejection fraction (HFrEF) identified a catalog of conserved fibroblast phenotypes across mouse models. Moreover, HFpEF-specific characteristics included induced metabolic, hypoxic and inflammatory transcription factors and pathways, including enhanced expression of Angiopoietin-like 4 (Angptl4) next to basement membrane compounds, such as collagen IV (Col4a1). Fibroblast activation was further dissected into transcriptional and compositional shifts and thereby highly responsive cell states for each HF model were identified. In contrast to HFrEF, where myofibroblast and matrifibrocyte activation were crucial features, we found that these cell states played a subsidiary role in early HFpEF. These disease-specific fibroblast signatures were corroborated in human myocardial bulk transcriptomes. Furthermore, we identified a potential cross-talk between macrophages and fibroblasts via SPP1 and TNFɑ with estimated fibroblast target genes including Col4a1 and Angptl4. Treatment with recombinant ANGPTL4 ameliorated the murine HFpEF phenotype and diastolic dysfunction by reducing collagen IV deposition from fibroblasts in vivo and in vitro. In line, ANGPTL4, was elevated in plasma samples of HFpEF patients and particularly high levels associated with a preserved global-longitudinal strain. Taken together, our study provides a comprehensive characterization of molecular fibroblast activation patterns in murine HFpEF, as well as the identification of Angiopoietin-like 4 as central mechanistic regulator with protective effects. [ABSTRACT FROM AUTHOR]

Aims/Introduction: The association between serum angiopoietin‐like 4 (ANGPTL4) levels and the severity of diabetic kidney disease (DKD) in patients with type 2 diabetes mellitus remains unclear. Methods: A total of 1,115 type 2 diabetes mellitus patients were analyzed in this cross‐sectional study. DKD index included DKD stages defined by estimated glomerular filtration rate, the albuminuria grades and DKD risk management grades. Serum levels of ANGPTL4 and other biomarkers were detected. Multivariable‐adjusted linear and logistic analyses were used to study the association between ANGPTL4 and DKD. The protein levels of ANGPTL4 were assessed in the kidney. Renal tubular cells were stimulated with glucose to study ANGPTL4 expression. Results: Compared with the participants in the third or fourth quantile of ANGPTL4, those in the first or second quantile of ANGPTL4 were younger, with lower glycated hemoglobin, triglycerides and urinary albumin creatinine ratio (all P < 0.05). There was a negative nonlinear relationship between ANGPTL4 and estimated glomerular filtration rate in type 2 diabetes mellitus patients. One standard deviation increased serum ANGPTL4 levels, the odds ratio of having DKD was 1.40 (95% confidence interval 1.08–1.80). The mediation analysis showed that triglycerides did not mediate the association between ANGPTL4 and DKD. Furthermore, ANGPTL4 could be the strongest among multiple panels of biomarkers in its association of DKD. Compared with mice at 8 weeks‐of‐age, db/db mice at 18 weeks‐of‐age had increased ANGPTL4 expression in glomeruli and tubular segments. In vitro, glucose could stimulate ANGPTL4 expression in tubular cells in a dose‐dependent manner. Conclusions: ANGPTL4 could be a potential marker and therapeutic target for DKD treatment. [ABSTRACT FROM AUTHOR]

Objective To explore the regulatory and functional mechanism of angiopoietin-like 4 (ANGPTL4) expression in gastric epithelial cells infected with Helicobacter pylori (H.pylori). Methods H.pylori-positive gastric specimens from Department of Gastroenterology of second Affiliated Hospital of Army Medical University in November 2021 and the gastric tissues of H.pylori-infected mice were collected, and the expression locations of ANGPTL4 in these gastric tissues were detected by immunofluorescence assay. After gastric epithelial cells and gastric organoids were infected with H.pylori, the expression of ANGPTL4 was detected by real-time PCR and Western blotting. The regulatory mechanism of H.pylori-induced ANGPTL4 expression was investigated by cellular models above. Recombinant ANGPTL4 was used to stimulate gastric epithelial cells to investigate the effect of ANGPTL4 on the claudin 1 (CLDN1) expression. Results H.pylori infection significantly increased the expression of ANGPTL4 in gastric epithelial cells in a CagA-dependent, bacterial concentration-dependent and infection time-dependent manner (P < 0.05). Compared to uninfected cells and CagA knockout (△cagA) H.pylori-induced ones, the ANGPTL4 expression was significantly increased in WT H.pylori-infected human and mouse gastric organoid (P < 0.05). Blocking NF-κB signaling pathway significantly inhibited the enhanced ANGPTL4 expression in H.pylori-infected gastric epithelial cells (P < 0.05). Recombinant ANGPTL4 activated ERK signaling pathway to down-regulate the CLDN1 expression in gastric epithelial cells (P < 0.05). Conclusion H.pylori activates NF-κB signaling pathway to up-regulate ANGPTL4 expression in gastric epithelial cells in a CagA-dependent manner, and ANGPTL4 activates ERK signaling pathway to down-regulate the CLDN1 expression in gastric epithelial cells, which may contributes to the development of gastric diseases induced by H.pylori infection.

High fat diets overwhelm the physiological mechanisms for absorption, storage, and utilization of triglycerides (TG); consequently TG, TG‐rich lipoproteins (TGRL), and TGRL remnants accumulate, circulate systemically, producing dyslipidemia. This associates with, or is causative for increased atherosclerotic cardiovascular risk, ischemic stroke, fatty liver disease, and pancreatitis. TGRL hydrolysis by endothelial surface‐bound lipoprotein lipase (LPL) generates metabolites like free fatty acids which have proinflammatory properties. While osteoblasts utilize fatty acids as an energy source, dyslipidemia is associated with negative effects on the skeleton. In this study we investigated the effects of TGRL lipolysis products (TGRL‐LP) on expression of a stress responsive transcription factor, termed activating transcription factor 3 (ATF3), reactive oxygen species (ROS), ATF3 target genes, and angiopoietin‐like 4 (Angptl4) in osteoblasts. As ATF3 negatively associates with osteoblast differentiation, we also investigated the skeletal effects of global ATF3 deletion in mice. TGRL‐LP increased expression of Atf3, proinflammatory proteins Ptgs2 and IL‐6, and induced ROS in MC3T3‐E1 osteoblastic cells. Angptl4 is an endogenous inhibitor of LPL which was transcriptionally induced by TGRL‐LP, while recombinant Angptl4 prevented TG‐driven Atf3 induction. Atf3 global knockout male mice demonstrated increased trabecular and cortical microarchitectural parameters. In summary, we find that TGRL‐LP induce osteoblastic cell stress as evidenced by expression of ATF3, which may contribute to the negative impact of dyslipidemia in the skeleton. Further, concomitant induction of Angptl4 in osteoblasts might play a protective role by reducing local lipolysis. [ABSTRACT FROM AUTHOR]

To investigate the functional differences of angiopoietin-related protein 4 (ANGPTL4) transcripts in hepatocellular carcinoma (HCC) cells. By transfecting ANGPTL4-Transcript 1 and ANGPTL4-Transcript 3 overexpression vectors into HepG2 and Huh7 cell lines with ANGPTL4 knockdown, the effects of overexpression of two transcripts on cell viability, invasion, migration, and apoptosis were analyzed. The expression of two transcripts was compared in human liver cancer tissue, and their effects on tumor development were validated in vivo experiments in mice. Compared with control, the overexpression of ANGPTL4-Transcript 1 had no significant effect on viability, invasion, healing, and apoptosis of HepG2 and Huh7 cells. However, these two cell lines overexpressing ANGPTL4-Transcript 3 showed remarkably enhanced cell viability, invasive and healing ability, and decreased apoptosis ability. Furthermore, the mRNA level of ANGPTL4-Transcript 3 was significantly increased in human HCC tissues and promoted tumor growth compared with Transcript 1. Different transcripts of gene ANGPTL4 have distinct effects on HCC. The abnormally elevated Transcript 3 with the specific ability of promoting HCC proliferation, infiltration, and migration is expected to become a new biological marker and more precise intervention target for HCC. [ABSTRACT FROM AUTHOR]

The infarcted heart undergoes irreversible pathological remodeling after reperfusion involving left ventricle dilation and excessive inflammatory reactions in the infarcted heart, frequently leading to fatal functional damage. Extensive attempts have been made to attenuate pathological remodeling in infarcted hearts using cardiac patches and anti-inflammatory drug delivery. In this study, we developed a paintable and adhesive hydrogel patch using dextran-aldehyde (dex-ald) and gelatin, incorporating the anti-inflammatory protein, ANGPTL4, into the hydrogel for sustained release directly to the infarcted heart to alleviate inflammation. We optimized the material composition, including polymer concentration and molecular weight, to achieve a paintable, adhesive hydrogel using 10% gelatin and 5% dex-ald, which displayed in-situ gel formation within 135 s, cardiac tissue-like modulus (40.5 kPa), suitable tissue adhesiveness (4.3 kPa), and excellent mechanical stability. ANGPTL4 was continuously released from the gelatin/dex-ald hydrogel without substantial burst release. The gelatin/dex-ald hydrogel could be conveniently painted onto the beating heart and degraded in vivo. Moreover, in vivo studies using animal models of acute myocardial infarction revealed that our hydrogel cardiac patch containing ANGPTL4 significantly improved heart tissue repair, evaluated by echocardiography and histological evaluation. The heart tissues treated with ANGPTL4-loaded hydrogel patches exhibited increased vascularization, reduced inflammatory macrophages, and structural maturation of cardiac cells. Our novel hydrogel system, which allows for facile paintability, appropriate tissue adhesiveness, and sustained release of anti-inflammatory drugs, will serve as an effective platform for the repair of various tissues, including heart, muscle, and cartilage.

Background and objective: The objective of this study was to investigate the potential of salidroside (SAL) (a major active compound in Rhodiola rosea L.) in regulating osteoclast differentiation and function by modulating the HIF-1 α pathway and its downstream target genes. Methods: The expression of HIF-1 α and its downstream target genes was examined at both mRNA and protein levels in osteoclasts treated with SAL. Immunofluorescence analysis was performed to assess the nuclear translocation and transcriptional activity of HIF-1 α in response to SAL. MTT, flow cytometry, qPCR, TRAP staining and bone resorption assays were used to evaluate the potential effect of salidroside on osteoclasts. Results: SAL enhanced the expression of HIF-1 α and its downstream target genes in osteoclasts. Immunofluorescence analysis confirmed the facilitation of HIF-1 α nuclear translocation and transcriptional activity by SAL. In addition, SAL enhanced osteoclast viability, differentiation and bone resorption activity in an autocrine manner through HIF-1 α /VEGF, IL-6 and ANGPTL4 pathways. Conclusion: SAL promotes osteoclast proliferation, differentiation and bone resorption through HIF-1 α /VEGF, IL-6 and ANGPTL4 pathways. [ABSTRACT FROM AUTHOR]

Proliferation and migration of epidermal stem cells (EpSCs) are essential for epithelialization during skin wound healing. Angiopoietin-like 4 (ANGPTL4) has been reported to play an important role in wound healing, but the mechanisms involved are not fully understood. Here, we investigate the contribution of ANGPTL4 to full-thickness wound re-epithelialization and the underlying mechanisms using Angptl4-knockout mice. Immunohistochemical staining reveals that ANGPTL4 is significantly upregulated in the basal layer cells of the epidermis around the wound during cutaneous wound healing. ANGPTL4 deficiency impairs wound healing. H&E staining shows that ANGPTL4 deficiency significantly reduces the thickness, length and area of the regenerated epidermis postwounding. Immunohistochemical staining for markers of EpSCs (α6 integrin and β1 integrin) and cell proliferation (PCNA) shows that the number and proliferation of EpSCs in the basal layer of the epidermis are reduced in ANGPTL4-deficient mice. In vitro studies show that ANGPTL4 deficiency impedes EpSC proliferation, causes cell cycle arrest at the G1 phase and reduces the expressions of cyclins D1 and A2, which can be reversed by ANGPTL4 overexpression. ANGPTL4 deletion suppresses EpSC migration, which is also rescued by ANGPTL4 overexpression. Overexpression of ANGPTL4 in EpSCs accelerates cell proliferation and migration. Collectively, our results indicate that ANGPTL4 promotes EpSC proliferation by upregulating cyclins D1 and A2 expressions and accelerating the cell cycle transition from G1 to S phase and that ANGPTL4 promotes skin wound re-epithelialization by stimulating EpSC proliferation and migration. Our study reveals a novel mechanism underlying EpSC activation and re-epithelialization during cutaneous wound healing.

Angiopoietin-like 4 (ANGPTL4) is a potent regulator of TAG metabolism, but knowledge of the mechanisms underlying ANGPTL4 transcription in response to fatty acids is still limited in teleost. In the current study, we explored the molecular characterisation of ANGPTL4 and regulatory mechanisms of ANGPTL4 in response to fatty acids in large yellow croaker (Larimichthys crocea). Here, croaker angptl4 contained a 1416 bp open reading frame encoding a protein of 471 amino acids with highly conserved 12-amino acid consensus motif. Angptl4 was widely expressed in croaker, with the highest expression in the liver. In vitro , oleic and palmitic acids (OA and PA) treatments strongly increased angptl4 mRNA expression in croaker hepatocytes. Moreover, angptl4 expression was positively regulated by PPAR family (PPAR- α , β and γ), and expression of PPAR γ was also significantly increased in response to OA and PA. Moreover, inhibition of PPAR γ abrogated OA- or PA-induced angptl4 mRNA expression. Beyond that, PA might increase angptl4 expression partly via the insulin signalling. Overall, the expression of ANGPTL4 is strongly upregulated by OA and PA via PPAR γ in the liver of croaker, which contributes to improve the understanding of the regulatory mechanisms of ANGPTL4 in fish. [ABSTRACT FROM AUTHOR]

Neonatal respiratory distress syndrome (NRDS) is the most common respiratory disease in preterm infants (PIs). The implication of Angiopoietin-like 4 (ANGPTL4) was reported in lung diseases. We delved into the role of serum ANGPTL4 in NRDS diagnosis/prognosis.Totally 256 PIs were prospectively selected, including 128 NRDS infants and 128 non-NRDS PIs. NRDS infants were assigned into Survival and Death groups. ANGPTL4 level in PIs and its diagnostic and prognostic value for NRDS were separately assessed by ELISA and receiver operating characteristic curve. The independent risk factors (IRFs) for death in NRDS infants were analysed by multivariate logistic regression.NRDS infants exhibited reduced gestational age, birth weight, and 5 min Apgar score. ANGPTL4 level rose in NRDS infants, and increased with NRDS severity. Serum ANGPTL4 level was negatively correlated with 5 min Apgar score in NRDS infants. The area under the curve of serum ANGPTL4 for the diagnosis of NRDS was 0.902, with 88.28% sensitivity, 86.72% specificity, and 255.98 ng/mL cut-off value; the AUC for the diagnosis of severe NRDS was 0.741, with 66.67% sensitivity, 79.52% specificity, and 625.5 ng/mL cut-off value. Low gestational age, birth weight and 5 min Apgar score, severe NRDS, and elevated serum ANGPTL4 levels were IRFs for death in NRDS infants. NRDS infants with increased serum ANGPTL4 level displayed decreased survival rate and short survival time.ANGPTL4 exhibited high diagnostic value and predictive value for death in NRDS, and it served as a biomarker for the diagnosis and prognosis of NRDS. [ABSTRACT FROM AUTHOR]

Background: The endogenous metabolism of polyunsaturated fatty acids is regulated by the fatty acid desaturase (FADS) gene cluster and is strongly associated with diseases such as atherosclerosis, dyslipidemia, and type 2 diabetes. However, the association between FADS and atherosclerosis remains a subject of debate. Methods: In this study, we specifically investigated the physiological role of Δ-5 fatty acid desaturase (FADS1) in aortic and peripheral vessel (namely, the femoral artery) atherosclerosis by targeting the selective knockdown of hepatic Fads1 in apolipoprotein E-null (ApoE-⁣/-) mice with antisense oligonucleotides (ASOs). Results: Knockdown of hepatic Fads1 in ApoE-⁣/- mice exacerbated aortic atherosclerosis and non-alcoholic fatty liver disease (NAFLD), resulting in weight loss. Upregulation of FADS1 mRNA expression in more severe atherosclerosis vascular tissues potentially caused the upregulation of angiopoietin-like 4 expression. Conclusions: Our study demonstrated that knockdown of hepatic Fads1 in ApoE-⁣/- mice aggravates spontaneous atherosclerosis and NAFLD but does not affect peripheral atherosclerosis (femoral artery) induced by vascular cuff combined with tandem stenosis.

Objective: To investigate the changes of serum ferritin, angiopoietin like protein 4 (ANGPTL4), procalcitonin to albumin ratio (PAR) and their evaluation value of prognosis in patients with acute respiratory distress syndrome (ARDS) with different conditions. Methods: 109 patients with ARDS who were admitted to the Department of Intensive Care Unit of The Fourth West China Hospital of Sichuan University from March 2019 to June 2022 were selected. According to the oxygenation index (PaO2/FiO2), the patients were divided into mild group (200 mmHg less than PaO2/FiO2 less than or equal to 300 mmHg, 38 cases), moderate group (100 mmHg less than PaO2/FiO2 less than or equal to 200 mmHg, 42 cases), severe group (29 cases ess than or equal to 100 mmHg). Results: The serum ferritin, ANGPTL4 levels and PAR of all patients with ARDS were detected, and the patients were divided into survival group (69 cases) and death group (40 cases) according to their survival status within 28 days after admission. Multivariate Logistic regression analysis of the risk factors of death in patients with ARDS within 28 days after admission. The predictive value of serum ferritin, ANGPTL4 and PAR in predicting prognosis of patients with ARDS was analyzed by receiver operating characteristic (ROC) curve. Serum ferritin, ANGPTL4, procalcitonin and PAR in the severe group were higher than those in the moderate group and mild group (P＜0.05), and serum albumin level was lower than that in moderate group and mild group (P＜0.05). Serum ferritin, ANGPTL4, procalcitonin and PAR in the death group were higher than those in the survival group (P＜0.05), and serum albumin level was lower than that in the survival group (P＜0.05). High SOFA score, high PAR and elevated serum ferritin and ANGPTL4 levels were risk factors for death in patients with ARDS within 28 days after admission (P＜0.05). The area under curve of united serum ferritin, ANGPTL4 and PAR to predict the prognosis of patients with ARDS was 0.867, which was higher than 0.775, 0.727 and 0.776 predicted by single indicators. Conclusion: Increased serum ferritin, ANGPTL4 levels and PAR in patients with ARDS are associated with exacerbation of the disease and poor prognosis. United detection of these three indicators is of high value in the prognosis assessment of patients with ARDS. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is one of the most common, heterogenous endocrine disorders and is the leading cause of ovulatory obstacle associated with abnormal folliculogenesis. Dysfunction of ovarian granulosa cells (GCs) is recognized as a major factor that underlies abnormal follicle maturation. Angiopoietin-like 4 (ANGPTL4) expression in GCs differs between patients with and without PCOS. However, the role and mechanism of ANGPTL4 in impaired follicular development are still poorly understood. Here, the case-control study was designed to investigate the predictive value of ANGPTL4 in PCOS while cell experiments in vitro were set for mechanism research. Results found that ANGPTL4 levels in serum and in follicular fluid, and its expression in GCs, were upregulated in patients with PCOS. In KGN and SVOG cells, upregulation of ANGPTL4 inhibited the proliferation of GCs by blocking G1/S cell cycle progression, as well as the molecular activation of the EGFR/JAK1/STAT3 cascade. Moreover, the STAT3-dependent CDKN1A(p21) promoter increased CDKN1A transcription, resulting in remarkable suppression effect on GCs. Together, our results demonstrated that overexpression of ANGPTL4 inhibited the proliferation of GCs through EGFR/JAK1/STAT3-mediated induction of p21, thus providing a novel epigenetic mechanism for the pathogenesis of PCOS. [ABSTRACT FROM AUTHOR]

To explore the effects of peanut allergy on CC027 mouse IEC function, RNA from PBS- and peanut-sensitized CC027 mouse-derived jejunal IECs before and after challenge with peanut (30, 60 and 120 min) was isolated for RNA-seq (Figure 2A). After intragastric FITC-dextran 4 kDa (FD4) administration during challenge, plasma FD4 levels in Peanut/Peanut CC027 mice were significantly higher compared with that of PBS/Peanut CC027 mice at 30, 60, 120 and 240 min after challenge (Figure 1H) but not in similarly treated C3H mice (Figure 1I). Keywords: allergy severity; angiopoietin-like 4; angptl4; barrier permeability; food allergy; intestinal epithelial cell; intestinal permeability; peanut allergy EN allergy severity angiopoietin-like 4 angptl4 barrier permeability food allergy intestinal epithelial cell intestinal permeability peanut allergy 210 215 6 02/20/23 20230201 NES 230201 Key Messages The genetically susceptible CC027/GeniUnc mouse and peanut-allergic pediatric patients demonstrate increased intestinal epithelial cell (IEC) permeability. Peanut/Peanut CC027 mouse-derived IECs, but not those from respective C3H mice, showed a significant I decrease i in TEER (Figure 1L) and I increase i in apical-to-basolateral FD4 (Figure 1M) compared with PBS/PBS CC027 mouse-derived jejunal IECs. [Extracted from the article]

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Angiopoietin‑like 4 (ANGPTL4), a member of the angiopoietin family, plays critical roles in angiogenesis, lipid metabolism and inflammation. It has been demonstrated that ANGPTL4 has significant influence on various diseases. Accumulating evidence has highlighted the impacts of ANGPTL4 on human malignancies. ANGPTL4 is commonly overexpressed in various types of cancer, such as breast, non‑small cell lung, gastric and colorectal cancer. Its upregulation promotes tumor growth, invasion, metastasis and angiogenesis, as well as metabolic reprogramming and resistance to programmed cell death, radiotherapy and chemotherapy. However, ANGPTL4 has also exhibited antitumor effects under certain conditions, indicating its complex roles in tumor biology. The transcriptional regulation of ANGPTL4 is influenced by multiple factors, such as HIF‑1, PPARs, TGF‑β and long non‑coding RNAs. In terms of signaling pathways, STATs, PI3K/AKT and COX-2/PGE2 are important in regulating cellular processes. The present review summarizes the biological functions of ANGPTL4 in tumors and its association with patient prognosis. Furthermore, the key molecular mechanisms and potential reasons for its dual roles in cancer are also discussed. In conclusion, ANGPTL4 is a valuable diagnostic biomarker and a potential therapeutic target for human cancers.

Angiopoietin-like 4 (ANGPTL4) is a secreted signaling protein that is implicated in cardiovascular disease, metabolic disorder, and cancer. Outside of its role in lipid metabolism, ANGPTL4 signaling remains poorly understood. Here, we identify ANGPTL4 as a Wnt signaling antagonist that binds to syndecans and forms a ternary complex with the Wnt co-receptor Lipoprotein receptor-related protein 6 (LRP6). This protein complex is internalized via clathrin-mediated endocytosis and degraded in lysosomes, leading to attenuation of Wnt/β-catenin signaling. Angptl4 is expressed in the Spemann organizer of Xenopus embryos and acts as a Wnt antagonist to promote notochord formation and prevent muscle differentiation. This unexpected function of ANGPTL4 invites re-interpretation of its diverse physiological effects in light of Wnt signaling and may open therapeutic avenues for human disease.

The Angiopoietin-like 4 (ANGPTL4) and ETS Variant Transcription Factor 4 (ETV4) are involved in the metabolic transition and carcinogenesis in the liver. However, the role of ETV4 in the development of non-alcoholic fatty liver disease (NAFLD) is currently unknown. Our study reveals that ETV4 expression was upregulated in the diet-induced non-alcoholic fatty liver disease, and plays a critical role in the dysregulated lipid metabolism. We demonstrate a mechanism by which ANGPTL4 regulates lipid homeostasis via involving the AMPK/ETV4 axis. Transient knockdown of ETV4 abolished the ANGPTL4-induced expression of Srebp1c, Acc and Fasn. Insulin treatment potentially increased the physical association of ETV4 with SREBP1, and promotes nuclear translocation and transcriptional activity of SREBP1. In addition, we show that combined therapy with omega-3 fatty acids and diacylglycerol O-acyltransferase inhibitor 1 (DGAT1) inhibitor (A-922500) counteracted the ANGPTL4-ETV4 axis-induced lipogenesis in vitro, and in vivo in obese mice via activation of GPR120-βarrestin2-AMPK pathway. Finally, we demonstrate that targeted pharmacologic therapy using GalNac-ETV4 siRNA that specifically inhibits ETV4 gene expression in the liver protects against diet-induced NAFLD, obesity and dyslipidemia. Hence, our study reveal previously unrecognized role of ETV4 in the NAFLD, and provides rationale targeting ETV4 to treat NAFLD. Exogenous supplementation of EPA and DHA in cells showed preferential incorporation of EPA and DHA in the neutral lipids rather than phospholipids. Inhibition of diacylglycerol O-acyltransferase 1 (DGAT1) shifted this accretion of EPA and DHA towards phospholipids. Aberrant expression of ANGPTL4-ETV4 contributes to metabolic dysfunction in NAFLD via inhibition of AMPK. Therapy with EPA, DHA and DGAT1 inhibitor activates AMPK and alleviates NAFLD via GPR120-βarrestin2 pathway. GalNac-ETV4 siRNA conjugate therapy attenuated NAFLD. [Display omitted] [ABSTRACT FROM AUTHOR]

Postnatal skeletal muscle differentiation from quiescent satellite cells is a highly regulated process, although our understanding of the contribution of nutritional factors in myogenesis is limited. Free fatty acids (FFAs) are known to cause detrimental effects to differentiated skeletal muscle cells by increasing oxidative stress which leads to muscle wasting and insulin resistance in skeletal muscle. In addition, FFAs are thought to act as inhibitors of skeletal muscle differentiation. However, the precise molecular mechanisms underlying the effects of FFAs on skeletal muscle differentiation remains to be elucidated. There is a clear relationship between dietary FFAs and their ability to suppress myogenesis and we propose the hypothesis that the FFA-mediated increase in angiopoietinlike protein 4 (ANGPTL4) may play a role in the inhibition of differentiation. This review discusses the role of FFAs in skeletal muscle differentiation to-date and proposes potential mechanisms of FFA-induced ANGPTL4 mediated inhibition of skeletal muscle differentiation. [ABSTRACT FROM AUTHOR]

Psoriasis, vitiligo and lichen planus (LP) are autoimmune skin diseases associated with metabolic syndrome. Angiopoietin-like 4 (ANGPTL4) is a member of angiopoietin-like proteins, which play an important role in lipid metabolism, and its serum concentration has been proposed as a biomarker of cardiometabolic complications, especially coronary artery disease (CAD). The study involved 56 patients with abovementioned dermatoses and 29 sex- and age-matched volunteers without dermatoses. ANGPTL4 serum concentration was measured by ELISA. ANGPTL4 concentration was statistically significantly higher in patients with LP compared to the control group (p < 0.01); moreover, it was significantly higher than in patients with psoriasis and vitiligo (p < 0.001, p < 0.01, respectively). There was no statistically significant difference in ANGPTL4 concentration between patients with psoriasis or vitiligo and controls. There was no correlation between ANGPTL4 concentration and age or BMI in all study groups. There was a positive correlation between ANGPTL4 concentration and fasting glucose (R = 0.43) and AST activity (R = 0.39) in psoriatic patients and ALT activity in patients with vitiligo (R = 0.44). ANGPTL4 could be a potential marker of metabolic complications in patients with LP, especially CAD. Perhaps patients with LP are more prone to CAD compared to the other two dermatoses, which requires further research. [ABSTRACT FROM AUTHOR]

Angiopoietin-like 4 (ANGPTL4) is involved in inflammation-associated diseases, such as rheumatoid arthritis, type 2 diabetes, atherosclerosis, and chronic obstructive pulmonary disease. The role of ANGPTL4 in the pathogenesis of inflammatory bowel disease (IBD) remains unknown. Here, the plasma ANGPTL4 levels peaked on days 3 and 5, and expression of ANGPTL4 of inflamed colons peaked on days 5 and 7 in mice with dextran sulfate sodium (DSS)-induced colitis. Simultaneously, CD8+T cells in the inflamed colons peaked at day 5 but declined at day 7. However, the ANGPTL4−/− mice treated with DSS exhibited exacerbated colitis with more CD8+T cells and macrophages infiltrating the colons. The exogenous ANGPTL4 protein protected the mice against DSS-induced colitis with less CD8+T cell and macrophage recruitment in the colons. In addition, recombinant ANGPTL4 directly downregulated the IFN-γ and NKG2D expression of CD8+T cells but had no effects on the CD86 expression and TNF-α secretion of macrophages ex vivo. Adding ANGPTL4 protein into ANGPTL4−/− mice almost blocked the onset of DSS-induced colitis. In parallel, the plasma ANGPTL4 levels were elevated in patients with Crohn's disease and ulcerative colitis at mild/moderate stage and restored to normal levels in IBD patients at a severe stage. The higher ANGPTL4 expression in the inflamed colons of patients with IBD was correlated with lower CD8+ cell infiltration, whereas no associations with macrophages. Our results demonstrated the compensatory protective effect of ANGPTL4 on IBD development at least via the downregulation of CD8+T cell activities. Adding the ANGPTL4 protein would have beneficial effects to retard the progression of IBD. • Expression of ANGPTL4 increased in mice treated with DSS. • ANGPTL4 reverses DSS-induced colitis in ANGPTL4−/− mice. • Elevated ANGPTL4 levels in early-stage of IBD patients. • ANGPTL4 inverse association with CD8+T cells in IBD patients. [ABSTRACT FROM AUTHOR]

Postnatal skeletal muscle differentiation from quiescent satellite cells is a highly regulated process, although our understanding of the contribution of nutritional factors in myogenesis is limited. Free fatty acids (FFAs) are known to cause detrimental effects to differentiated skeletal muscle cells by increasing oxidative stress which leads to muscle wasting and insulin resistance in skeletal muscle. In addition, FFAs are thought to act as inhibitors of skeletal muscle differentiation. However, the precise molecular mechanisms underlying the effects of FFAs on skeletal muscle differentiation remains to be elucidated. There is a clear relationship between dietary FFAs and their ability to suppress myogenesis and we propose the hypothesis that the FFA-mediated increase in angiopoietin-like protein 4 (ANGPTL4) may play a role in the inhibition of differentiation. This review discusses the role of FFAs in skeletal muscle differentiation to-date and proposes potential mechanisms of FFA-induced ANGPTL4 mediated inhibition of skeletal muscle differentiation.

Angiopoietin-like 4 (ANGPTL4) is an important regulator of plasma triglyceride (TG) levels and an attractive pharmacological target for lowering plasma lipids and reducing cardiovascular risk. Here, we aimed to study the efficacy and safety of silencing ANGPTL4 in the livers of mice using hepatocyte-targeting GalNAc-conjugated antisense oligonucleotides (ASOs). Compared with injections with negative control ASO, four injections of two different doses of ANGPTL4 ASO over 2 weeks markedly downregulated ANGPTL4 levels in liver and adipose tissue, which was associated with significantly higher adipose LPL activity and lower plasma TGs in fed and fasted mice, as well as lower plasma glucose levels in fed mice. In separate experiments, injection of two different doses of ANGPTL4 ASO over 20 weeks of high-fat feeding reduced hepatic and adipose ANGPTL4 levels but did not trigger mesenteric lymphadenopathy, an acute phase response, chylous ascites, or any other pathological phenotypes. Compared with mice injected with negative control ASO, mice injected with ANGPTL4 ASO showed reduced food intake, reduced weight gain, and improved glucose tolerance. In addition, they exhibited lower plasma TGs, total cholesterol, LDL-C, glucose, serum amyloid A, and liver TG levels. By contrast, no significant difference in plasma alanine aminotransferase activity was observed. Overall, these data suggest that ASOs targeting ANGPTL4 effectively reduce plasma TG levels in mice without raising major safety concerns.

Abstract Background As a tissue-specific proangiogenic or antiangiogenic agent, angiopoietin-like 4 (ANGPTL4) has recently gained attention in many diseases, such as metabolic syndrome, cardiovascular disease and cancer. However, the roles of ANGPTL4 in angiogenesis and tumor growth in epithelial ovarian cancer, the most lethal gynecologic malignancy, remain unclear. Objective To identify a novel mechanism of ANGPTL4 inhibition in epithelial ovarian cancer. Methods Western blot, quantitative reverse transcription PCR, and immunofluorescence analyses were applied to evaluate ANGPTL4 expression in ovarian cancer cell lines. Cell proliferation, migration, and invasion were investigated through 5-ethynyl-2′-deoxyuridine (EdU) incorporation, CCK-8 and Transwell assays. The expression of epithelial-mesenchymal transition (EMT)-related proteins in ovarian cancer cells and tumor-bearing mice was evaluated. CD31 staining was used to identify tumor angiogenesis. Immunoprecipitation was performed to examine the regulatory relationship between ANGPTL4 and the vascular endothelial growth factor receptor 2 (VEGFR2)/vascular endothelial (VE)-cadherin/Src complex. VEGFR2 phosphorylation at Y949 and VE-cadherin expression were assessed by western blotting. Inactivation of VEGFR2 Y949 phosphorylation was achieved in a MISIIR-TAg VEGFR2 Y949F/Y949F mouse model. Results Here, we demonstrated that ANGPTL4 was overexpressed in A2780 and CAOV3 ovarian cancer cells. In vitro assays indicated that inhibition of ANGPTL4 by lentiviral small interfering RNA does not alter ovarian cancer cell proliferation, migration, invasion, and EMT, while ANGPTL4 silencing exhibited significant inhibitory effects on tumor angiogenesis, growth, and metastasis in vivo. Immunoprecipitation analysis showed that suppression of ANGPTL4 was accompanied by dissociation of the VEGFR2/VE-cadherin/Src complex and phosphorylation of VEGFR2 Y949 in A2780 and CAOV3 ovarian tumors. Inactivation of VEGFR2 Y949 phosphorylation in MISIIR-TAg VEGFR2 Y949F/Y949F mice abolished all tumor-suppressive effects of ANGPTL4 inhibition in spontaneous ovarian carcinoma. Conclusions Overall, our results indicate that ANGPLT4 silencing delays tumor progression in specific types of ovarian cancer and may be a potential target for individualized treatment of ovarian cancer.

Background: The role of Lactobacillus casei on human health is well documented. However, little is known about their protein effects on food digestion. Therefore, in the present study, we aimed to investigate the efficacy of three proteins of L. casei on lipid and carbohydrate digestion that was identified at acidic pH in our previous study. Methods: Chaperonin (Ch), metal-dependent hydrolase (HYD), and lysozyme (LYS) proteins that were expressed by cultivated L. casei at pH 5 were extracted. HepG2 cell line was used to elucidate the effect of the considered three proteins on gene expression related to fat and glucose metabolism. The target genes were determined by Kyoto Encyclopedia of Genes and Genomes pathway analysis and extracted proteins were transfected into HepG2 cells. After 48 and 120 h, the mRNA expression of the following genes was analyzed using real-time polymerase chain reaction, insulin receptor substrate 2, (IRS-2), Protein kinase Bβ (AKT2), insulin-like growth factor 1, angiopoietin-like 4 (Angptl-4), and lipoprotein lipase (LPL). Results: The expression of all of the genes was significantly increased in comparison to control under the effect of Ch and metal-dependent HYDs after 48 h of culture. By increasing the duration of transfection from 48 h to 120 h, the expression of Angptl-4 from Ch and metal-dependent HYDs was reduced significantly, whereas the expression of LPL and Angptl-4 genes after 5 days was significantly increased in LYS compared to the last 3 days. Conclusions: L. casei secrets acidic proteins such as Ch, metal-dependent HYD, and LYS in bloodstream are involved in the digestion of carbohydrates and fats in the liver.

COVID‐19 is associated with encephalitis in critically ill patients and endothelial dysfunction seems to contribute to this life‐threatening complication. Our objective was to determine the hallmark of endothelial activation in COVID‐19‐related encephalitis. In an observational study in intensive care unit (ICU), we compared vascular biomarkers of critically ill COVID‐19 patients with or without encephalitis. To be classified in the encephalitis group, patients had to have new onset of central neurologic symptom, and pathological findings on either brain magnetic resonance imaging (MRI) and/or electroencephalogram (EEG). Among the 32 critically ill COVID‐19 consecutive patients, 21 were categorized in the control group and 11 in the encephalitis group. Encephalitis patients had a longer ICU stay than control patients (median length [25th–75th percentile] of 52 [16–79] vs. 20.5 [11–44] days, respectively, p = 0.04). Nine‐month overall follow‐up mortality reached 21% (7/32 patients), with mortality rates in the encephalitis group and the control group of 27% and 19%, respectively. Encephalitis was associated with significant higher release of soluble endothelial activation markers (sE‐selectin, tumor necrosis factor‐α (TNF‐α), interleukin 6, placental growth factor, and thrombomodulin), but these increases were correlated with TNF‐α plasmatic levels. The hypoxia‐inducible protein angiopoietin‐like 4 (ANGPTL4) was at significantly higher levels in encephalitis patients compared to control patients (p = 0.0099), and in contrary to the other increased factors, was not correlated with TNF‐α levels (r = 0.2832, p = 0.1163). Our findings suggest that COVID‐19‐related encephalitis is a cytokine‐associated acute brain dysfunction. ANGPTL4 was the only elevated marker found in encephalitis patients, which was not correlated with systemic inflammation, suggesting that ANGPTL4 might be a relevant factor to predict encephalitis in critically ill COVID‐19 patients. [ABSTRACT FROM AUTHOR]

Purpose: Angiopoietin-like 4 (ANGPTL4) was recently shown to be associated with cancer progression but little is known about its contribution to cancer metabolism. The purpose of this study was to elucidate the role of ANGPTL4 in glucose metabolism in colorectal cancer (CRC). Methods: Immunohistochemical staining of CRC specimens classified 84 patients into two groups according to ANGPTL4 expression. Clinicopathological characteristics, gene mutation status obtained by next-generation sequencing, and fluorodeoxyglucose (FDG) uptake measured by positron emission tomography/computed tomography (PET/CT) were compared between the two groups. Furthermore, the impact of ANGPTL4 expression on cancer metabolism was investigated by a subcutaneous xenograft mouse model using the ANGPTL4 knockout CRC cell line, and glucose transporter (GLUT) expression was evaluated. Results: There were significantly more cases of T3/4 tumours (94.3% vs. 57.1%, P < 0.001) and perineural invasion (42.9% vs. 22.4%, P = 0.046) in the ANGPTL4-high group than in the low group. Genetic exploration revealed a higher frequency of KRAS mutation (54.3% vs. 22.4%, P = 0.003) in the ANGPTL4-high tumours. All the FDG uptake parameters were significantly higher in ANGPTL4-high tumours. In vivo analysis showed a significant reduction in tumour size due to ANGPTL4 knockout with lower expression of GLUT1 and GLUT3, and suppression of AKT phosphorylation. Conclusion: ANGPTL4 regulates the expression of GLUTs by activating the PI3K–AKT pathway and thereby promoting glucose metabolism in CRC. These findings establish a new functional role of ANGPTL4 in cancer progression and lay the foundation for developing a novel therapeutic target. [ABSTRACT FROM AUTHOR]

Objective: To detect whether Danlou Tablet (DLT) regulates the hypoxia-induced factor (HIF)-1α-angiopoietin-like 4 (Angptl4) mRNA signaling pathway and explore the role of DLT in treating chronic intermittent hypoxia (CIH)-induced dyslipidemia and arteriosclerosis. Methods: The mature adipocytes were obtained from 3T3-L1 cell culturation and allocated into 8 groups including control groups (Groups 1 and 5, 0.1 mL of cell culture grade water); DLT groups (Groups 2 and 6, 0.1 mL of 1,000 µg/mL DLT submicron powder solution); dimethyloxalylglycine (DMOG) groups (Groups 3 and 7, DMOG and 0.1 mL of cell culture grade water); DMOG plus DLT groups (Groups 4 and 8, DMOG and 0.1 mL of 1,000 µg/mL DLT submicron powder solution). Groups 1–4 used mature adipocytes and groups 5–8 used HIF-1 α-siRNA lentivirus-transfected mature adipocytes. After 24-h treatment, real-time polymerase chain reaction and Western blot were employed to determine the mRNA and protein expression levels of HIF-1 α and Angptl4. In animal experiments, the CIH model in ApoE−/− mice was established. Sixteen mice were complete randomly divided into 4 groups including sham group, CIH model group [intermittent hypoxia and normal saline (2 mL/time) gavage once a day]; Angptl4 Ab group [intermittent hypoxia and Angptl4 antibody (30 mg/kg) intraperitoneally injected every week]; DLT group [intermittent hypoxia and DLT (250 mg/kg) once a day], 4 mice in each group. After 4-week treatment, enzyme linked immunosorbent assay was used to detect the expression levels of serum total cholesterol (TC) and triglyceride (TG). Hematoxylin-eosin and CD68 staining were used to observe the morphological properties of arterial plaques. Results: Angptl4 expression was dependent on HIF-1 α, with a reduction in mRNA expression and no response in protein level to DMOG or DLT treatment in relation to siHIF-1 α -transfected cells. DLT inhibited HIF-1 α and Angptl4 mRNA expression (P<0.05 or P<0.01) and reduced HIF-1 α and Angptl4 protein expressions with DMOG in mature adipocytes (all P<0.01), as the effect on HIF-1 α protein also existed in the presence of siHIF-1 α (P<0.01). ApoE−/− mice treated with CIH had increased TG and TC levels (all P<0.01) and atherosclerotic plaque. Angptl4 antibody and DLT both reduce TG and TC levels (all P<0.01), as well as reducing atherosclerotic plaque areas, narrowing arterial wall thickness and alleviating atherosclerotic lesion symptoms to some extent. Conclusion: DLT had positive effects in improving dyslipidemia and arteriosclerosis by inhibiting Angptl4 protein level through HIF-1 α-Angptl4 mRNA signaling pathway. [ABSTRACT FROM AUTHOR]

Epicardial adipose tissue (EAT) is known to affect atherosclerosis and coronary artery disease (CAD) pathogenesis, persistently releasing pro-inflammatory adipokines that affect the myocardium and coronary arteries. Angiopoietin-like 4 (ANGPTL4) is a protein secreted from adipose tissue and plays a critical role in the progression of atherosclerosis. Here, the expression of ANGPTL4 in EAT was investigated in CAD subjects. Thirty-four consecutive patients (13 patients with significant CAD; 21 patients without CAD) undergoing elective open-heart surgery were recruited. EAT and pericardial fluid were obtained at the time of surgery. mRNA expression and ANGPTL4 and IL-1β levels were evaluated by qRT-PCR and ELISA. The expression of ANGPTL4 (p = 0.0180) and IL-1β (p < 0.0001) in EAT significantly increased in the CAD group compared to that in the non-CAD group and positively correlated (p = 0.004). Multiple regression analysis indicated that CAD is a contributing factor for ANGPTL4 expression in EAT. IL-1β level in the pericardial fluid was significantly increased in patients with CAD (p = 0.020). Moreover, the expression of ANGPTL4 (p = 0.004) and IL-1β (p < 0.001) in EAT was significantly increased in non-obese patients with CAD. In summary, ANGPTL4 expression in EAT was increased in CAD patients. [ABSTRACT FROM AUTHOR]

Selective intrauterine growth restriction (sIUGR) is a severe complication in monochorionic (MC) twin pregnancies, and it carries increased risks of poor prognosis. Current data suggest that vascular anastomoses and unequal placental sharing may be the key contributor to discordant foetal growth. While MC twins derive from a single zygote and have almost identical genetic information, the precise mechanisms remain unknown. DNA hydroxymethylation is a newly discovered epigenetic feature associated with gene regulation and modification. Here, we investigate discordant hydroxymethylation patterns between two placental shares of sIUGR and analyse the potential role of aberrant hydroxymethylation of angiopoietin-like 4 (ANGPTL4) in placental dysplasia. Hydroxymethylation DNA immunoprecipitation (hMeDIP)-chip and mRNA sequencing were performed to identify hydroxymethylation-associated genes. Real-time qPCR, western blotting, and immunohistochemistry were used to confirm ANGPTL4 expression. The mechanisms regulating ANGPTL4 were investigated by cell migration assay, invasion assay, viability assay, and apoptotic ratio assays, western blotting and hMeDIP-qPCR. Decreased ANGPTL4 was detected in the smaller placental shares of sIUGR. ANGPTL4 knockdown suppressed trophoblast invasiveness and migration, which possibly occurred through hypoxia inducible factor 1α (HIF-1α) and HIF-1 signalling pathway. Hypoxia leads to aberrant expression of ANGPTL4 and HIF-1α, positively correlated with their aberrant hydroxymethylation levels in promoter regions. Aberrant hydroxymethylation of ANGPTL4 may contribute to placental impairment by the HIF-1 signalling pathway in smaller placental shares of sIUGR.

Objective: Brown adipose tissue (BAT) burns fatty acids (FAs) to produce heat, and shows diurnal oscillation in glucose and triglyceride (TG)-derived FA-uptake, peaking around wakening. Here we aimed to gain insight in the diurnal regulation of metabolic BAT activity. Methods: RNA-sequencing, chromatin immunoprecipitation (ChIP)-sequencing, and lipidomics analyses were performed on BAT samples of wild type C57BL/6J mice collected at 3-hour intervals throughout the day. Knockout and overexpression models were used to study causal relationships in diurnal lipid handling by BAT. Results: We identified pronounced enrichment of oscillating genes involved in extracellular lipolysis in BAT, accompanied by oscillations of FA and monoacylglycerol content. This coincided with peak lipoprotein lipase (Lpl) expression, and was predicted to be driven by peroxisome proliferator-activated receptor gamma (PPARγ) activity. ChIP-sequencing for PPARγ confirmed oscillation in binding of PPARγ to Lpl. Of the known LPL-modulators, angiopoietin-like 4 (Angptl4) showed the largest diurnal amplitude opposite to Lpl, and both Angptl4 knockout and overexpression attenuated oscillations of LPL activity and TG-derived FA-uptake by BAT. Conclusions: Our findings highlight involvement of PPARγ and a crucial role of ANGPTL4 in mediating the diurnal oscillation of TG-derived FA-uptake by BAT, and imply that time of day is essential when targeting LPL activity in BAT to improve metabolic health.

Angiopoietin-like (ANGPTL) 8 is a secreted inhibitor of LPL, a key enzyme in plasma triglyceride metabolism. It was previously reported that ANGPTL8 requires another member of the ANGPTL family, ANGPTL3, to act on LPL. ANGPTL3, much like ANGPTL4, is a physiologically relevant regulator of LPL activity, which causes irreversible inactivation of the enzyme. Here, we show that ANGPTL8 can form complexes with either ANGPTL3 or ANGPTL4 when the proteins are refolded together from their denatured states. In contrast to the augmented inhibitory effect of the ANGPTL3/ANGPTL8 complex on LPL activity, the ANGPTL4/ANGPTL8 complex is less active compared with ANGPTL4 alone. In our experiments, all three members of the ANGPTL family use the same mechanism to inactivate LPL, which involves dissociation of active dimeric LPL to monomers. This inactivation can be counteracted by the presence of glycosylphosphatidylinositol-anchored HDL binding protein 1, the endothelial LPL transport protein previously known to protect LPL from spontaneous and ANGPTL4-catalyzed inactivation. Our data demonstrate that ANGPTL8 may function as an important metabolic switch, by forming complexes with ANGPTL3, or with ANGPTL4, in order to direct the flow of energy from triglycerides in blood according to the needs of the body.

Objective To study the relationship between angiopoietin-like 4 (Angptl4) and podocyte injury and protein uria in renal tissues of patients with primary nephrotic syndrome. Methods Patients with primary nephrotic syndrome, including 11 cases of minimal change disease (MCD), 11 cases of membranous nephropathy (MN) and 9 cases of mesangial proliferative glomerulonephritis (MsPGN) were enrolled in this study. Renal tissues were collected during biopsy for Angptl4 and podocyte injury marker desmin immunofluorescence stainning, and fluorescence intensity was calculated. Angptl4 was double stained with podocyte marker synaptopodin, mesangial cell marker CD90, endothelial cell marker CD31 and glomerular basement membrane marker laminin to study the overlap rate of Angptl4 with various types of cells. The basic information of gender, age, 24 h-urine protein, serum triglycerides, serum total cholesterol and serum creatinine were collected. The correlation between the expression of Angptl4 and desmin immunofluorescence intensity, 24 h protein uria, serum triglycerides and serum total cholesterol was analyzed. Results There were no significant differences in gender, age, serum creatinine and 24 h-urine protein between the three groups (P＞0.05). Compared with MsPGN patients, the glomerular Angptl4 expression increased in MCD and MN patients (P＜0.05), and the increased Angptl4 had the highest degree of overlap with podocytes rather than mesangial cells, endothelial cells or endothelial cells. Angptl4 expression in glomeruli was correlated with glomerular desmin expression and 24 h-urine protein (P＜0.01), but not correlated with the serum triglycerides and serum total cholesterol (P＞0.05). Conclusion In patients with primary MCD and MN, the expression of glomerular Angptl4 is increased, which is associated with podocyte injury and proteinuria, but not with blood lipids. [ABSTRACT FROM AUTHOR]

Psoriasis, vitiligo and lichen planus (LP) are autoimmune skin diseases associated with metabolic syndrome. Angiopoietin-like 4 (ANGPTL4) is a member of angiopoietin-like proteins, which play an important role in lipid metabolism, and its serum concentration has been proposed as a biomarker of cardiometabolic complications, especially coronary artery disease (CAD). The study involved 56 patients with abovementioned dermatoses and 29 sex- and age-matched volunteers without dermatoses. ANGPTL4 serum concentration was measured by ELISA. ANGPTL4 concentration was statistically significantly higher in patients with LP compared to the control group (p < 0.01); moreover, it was significantly higher than in patients with psoriasis and vitiligo (p < 0.001, p < 0.01, respectively). There was no statistically significant difference in ANGPTL4 concentration between patients with psoriasis or vitiligo and controls. There was no correlation between ANGPTL4 concentration and age or BMI in all study groups. There was a positive correlation between ANGPTL4 concentration and fasting glucose (R = 0.43) and AST activity (R = 0.39) in psoriatic patients and ALT activity in patients with vitiligo (R = 0.44). ANGPTL4 could be a potential marker of metabolic complications in patients with LP, especially CAD. Perhaps patients with LP are more prone to CAD compared to the other two dermatoses, which requires further research.

Abstract Overcoming multidrug resistance has always been a major challenge in cancer treatment. Recent evidence suggested epithelial-mesenchymal transition plays a role in MDR, but the mechanism behind this link remains unclear. We found that the expression of multiple ABC transporters was elevated in concordance with an increased drug efflux in cancer cells during EMT. The metastasis-related angiopoietin-like 4 (ANGPTL4) elevates cellular ATP to transcriptionally upregulate ABC transporters expression via the Myc and NF-κB signaling pathways. ANGPTL4 deficiency reduced IC50 of anti-tumor drugs and enhanced apoptosis of cancer cells. In vivo suppression of ANGPTL4 led to higher accumulation of cisplatin-DNA adducts in primary and metastasized tumors, and a reduced metastatic tumor load. ANGPTL4 empowered cancer cells metabolic flexibility during EMT, securing ample cellular energy that fuels multiple ABC transporters to confer EMT-mediated chemoresistance. It suggests that metabolic strategies aimed at suppressing ABC transporters along with energy deprivation of EMT cancer cells may overcome drug resistance.

Background: As a tissue-specific proangiogenic or antiangiogenic agent, angiopoietin-like 4 (ANGPTL4) has recently gained attention in many diseases, such as metabolic syndrome, cardiovascular disease and cancer. However, the roles of ANGPTL4 in angiogenesis and tumor growth in epithelial ovarian cancer, the most lethal gynecologic malignancy, remain unclear. Objective: To identify a novel mechanism of ANGPTL4 inhibition in epithelial ovarian cancer. Methods: Western blot, quantitative reverse transcription PCR, and immunofluorescence analyses were applied to evaluate ANGPTL4 expression in ovarian cancer cell lines. Cell proliferation, migration, and invasion were investigated through 5-ethynyl-2′-deoxyuridine (EdU) incorporation, CCK-8 and Transwell assays. The expression of epithelial-mesenchymal transition (EMT)-related proteins in ovarian cancer cells and tumor-bearing mice was evaluated. CD31 staining was used to identify tumor angiogenesis. Immunoprecipitation was performed to examine the regulatory relationship between ANGPTL4 and the vascular endothelial growth factor receptor 2 (VEGFR2)/vascular endothelial (VE)-cadherin/Src complex. VEGFR2 phosphorylation at Y949 and VE-cadherin expression were assessed by western blotting. Inactivation of VEGFR2 Y949 phosphorylation was achieved in a MISIIR-TAg VEGFR2Y949F/Y949F mouse model. Results: Here, we demonstrated that ANGPTL4 was overexpressed in A2780 and CAOV3 ovarian cancer cells. In vitro assays indicated that inhibition of ANGPTL4 by lentiviral small interfering RNA does not alter ovarian cancer cell proliferation, migration, invasion, and EMT, while ANGPTL4 silencing exhibited significant inhibitory effects on tumor angiogenesis, growth, and metastasis in vivo. Immunoprecipitation analysis showed that suppression of ANGPTL4 was accompanied by dissociation of the VEGFR2/VE-cadherin/Src complex and phosphorylation of VEGFR2 Y949 in A2780 and CAOV3 ovarian tumors. Inactivation of VEGFR2 Y949 phosphorylation in MISIIR-TAg VEGFR2Y949F/Y949F mice abolished all tumor-suppressive effects of ANGPTL4 inhibition in spontaneous ovarian carcinoma. Conclusions: Overall, our results indicate that ANGPLT4 silencing delays tumor progression in specific types of ovarian cancer and may be a potential target for individualized treatment of ovarian cancer. [ABSTRACT FROM AUTHOR]

• ANGPTL4 reduced infarct volume at day 3 post-stroke. • ANGPTL4 enhanced angiogenesis and neurogenesis in the ischemic brain by promoted the phosphorylation of AKT. • ANGPTL4 inhibited inflammatory response in the ischemic brain by decreasing the expression of Fas and FasL. The purpose of the present study is to investigate whether angiopoietin-like 4 (ANGPTL4) can promote angiogenesis and neurogenesis following stroke, as well as to explore the potential underlying mechanisms. ANGPTL4 (40 μg/kg) or a vehicle was administered via tail vein beginning 5 min prior to electrocoagulation-induced stroke in male C57/B6 J mice. Infarct volume was measured via Nissl staining at day 3 post-stroke. Angiogenesis, neurogenesis and activation of microglia were evaluated by immunofluorescence co-labelling bromodeoxyuridine (BrdU) with von Willebrand factor (vWF), doublecortin (DCX), neuronal nuclei (NeuN) and Iba1 at day 7 post-stroke. The levels of p-AKT, T-AKT, VEGF, MPO, Fas and FasL in the ipsilesional brain were detected by Western blot analysis at day 1 post-stroke. Compared with the Vehicle group, ANGPTL4 reduced infarct volume significantly at day 3 post-stroke. ANGPTL4 significantly increased the number of BrdU+, BrdU+/vWF+and BrdU+/DCX+ cells in the peri-infarct zone, subventricular zone and subgranular zone and inhibited BrdU+/Iba1+ cells in the peri-infarct zone at day 7 post-stroke. The level of p-AKT and the ratio of phospho-AKT to total-AKT in the ipsilesional brain were significantly elevated, the levels of MPO, Fas and FasL were significantly declined; however, there was no significant difference at day 1 post-stroke between the VEGF and total-AKT levels in both groups. ANGPTL4 enhances angiogenesis and neurogenesis post-stroke by upregulating the phosphorylation of AKT, reduces neuronal death and inhibits inflammatory response, which resultes from the inhibition of FasL/Fas expression and its downstream pathway. [ABSTRACT FROM AUTHOR]

The aim of the present study was to analyze the expression levels of angiopoietin-like 4 (ANGPTL4) in breast cancer to investigate the association between ANGPTL4 and breast cancer. Immunohistochemistry was performed on formalin-fixed paraffin-embedded tissues, including 205 invasive ductal carcinoma (IDC) of no special type, 40 normal breast, 40 atypical ductal hyperplasia (ADH) and 40 ductal carcinomas in situ (DCIS) tissues. The non-parametric Kruskal-Wallis test was used to evaluate the differential expression of ANGPTL4 and clinicopathological parameters in breast cancer. Kaplan-Meier analysis and Cox regression analysis were used to evaluate the association between the expression levels of ANGPTL4 and the prognosis of breast cancer. The results revealed that ANGPTL4 expression was higher in IDC (63.4%; 130/205) compared with in normal breast tissues (17.5%; 7/40), ADH (30%; 12/40) and DCIS (37.5%; 15/40). The clinical significance of ANGPTL4 expression was analyzed in a total of 205 IDC tissues, and high expression levels of ANGPTL4 were positively associated with pathological stage (P<0.001), tumor size (P<0.001), histological grade (P<0.001), lymph node metastasis (P<0.001), distant metastasis (P<0.001) and local recurrence (P<0.001). Kaplan-Meier analysis revealed that patients with high ANGPTL4 expression had a shorter overall survival (OS; P<0.001) and disease-free survival (DFS; P<0.001) compared with patients with low ANGPTL4 expression. Multivariate Cox regression analysis revealed that ANGPTL4 was an independent prognostic factor for breast cancer OS (P=0.034) and DFS (P=0.011). The results of the present study demonstrated that ANGPLT4 was associated with malignant progression and poor prognosis of breast cancer, suggesting that ANGPLT4 may be a novel therapeutic target for breast cancer. [ABSTRACT FROM AUTHOR]

Selective intrauterine growth restriction (sIUGR) is a severe complication in monochorionic (MC) twin pregnancies, and it carries increased risks of poor prognosis. Current data suggest that vascular anastomoses and unequal placental sharing may be the key contributor to discordant foetal growth. While MC twins derive from a single zygote and have almost identical genetic information, the precise mechanisms remain unknown. DNA hydroxymethylation is a newly discovered epigenetic feature associated with gene regulation and modification. Here, we investigate discordant hydroxymethylation patterns between two placental shares of sIUGR and analyse the potential role of aberrant hydroxymethylation of angiopoietin-like 4 (ANGPTL4) in placental dysplasia. Hydroxymethylation DNA immunoprecipitation (hMeDIP)-chip and mRNA sequencing were performed to identify hydroxymethylation-associated genes. Real-time qPCR, western blotting, and immunohistochemistry were used to confirm ANGPTL4 expression. The mechanisms regulating ANGPTL4 were investigated by cell migration assay, invasion assay, viability assay, and apoptotic ratio assays, western blotting and hMeDIP-qPCR. Decreased ANGPTL4 was detected in the smaller placental shares of sIUGR. ANGPTL4 knockdown suppressed trophoblast invasiveness and migration, which possibly occurred through hypoxia inducible factor 1α (HIF-1α) and HIF-1 signalling pathway. Hypoxia leads to aberrant expression of ANGPTL4 and HIF-1α, positively correlated with their aberrant hydroxymethylation levels in promoter regions. Aberrant hydroxymethylation of ANGPTL4 may contribute to placental impairment by the HIF-1 signalling pathway in smaller placental shares of sIUGR. [ABSTRACT FROM AUTHOR]

The mTOR pathway is one of the key signal cascades in the pathogenesis of idiopathic pulmonary fibrosis. Previous studies have mainly focused on this pathway in the fibroblasts and/or myofibroblasts, but not in the epithelial cells. In this study, we sought to investigate the role of the mTOR pathway in lung epithelial cells in lung fibrosis. Using Sftpc-mTORSL+IT transgenic mice, in which activemTORis conditionally expressed in lung epithelial cells, we assessed the effects of chronically activated mTOR in lung epithelial cells on lung phenotypes as well as bleomycin-induced lung fibrosis. Furthermore, we isolated alveolar epithelial cell type 2 frommice and performed RNA sequencing. Sftpc-mTORSL11IT transgenic mice had no obvious abnormal findings, but, after bleomycin administration, showed more severe fibrotic changes and lower lung compliance than control mice. RNA sequencing revealed Angptl4 (angiopoietin-like protein 4) as a candidate downstream gene of the mTOR pathway. In vitro studies revealed that ANGPTL4, as well as mTOR, promoted tight junction vulnerability and epithelial-mesenchymal transition. mTOR activation in lung epithelial cells promoted lung fibrosis and the expression of ANGPTL4, a novel downstream target of the mTOR pathway, which could be related to the etiology of fibrosis. [ABSTRACT FROM AUTHOR]

Angiopoietin-like 4 (ANGPTL4) is a target of hypoxia that accumulates in the endothelial extracellular matrix. While ANGPTL4 is known to regulate angiogenesis and vascular permeability, its context-dependent role related to vascular endothelial growth factor (VEGF) has been suggested in capillary morphogenesis. We here thus develop in vitro 3D models coupled to imaging and morphometric analysis of capillaries to decipher ANGPTL4 functions either alone or in the presence of VEGF. ANGPTL4 induces the formation of barely branched and thin endothelial capillaries that display linear adherens junctions. However, ANGPTL4 counteracts VEGF-induced formation of abundant ramified capillaries presenting cell–cell junctions characterized by VE-cadherin containing reticular plaques and serrated structures. We further deciphered the early angiogenesis steps regulated by ANGPTL4. During the initial activation of endothelial cells, ANGPTL4 alone induces cell shape changes but limits the VEGF-induced cell elongation and unjamming. In the growing sprout, ANGPTL4 maintains cohesive VE-cadherin pattern and sustains moderate 3D cell migration but restricts VEGF-induced endothelium remodeling and cell migration. This effect is mediated by differential short- and long-term regulation of P-Y1175-VEGFR2 and ERK1-2 signaling by ANGPTL4. Our in vitro 3D models thus provide the first evidence that ANGPTL4 induces a specific capillary morphogenesis but also overcomes VEGF effect.

Angiopoietin-like 4 (ANGPTL4) regulates plasma triglyceride levels by inhibiting LPL. Inactivation of ANGPTL4 decreases plasma triglycerides and reduces the risk of coronary artery disease. Unfortunately, targeting ANGPTL4 for the therapeutic management of dyslipidemia and atherosclerosis is hampered by the observation that mice and monkeys in which ANGPTL4 is inactivated exhibit lipid accumulation in the mesenteric lymph nodes (MLNs). In mice these pathological events exclusively unfold upon feeding a high saturated FA diet and are followed by an ultimately lethal pro-inflammatory response and chylous ascites. Here, we show that Angptl4−/− mice fed a diet rich in trans FAs develop numerous lipid-filled giant cells in their MLNs, yet do not have elevated serum amyloid and haptoglobin, do not exhibit ascites, and survive, unlike Angptl4−/− mice fed a saturated FA-rich diet. In RAW264.7 macrophages, the saturated FA, palmitate, markedly increased markers of inflammation and the unfolded protein response, whereas the trans-unsaturated elaidate and the cis-unsaturated oleate had the opposite effect. In conclusion, trans and saturated FAs have very distinct biological effects in macrophages. Furthermore, lipid accumulation in MLNs is uncoupled from activation of an acute-phase response and chylous ascites, suggesting that ANGPTL4 should not be fully dismissed as target for dyslipidemia.

Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS) characterized by the presence of focal demyelinated plaques. Sufficient clearance of myelin and cellular debris is one of the requirements for proper tissue repair and remyelination. The mechanisms underlying the clearance of such debris by phagocytes are not fully understood, but recent findings suggest a prominent role for lipoprotein-lipase (LPL) in this process. Here, we demonstrate that angiopoietin-like 4 (ANGPTL4), a potent inhibitor of LPL, is abundantly expressed in astrocytes in control white matter tissue and its expression is markedly reduced in active MS lesions. We provide evidence that ANGPTL4 inhibits the uptake of myelin-derived lipids by LPL-immunoreactive phagocytes. Taken together, our data suggest that the strong reduction in astrocytic ANGPTL4 expression in active demyelinating MS lesions enables phagocytes to adequately clear myelin debris, setting the stage for remyelination.