Silencing HIF-1α aggravates non-alcoholic fatty liver disease in vitro through inhibiting PPAR-α/ANGPTL4 singling pathway / El silenciamiento de HIF-1α agrava la esteatohepatitis no alcohólica in vitro a través de la inhibición de la vía de señalización PPAR-α/ANGPTL4

Objective: Non-alcoholic fatty liver disease (NAFLD) is an aberrant lipid metabolism disease. Hypoxia inducible factor-1 (HIF-1α) is a transcription factor which plays an important part in adapting lower oxygen condition. Here, we aimed to clarify the relationship between HIF-1α and NAFLD. Methods: HepG2 cells was stimulated by oleic acid (OA) and palmitic acid (PA) to establish in vitro model of NAFLD. The expression of lipid metabolism-related genes, the binding of PPARα to HIF-1α promoter, the lipid deposition, and oxidative stress were detected by qRT-PCR, western blot, Chip assay, Oil Red O staining and ELISA assays, respectively. Results: HIF-1α silence promoted lipid accumulation in NAFLD cells, accompanying by the significantly increased contents of TG (triglyceride) and ApoB (apolipoprotein B). In HepG2 cells treated with OA/PA, the expression of lipid metabolism-related genes and proteins, including APOE, A2m, TNFRSF11B, LDLr, and SREBP2, and the intracellular lipid deposition were up-regulated and further aggravated after silencing HIF-1α. In addition, the loss of HIF-1α could remarkably elevate MDA contents while inhibit the activities of beneficial antioxidant enzymes SOD and GSH-Px to activate oxidative stress, and promote the secretion of pro-inflammatory IL-6 and TNF-α to aggravate inflammation in NDFLD cells. PPARα positively bound to HIF-1α promoter. The silence of PPARα aggravated lipid deposition under normal or hypoxic environment in NAFLD cells. In addition, PPAR-α silence could decrease the expression of HIF-1α and ANGPTL4 in NAFLD cell model; moreover, the expression of APOE, A2m and TNFRSF11B and the production of TG and MDA were increased by PPAR-α suppression. Conclusion: HIF-1α plays a crucial role in the regulation of lipid metabolism through activating PPAR-α/ANGPTL4 signaling pathway in NAFLD.(AU)

Objetivo: La esteatohepatitis no alcohólica (EHNA) es una enfermedad del metabolismo aberrante de los lípidos. El factor inducible por hipoxia 1 (HIF-1α) es un factor de transcripción que desempeña una función importante en la adaptación de la afección de nivel de oxígeno bajo. En el presente documento, intentamos aclarar la relación entre HIF-1α y la EHNA. Métodos: Las células HepG2 se estimularon con ácido oleico (OA) y ácido palmítico (PA) para establecer un modelo in vitro de la EHNA. La expresión de los genes relacionados con el metabolismo de los lípidos, la unión de PPARα al promotor HIF-1α, el depósito de lípidos y el estrés oxidativo se detectaron mediante ensayos de qRT-PCR, inmunoelectrotransferencia, ensayo de inmunoprecipitación de cromatina (ChIP), ensayos de tinción de rojo aceite O y ELISA, respectivamente. Resultados: El silencio de HIF-1α promovió la acumulación de lípidos en las células de la EHNA, acompañada de un aumento significativo del contenido de triglicéridos (TG) y apolipoproteína B (ApoB). En las células HepG2 tratadas con OA/PA, la expresión de genes y proteínas relacionados con el metabolismo lipídico, incluidos APOE, A2m, TNFRSF11B, LDLr y SREBP2, y el depósito de lípidos intracelular se regularon al alza y se agravaron aún más después de silenciar HIF-1α. Además, la pérdida de HIF-1α podría elevar notablemente el contenido de MDA e inhibir las actividades de las enzimas antioxidantes beneficiosas SOD y GSH-Px para activar el estrés oxidativo, y promover la secreción de IL-6 pro-inflamatoria y TNF-α para agravar la inflamación en las células de la EHNA. PPARα se unió positivamente al promotor HIF-1α. El silencio de PPARα agravó el depósito de lípidos en un ambiente normal o hipóxico en las células de la EHNA. Además, el silencio de PPAR-α pudo disminuir la expresión de HIF-1α y ANGPTL4 en el modelo de células de la EHNA; por otra parte, la expresión de APOE, A2m y TNFRSF11B, y la producción de TG y MDA aumentaro,

Silencing HIF-1α aggravates non-alcoholic fatty liver disease in vitro through inhibiting PPAR-α/ANGPTL4 singling pathway.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is an aberrant lipid metabolism disease. Hypoxia inducible factor-1 (HIF-1α) is a transcription factor which plays an important part in adapting lower oxygen condition. Here, we aimed to clarify the relationship between HIF-1α and NAFLD. METHODS: HepG2 cells was stimulated by oleic acid (OA) and palmitic acid (PA) to establish in vitro model of NAFLD. The expression of lipid metabolism-related genes, the binding of PPARα to HIF-1α promoter, the lipid deposition, and oxidative stress were detected by qRT-PCR, western blot, Chip assay, Oil Red O staining and ELISA assays, respectively. RESULTS: HIF-1α silence promoted lipid accumulation in NAFLD cells, accompanying by the significantly increased contents of TG (triglyceride) and ApoB (apolipoprotein B). In HepG2 cells treated with OA/PA, the expression of lipid metabolism-related genes and proteins, including APOE, A2m, TNFRSF11B, LDLr, and SREBP2, and the intracellular lipid deposition were up-regulated and further aggravated after silencing HIF-1α. In addition, the loss of HIF-1α could remarkably elevate MDA contents while inhibit the activities of beneficial antioxidant enzymes SOD and GSH-Px to activate oxidative stress, and promote the secretion of pro-inflammatory IL-6 and TNF-α to aggravate inflammation in NDFLD cells. PPARα positively bound to HIF-1α promoter. The silence of PPARα aggravated lipid deposition under normal or hypoxic environment in NAFLD cells. In addition, PPAR-α silence could decrease the expression of HIF-1α and ANGPTL4 in NAFLD cell model; moreover, the expression of APOE, A2m and TNFRSF11B and the production of TG and MDA were increased by PPAR-α suppression. CONCLUSION: HIF-1α plays a crucial role in the regulation of lipid metabolism through activating PPAR-α/ANGPTL4 signaling pathway in NAFLD.

Adipocytes induce the resistance of ovarian cancer to carboplatin through ANGPTL4.

The resistance of cancer cells to carboplatin restricts their efficacy in the clinical setting, and a solution to reverse the resistance is urgently required for the treatment of ovarian cancer. An increasing number of studies have found associations between obesity and the incidence, and mortality rates of female cancer. However, the association between adipocytes and the resistance of ovarian cancer has rarely been reported. Based on this, the present study first revealed the inductive effect of adipocytes on the resistance of ovarian cancer to carboplatin using in vivo and in vitro experiments. Subsequently, it was identified that the angiopoietin­like 4 (ANGPTL4) secreted by adipocytes played a vital role in the resistance of ovarian cancer using bioinformatics analysis, cellular and molecular biological experiments, as well as forward and backward validation. The glycosylated ANGPTL4 protein could bind with integrin α5ß1 on the surface of ovarian cancer cells; following which, it could activate the c­myc/NF­κB pathway and stimulate the expression of the antiapoptotic protein Bcl­xL, as well as the ABC transporter family members ABCB1, ABCC1 and ABCG2. Thus, inducing the resistance of ovarian cancer to carboplatin. In conclusion, targeting the adipocyte­derived ANGPTL4 combined with the application of carboplatin contributes to the clinical treatment for ovarian cancer.

Pharmacological aspects of ANGPTL3 and ANGPTL4 inhibitors: New therapeutic approaches for the treatment of atherogenic dyslipidemia.

Among the determinants of atherosclerotic cardiovascular disease (ASCVD), genetic and experimental evidence has provided data on a major role of angiopoietin-like proteins 3 and 4 (ANGPTL3 and ANGPTL4) in regulating the activity of lipoprotein lipase (LPL), antagonizing the hydrolysis of triglycerides (TG). Indeed, beyond low-density lipoprotein cholesterol (LDL-C), ASCVD risk is also dependent on a cluster of metabolic abnormalities characterized by elevated fasting and post-prandial levels of TG-rich lipoproteins and their remnants. In a head-to-head comparison between murine models for ANGPTL3 and ANGPTL4, the former was found to be a better pharmacological target for the treatment of hypertriglyceridemia. In humans, loss-of-function mutations of ANGPTL3 are associated with a marked reduction of plasma levels of VLDL, low-density lipoprotein (LDL) and high-density lipoprotein (HDL). Carriers of loss-of-function mutations of ANGPTL4 show instead lower TG-rich lipoproteins and a modest but significant increase of HDL. The relevance of ANGPTL3 and ANGPTL4 as new therapeutic targets is proven by the development of monoclonal antibodies or antisense oligonucleotides. Studies in animal models, including non-human primates, have demonstrated that short-term treatment with monoclonal antibodies against ANGPTL3 and ANGPTL4 induces activation of LPL and a marked reduction of plasma TG-rich-lipoproteins, apparently without any major side effects. Inhibition of both targets also partially reduces LDL-C, independent of the LDL receptor. Similar evidence has been observed with the antisense oligonucleotide ANGPTL3-LRX. The genetic studies have paved the way for the development of new ANGPTL3 and 4 antagonists for the treatment of atherogenic dyslipidemias. Conclusive data of phase 2 and 3 clinical trials are still needed in order to define their safety and efficacy profile.

LMX1A inhibits C-Myc expression through ANGPTL4 to exert tumor suppressive role in gastric cancer.

Our research group has showed that the LIM homeobox transcription factor 1 alpha (LMX1A) is inactivated in gastric cancers. Overexpression of LMX1A inhibits tumor growth. However, the mechanisms remains unclear. Considering LMX1A as a transcription factor, a comparison of RNA-seq between gastric cancer cells (GCCs) and GCCs with LMX1A overexpressed was performed to identify genes transcriptionally activated by LMX1A. Among the potential LMX1A target genes, angiopoietin-like 4 (ANGPTL4) has been reported to be an important tumor suppressor and thus was selected for further validation and research. Both LMX1A and ANGPTL4 showed downregulated expression in gastric cancer samples. More importantly, the expression of LMX1A is positively correlated with ANGPTL4, without including other family members in gastric cancer cell lines. What's more, knockdown of ANGPTL4 rescued the tumor suppressive phenotype of LMX1A overexpression, which indicated that LMX1A upregulates ANGPTL4 to exert its role. Mechanistically, we found that LMX1A inhibited the expression of the oncogene C-Myc, which is alleviated by ANGPTL4 knockdown. In general, our results showed that LMX1A exerts its tumor suppressive role by activating ANGPTL4 to inhibit C-Myc.

Antibody Treatment against Angiopoietin-Like 4 Reduces Pulmonary Edema and Injury in Secondary Pneumococcal Pneumonia.

Secondary bacterial lung infection by Streptococcus pneumoniae (S. pneumoniae) poses a serious health concern, especially in developing countries. We posit that the emergence of multiantibiotic-resistant strains will jeopardize current treatments in these regions. Deaths arising from secondary infections are more often associated with acute lung injury, a common consequence of hypercytokinemia, than with the infection per se Given that secondary bacterial pneumonia often has a poor prognosis, newer approaches to improve treatment outcomes are urgently needed to reduce the high levels of morbidity and mortality. Using a sequential dual-infection mouse model of secondary bacterial lung infection, we show that host-directed therapy via immunoneutralization of the angiopoietin-like 4 c-isoform (cANGPTL4) reduced pulmonary edema and damage in infected mice. RNA sequencing analysis revealed that anti-cANGPTL4 treatment improved immune and coagulation functions and reduced internal bleeding and edema. Importantly, anti-cANGPTL4 antibody, when used concurrently with either conventional antibiotics or antipneumolysin antibody, prolonged the median survival of mice compared to monotherapy. Anti-cANGPTL4 treatment enhanced immune cell phagocytosis of bacteria while restricting excessive inflammation. This modification of immune responses improved the disease outcomes of secondary pneumococcal pneumonia. Taken together, our study emphasizes that host-directed therapeutic strategies are viable adjuncts to standard antimicrobial treatments.IMPORTANCE Despite extensive global efforts, secondary bacterial pneumonia still represents a major cause of death in developing countries and is an important cause of long-term functional disability arising from lung tissue damage. Newer approaches to improving treatment outcomes are needed to reduce the significant morbidity and mortality caused by infectious diseases. Our study, using an experimental mouse model of secondary S. pneumoniae infection, shows that a multimodal treatment that concurrently targets host and pathogen factors improved lung tissue integrity and extended the median survival time of infected mice. The immunoneutralization of host protein cANGPTL4 reduced the severity of pulmonary edema and damage. We show that host-directed therapeutic strategies as well as neutralizing antibodies against pathogen virulence factors are viable adjuncts to standard antimicrobial treatments such as antibiotics. In view of their different modes of action compared to antibiotics, concurrent immunotherapies using antibodies are potentially efficacious against secondary pneumococcal pneumonia caused by antibiotic-resistant pathogens.

RREB1-induced upregulation of the lncRNA AGAP2-AS1 regulates the proliferation and migration of pancreatic cancer partly through suppressing ANKRD1 and ANGPTL4.

Long noncoding RNAs (lncRNAs) have been reported to be involved in a variety of human diseases, including cancers. However, their mechanisms have not yet been fully elucidated. We investigated lncRNA changes that may be associated with pancreatic cancer (PC) by analyzing published microarray data, and identified AGAP2-AS1 as a relatively overexpressed lncRNA in PC tissues. qRT-PCR assays were performed to examine expression levels of AGAP2-AS1. MTT assays, colony formation assays, and EdU assays were used to determine the proliferative capacity of cells. Flow cytometry and TUNEL assays were used to study the regulation of AGAP2-AS1 in the cell cycle and apoptosis. Transwell experiments were used to study changes in cell invasion and metastasis, and a nude mouse model was established to assess the effects of AGAP2-AS1 on tumorigenesis in vivo. RNA sequencing was performed to probe AGAP2-AS1-related pathways. Subcellular fractionation and FISH assays were used to determine the distribution of AGAP2-AS1 in PC cells, and RIP and ChIP were used to determine the molecular mechanism of AGAP2-AS1-mediated regulation of potential target genes. Increased expression of AGAP2-AS1 was associated with tumor size and pathological stage progression in patients with PC. RREB1 was found to activate transcription of AGAP2-AS1 in PC cells. AGAP2-AS1 affected proliferation, apoptosis, cycle arrest, invasion, and metastasis of PC cells in vitro, and AGAP2-AS1 regulated PC proliferation in vivo. Furthermore, AGAP2-AS1 epigenetically inhibited the expression of ANKRD1 and ANGPTL4 by recruiting zeste homolog 2 (EZH2), thereby promoting PC proliferation and metastasis. In summary, our data show that RREB1-induced upregulation of AGAP2-AS1 regulates cell proliferation and migration in PC partly through suppressing ANKRD1 and ANGPTL4 by recruiting EZH2. AGAP2-AS1 represents a potential target for the diagnosis and treatment of PC in the future.

Targeting metabolic flexibility via angiopoietin-like 4 protein sensitizes metastatic cancer cells to chemotherapy drugs.

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.

Knockdown of angiopoietin-like 4 inhibits the development of human gastric cancer.

Human gastric cancer (GC) is the second most common cause of cancer-related deaths worldwide and is one of the most common metastatic cancers. Tumor proliferation, apoptosis, metastasis and invasion are important predictors of the invasiveness of GC and are key factors in cancer-induced death. Angiopoietin-like 4 (ANGPTL4) is a secreted protein that belongs to the angiopoietin (ANGPTL) family and is involved in the regulation of cancer metastasis. However, whether ANGPTL4 plays a role in the progression of GC remain unclear. In the present study, immunoreactivity of ANGPTL4 demonstrated that ANGPTL4 expression was upregulated in GC tissues with the development of GC. The siRNA targeting ANGPTL4 effectively knocked down ANGPTL4 in the SNU­1 and BGC823 cell lines at the mRNA and protein levels. Following ANGPTL4 downregulation, the proliferation and invasion abilities of GC cell lines were suppressed as determined by MTT and Transwell assays, and cell apoptosis level and sensitivity to cisplatin were increased as determined by flow cytometry and MTT assay. In conclusion, these findings suggest that ANGPTL4 may be a new potential therapeutic target for GC.

Cholecystokinin is involved in triglyceride fatty acid uptake by rat adipose tissue.

The incorporation of plasma triglyceride (TG) fatty acids to white adipose tissue (WAT) depends on lipoprotein lipase (LPL), which is regulated by angiopoietin-like protein-4 (ANGPTL-4), an unfolding molecular chaperone that converts active LPL dimers into inactive monomers. The production of ANGPTL-4 is promoted by fasting and repressed by feeding. We hypothesized that the postprandial hormone cholecystokinin (CCK) facilitates the storage of dietary TG fatty acids in WAT by regulating the activity of the LPL/ANGPTL-4 axis and that it does so by acting directly on CCK receptors in adipocytes. We report that administration of CCK-8 (a bioactive fragment of CCK) to rats: (i) reduces plasma ANGTPL-4 levels; (ii) represses Angptl-4 expression in WAT and (iii) simultaneously enhances LPL activity in this tissue without inducing Lpl expression. In vivo CCK-8 effects are specifically antagonized by the CCK-2 receptor (CCK-2R) antagonist, L-365,260. Moreover, CCK-8 downregulates Angptl-4 expression in wild-type pre-adipocytes, an effect that is not observed in engineered pre-adipocytes lacking CCK-2R. These effects have functional consequences as CCK-8 was found to promote the uptake of dietary fatty acids by WAT, as demonstrated by means of proton nuclear magnetic resonance (1H-NMR). The efficacy of acute CCK-8 administration was not reduced after chronic CCK-8 treatment. Moreover, the effects of CCK-8 on WAT were not associated to the increase of circulating insulin. Our results show that cholecystokinin promotes lipid storage in WAT by acting on adipocyte CCK-2R, suggesting a pivotal role for CCK in TG homeostasis.

Buyang Huanwu Tang improves denervation-dependent muscle atrophy by increasing ANGPTL4, and increases NF-κB and MURF1 levels.

Denervated-dependent skeletal muscle atrophy (DSMA) is a disorder caused by the peripheral neuro­disconnection of skeletal muscle. The current study aimed to investigate the molecular mechanism and potential therapeutic strategies for the DSMA. A DSMA rat model was established. A lentiviral vector expressing small interfering RNA (siRNA) targeting angiopoietin­like protein 4 (ANGPTL4) was generated and injected into the rats that were also treated with Buyang Huanwu Tang (BYHWT). Reverse transcription­quantitative polymerase chain reaction was performed to examine ANGPTL4 mRNA expression in anterior cervical muscle samples. Western blot assay was used to evaluate ANGPTL4, nuclear factor­κB (NF­κB) and muscle RING­finger protein­1 (MURF1) expression. The ultrastructure of muscle tissues was viewed using transmission electron microscopy. The cell apoptosis in muscle tissues was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling. The results indicated that BYHWT treatment increased ANGPTL4 mRNA and protein levels in muscle tissues. The suppression of ANGPTL4 using siRNA significantly increased inflammatory cells compared with the control siRNA group. BYHWT protected the ultrastructure muscle tissues and inhibited cell apoptosis in the DSMA model. The protective effect of BYHWT protected may be mediated by increased expression of NF­κB p65 and MURF1. In conclusion, BYHWT may improve denervation­dependent muscle atrophy by increasing ANGPTL4 expression, involving NF­κB and MURF1 signaling.

Paeoniflorin ameliorates Adriamycin-induced nephrotic syndrome through the PPARγ/ANGPTL4 pathway in vivo and vitro.

Paeoniflorin (PF), an effective composition that is extracted from Radix Paeoniae Alba, plays a role in protecting against various kidney diseases. However, the mechanism of PF on nephrotic syndrome (NS) remains unclear. The aim of this study was to investigate the protective role of PF on Adriamycin (ADR)-induced NS in vivo and vitro as well as its potential mechanism. In animal study, PF significantly decreased the levels of 24-h urine protein, blood urea nitrogen, serum creatinine, total cholesterol and triglycerides in NS rats, but increased the total protein and albumin levels. Hematoxylin-eosin (HE) staining revealed that the kidney lesion was resolved upon PF treatment. After treatment with PF, the morphology and number of podocytes in renal tissue were restored to normal. PF increased expression of synaptopodin and decreased expression of desmin, demonstrating a protective effect in podocyte injury. Further studies revealed that PF upregulated Peroxisome proliferator-activated receptor gamma (PPARγ) and restrained Angiopointin-like 4 (ANGPTL4) in kidney tissue. In vitro study, PF reduced Caspase3 and Bax and increased Bcl-2, indicating that the apoptosis rate of podocytes induced by ADR was reduced by PF. Furthermore, PF ameliorated podocyte injury by upregulating synaptopodin and reducing desmin. In accordance with animal study, PF downregulated ANGPTL4 by activating PPARγ. However, the therapeutic effects of PF were reversed by GW9662 (PPARγ inhibitor), likely by suppressing ANGPTL4 degradation. In general, these results demonstrate that PF has a good therapeutic effect on NS by activating PPARγ and subsequently inhibiting ANGPTL4.

Hypoxia-induced angiopoietin-like protein 4 as a clinical biomarker and treatment target for human prostate cancer.

Angiopoietin-like protein 4 (ANGPTL4) is a multifunctional protein, playing roles in glucose and lipid metabolism, inflammation, angiogenesis, and tumorigenesis. Recent research suggests that ANGPTL4 is induced by hypoxia and is a useful diagnostic or prognostic marker for various cancers. However, it remains unclear whether ANGPTL4 expression influences prostate cancer. Here we examined the biological and clinical relevance of ANGPTL4 expression in prostate cancer. Firstly we examined ANGPTL4 expression in the prostate cancer cell lines LNCaP and LNCaP/CH incubated at 1% O2 for at least 6 months. We compared cellular proliferation, migration, and ANGPTL4 secretion in a culture medium between these cell lines. In addition, we investigated the effect of various concentrations of recombinant ANGPTL4 protein (rANGPTL4) on cellular proliferation and intracellular signaling pathways. Moreover, we used ANGPTL4 knockdown by RNA interference to investigate the influence of ANGPTL4 expression on these cell lines. Finally, we investigated the correlation between ANGPTL4 expression in prostate cancer specimens and clinicopathological parameters using immunohistochemistry. Our data suggested that the expression of ANGPTL4 in hypoxic conditions was 14.4-fold higher than that in normoxic condition. ANGPTL4 secretion in the culture medium increased 7.0-fold. In addition, rANGPTL4 increased cellular proliferation 1.72-fold via Akt activation. Moreover, ANGPTL4 knockdown decreased cell growth and its secretion by 25.7 and 41.4%, respectively, compared with the control. A multivariate analysis showed that positive ANGPTL4 expression in the resected specimens was an independent prognostic indicator of biochemical recurrence (P=0.03, hazard ratio = 2.02). Our results show that ANGPTL4 is induced by hypoxia and promotes cancer progression via the activated PI3K/Akt pathway. Moreover, ANGPTL4 can be used as a prognostic marker for prostate cancer patients undergoing radical prostatectomy.