ANGPTL3 Downregulation Increases Intracellular Lipids by Reducing Energy Utilization.

BACKGROUND: ANGPTL3 (angiopoietin-like protein 3) is a circulating protein with a key role in maintaining lipoprotein homeostasis. A monoclonal antibody against ANGPTL3 is an approved and well-tolerated treatment to reduce lipoproteins in familial hypercholesterolemia homozygotes. However, the reduction of hepatic ANGPTL3 synthesis using an antisense oligonucleotide unexpectedly resulted in a dose-dependent increase in liver lipid content and circulating transaminases, resulting in the termination of the clinical trial. Meanwhile, the use of silencing RNAs remains an area of active investigation. Our study sought to investigate whether intracellular downregulation of ANGPTL3 may lead to a primary increase in neutral lipids within the hepatocyte. METHODS: We downregulated ANGPTL3 by silencing RNA in primary human hepatocytes 3-dimensional spheroids, HepG2/LX-2 3-dimensional spheroids, and in HepG2, Hep3B2, and Huh7 cultured in 2 dimensions. RESULTS: ANGPTL3 downregulation increased neutral lipids in all models investigated. Interestingly, ANGPTL3 induced lower intracellular deiodinase type 1 protein levels resulting in a reduction in beta-oxidation and causing an increase in triglycerides stored in lipid droplets. CONCLUSIONS: In conclusion, intracellular ANGPTL3 downregulation by silencing RNA led to an increase in triglycerides content due to a reduction in energy substrate utilization resembling a primary intracellular hepatocyte hypothyroidism.

The Fibrinogen-like Domain of ANGPTL3 Facilitates Lipolysis in 3T3-L1 Cells by Activating the Intracellular Erk Pathway.

BACKGROUND: ANGPTL3 stimulates lipolysis in adipocytes, but the underlying molecular mechanism is yet unknown. The C-terminal fibrinogen-like domain of ANGPTL3 (ANGPTL3-Fld) activates the AKT pathway in endothelial cells. Hence, we evaluated whether ANGPTL3-Fld stimulates lipolysis in adipocytes through the MAPK kinase pathway. MATERIALS AND METHODS: 3T3-L1 adipocytes were treated with isoproterenol (ISO), ANGPTL3-Fld, or both. Lipolysis was evaluated through the release of free fatty acids (FFAs) in the culture medium. The activation status of intracellular kinases was evaluated with and without the inhibition of the BRAF-ERK arm of the MAPK pathway. RESULTS: ANGPTL3-Fld alone was not able to activate lipolysis, while the combination of ANGPTL3-Fld and ISO determined a 10-fold enrichment of the FFA concentration in the culture medium with an incremental effect (twofold) when compared with ISO alone. ANGPTL3-Fld alone inhibited hormone-sensitive lipase (HSL), whereas the treatment with ISO induced the activation of HSL. The net balance of ANGPTL3-Fld and ISO cotreatment resulted in HSL activation. The results indicate that ANGPTL3-Fld generated an intracellular activation signal involving the MAPK-ERK pathway, possibly through the PDGFRß-PLCγ-AMPK axis. CONCLUSION: ANGPTL3-Fld appears to act as a facilitator of lipolysis in adipocytes, and this effect was driven by a signal mediated by a pathway that is different from the canonical ß-adrenergic stimulus.

ANGPTL3 impacts proteinuria and hyperlipidemia in primary nephrotic syndrome.

BACKGROUND: It is unclear why primary nephrotic syndrome (PNS) patients often have dyslipidemia. Recent studies have shown that angiopoietin-like protein 3 (ANGPTL3) is an important regulator of lipid metabolism. In this study, we explored how ANGPTL3 impacts dyslipidemia during PNS development. METHODS: We measured the serum levels of ANGPTL3 in PNS patients (n=196). Furthermore, the degree of proteinuria and lipid metabolism were examined in angptl3-overexpressing transgenic (angptl3-tg) mice at different ages. Moreover, in this study, we used the clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system to create angptl3-knockout (angptl3-/-) mice to investigate lipopolysaccharide (LPS)-induced nephrosis. RESULTS: Compared with that in the healthy group, the serum level of ANGPTL3 in the PNS group was significantly increased (32 (26.35-39.66) ng/ml vs. 70.44 (63.95-76.51) ng/ml, Z =-4.81, P < 0.001). There were significant correlations between the serum level of ANGPTL3 and the levels of cholesterol (r=0.34, P < 0.001), triglycerides (r= 0.25, P = 0.001) and low-density lipoprotein (r= 0.50, P < 0.001) in PNS patients. With increasing age, angptl3-tg mice exhibited increasingly severe hypertriglyceridemia and proteinuria. The pathological features of angptl3-tg mice included rich lipid droplet deposition in hepatocytes and diffuse podocyte effacement. Compared to wild-type mice, angptl3-/- mice showed significantly lower degrees of lipid dysfunction and proteinuria after stimulation with LPS. The effects of ANGPTL3 on nephrotic dyslipidemia were confirmed in cultured hepatocytes subjected to angptl3 knockdown or overexpression. Finally, significant alterations in lipoprotein lipase (LPL) levels were observed in liver tissues from Angptl3-/- and wild-type mice stimulated with LPS. CONCLUSIONS: ANGPTL3 could be involved in the development of dyslipidemia, as well as proteinuria, during PNS pathogenesis. Inhibition of LPL expression may the mechanism by which ANGPTL3 induces hyperlipidemia in PNS.

Addressing dyslipidemic risk beyond LDL-cholesterol.

Despite the success of LDL-lowering drugs in reducing cardiovascular disease (CVD), there remains a large burden of residual disease due in part to persistent dyslipidemia characterized by elevated levels of triglyceride-rich lipoproteins (TRLs) and reduced levels of HDL. This form of dyslipidemia is increasing globally as a result of the rising prevalence of obesity and metabolic syndrome. Accumulating evidence suggests that impaired hepatic clearance of cholesterol-rich TRL remnants leads to their accumulation in arteries, promoting foam cell formation and inflammation. Low levels of HDL may associate with reduced cholesterol efflux from foam cells, aggravating atherosclerosis. While fibrates and fish oils reduce TRL, they have not been uniformly successful in reducing CVD, and there is a large unmet need for new approaches to reduce remnants and CVD. Rare genetic variants that lower triglyceride levels via activation of lipolysis and associate with reduced CVD suggest new approaches to treating dyslipidemia. Apolipoprotein C3 (APOC3) and angiopoietin-like 3 (ANGPTL3) have emerged as targets for inhibition by antibody, antisense, or RNAi approaches. Inhibition of either molecule lowers TRL but respectively raises or lowers HDL levels. Large clinical trials of such agents in patients with high CVD risk and elevated levels of TRL will be required to demonstrate efficacy of these approaches.

Angiopoietin-like 3 (ANGPTL3) drives cell proliferation, migration and angiogenesis in cervical cancer via binding to integrin alpha v beta 3.

Angiopoietin-like 3 (ANGPTL3) has been uncovered to play an oncogenic role in several kinds of human malignancies. Nevertheless, whether ANGPTL3 functions in cervical cancer (CC) has not yet been reported. This paper is intended to explore the impact of ANGPTL3 on CC cells and elucidate the potential mechanism. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were performed to analyze the ANGPTL3 expression. Western blot was also performed to examine integrin αvß3 protein level. Cell proliferation was evaluated by MTT assay, EdU staining and Western blot analysis. In addition, the migratory and invasive abilities of cells were, respectively, estimated by wound healing and transwell assays. Tube formation assay was performed to determine endothelial cell angiogenesis. Levels of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) were measured by ELISA. As a result, ANGPTL3 expression was significantly higher in CC cells relative to that in normal cervical cells. Silencing of ANGPTL3 suppressed cell proliferation, migration and invasion. Besides, downregulation of ANGPTL3 inhibited human umbilical vein endothelial cell (HUVEC) angiogenesis and repressed protein level of integrin alpha v beta 3 (αvß3). Upregulation of αvß3 offsets the inhibitory effect of ANGPTL3 on proliferation, migration and invasion in CC cells. Upregulated expression of αvß3 promoted blood vessel formation and secretions of VEGF and VEGFR2. In conclusion, ANGPTL3 silencing may serve as a tumor suppressor in CC through integrin αvß3, which provides a potentially novel therapeutic target for patients with CC.

Selective targeting of angiopoietin-like 3 (ANGPTL3) with vupanorsen for the treatment of patients with familial partial lipodystrophy (FPLD): results of a proof-of-concept study.

BACKGROUND: Familial partial lipodystrophy (FPLD) is a rare disease characterized by selective loss of peripheral subcutaneous fat, associated with dyslipidemia and diabetes mellitus. Reductions in circulating levels of ANGPTL3 are associated with lower triglyceride and other atherogenic lipids, making it an attractive target for treatment of FPLD patients. This proof-of-concept study was conducted to assess the efficacy and safety of targeting ANGPTL3 with vupanorsen in patients with FPLD. METHODS: This was an open-label study. Four patients with FPLD (two with pathogenic variants in LMNA gene, and two with no causative genetic variant), diabetes (HbA1c ≥ 7.0 % and ≤ 12 %), hypertriglyceridemia (≥ 500 mg/dL), and hepatic steatosis (hepatic fat fraction, HFF ≥ 6.4 %) were included. Patients received vupanorsen subcutaneously at a dose of 20 mg weekly for 26 weeks. The primary endpoint was the percent change from baseline in fasting triglycerides at Week 27. Other endpoints analyzed at the same time point included changes in ANGPTL3, fasting lipids and lipoproteins, insulin secretion/sensitivity, postprandial lipids, and glycemic changes in response to a mixed meal test, HFF measured by MRI, and body composition measured by dual-energy absorptiometry (DEXA). RESULTS: Baseline mean ± SD fasting triglyceride level was 9.24 ± 4.9 mmol/L (817.8 ± 431.9 mg/dL). Treatment resulted in reduction in fasting levels of triglycerides by 59.9 %, ANGPTL3 by 54.7 %, and in several other lipoproteins/lipids, including very low-density lipoprotein cholesterol by 53.5 %, non-high-density lipoprotein cholesterol by 20.9 %, and free fatty acids (FFA) by 41.7 %. The area under the curve for postprandial triglycerides, FFA, and glucose was reduced by 60 %, 32 %, and 14 %, respectively. Treatment with vupanorsen also resulted in 55 % reduction in adipose tissue insulin resistance index, while other insulin sensitivity indices and HbA1c levels were not changed. Additional investigations into HFF and DEXA parameters suggested dynamic changes in fat partitioning during treatment. Adverse events observed were related to common serious complications associated with diabetes and FPLD. Vupanorsen was well tolerated, and there was no effect on platelet count. CONCLUSIONS: Although limited, these results suggest that targeting ANGPTL3 with vupanorsen could address several metabolic abnormalities in patients with FPLD.

Vaccine targeting ANGPTL3 ameliorates dyslipidemia and associated diseases in mouse models of obese dyslipidemia and familial hypercholesterolemia.

Dyslipidemia is a risk factor for cardiovascular disease (CVD), a major cause of death worldwide. Angiopoietin-like protein 3 (ANGPTL3), recognized as a new therapeutic target for dyslipidemia, regulates the metabolism of low-density lipoprotein-cholesterol (LDL-C) and triglycerides. Here, we design 3 epitopes (E1-E3) for use in development of a peptide vaccine targeting ANGPTL3 and estimate effects of each on obesity-associated dyslipidemia in B6.Cg-Lepob /J (ob/ob) mice. Vaccination with the E3 (32EPKSRFAMLD41) peptide significantly reduces circulating levels of triglycerides, LDL-C, and small dense (sd)-LDL-C in ob/ob mice and decreases obese-induced fatty liver. Moreover, E3 vaccination does not induce cytotoxicity in ob/ob mice. Interestingly, the effect of E3 vaccination on dyslipidemia attenuates development of atherosclerosis in B6.KOR/StmSlc-Apoeshl mice fed a high-cholesterol diet, which represent a model of severe familial hypercholesterolemia (FH) caused by ApoE loss of function. Taken together, ANGPTL3 vaccination could be an effective therapeutic strategy against dyslipidemia and associated diseases.

Human Angiopoietin-like Protein 3/ANGPTL3 Antibodies: Adding to the Armamentarium in the Management of Dyslipidemia.

ABSTRACT: Cardiovascular (CV) disease remains the leading cause of death in the United States. In addition to lifestyle modifications, current guidelines primarily focus on lowering low-density lipoprotein cholesterol (LDL-C) to reduce atherosclerotic CV disease risk. However, despite aggressive management, a degree of residual risk remains, suggesting that focusing on lowering LDL-C alone may be inadequate and that other lipid parameters may need to be targeted. In patients who remain at high risk despite current pharmacologic options either because of inadequate response, elevated levels of other lipoproteins, or those who have genetic variants predisposing them to atherosclerotic CV disease, additional treatment strategies continue to be sought. One such group is the homozygous familial hypercholesterolemia population, especially those patients carrying the null low-density lipoprotein receptor mutation as they often fail to derive the same benefit from traditional LDL-C lower strategies such as statins and proprotein convertase subtilisin/kexin type 9 inhibitors that work by upregulating the LDL receptor. Emerging data also suggest that patients with increased levels of triglyceride-rich lipoproteins are also at increased risk as elevated levels are proposed to have a role in various pathways promoting atherogenesis. Angiopoietin-life protein 3 (ANGLTPL3) has recently become a target of interest because of the discovery that inhibiting its action leads to reductions in lipid parameters. Although the mechanism of action of ANGLTPL3 inhibitors is independent of the LDL receptor, their ability to significantly lower triglycerides and LDL-C make them an attractive option particularly in patients with homozygous familial hypercholesterolemia and hypertriglyceridemia. The efficacy and safety of 2 ANGLTPL3 inhibitor agents have been evaluated in clinical trials. In this review, the lipid lowering, metabolic effects, and safety are reported. Ongoing trials assessing CV outcomes as well as long-term safety data are still needed to provide a more definitive role for these agents in the overall management in these populations.

ANGPTL3 Overexpression Suppresses the Development of Oncogenic Properties in Renal Cell Carcinoma via the Wnt/ß-Catenin Signaling Pathway and Predicts Good Prognosis.

Angiopoietin-like 3 (ANGPTL3), which is involved in new blood vessel growth, has been reported to exhibit an abnroaml expression in many different cancers. However, the expressing pattern and functions of ANGPTL3 renal cell carcinoma (RCC) were rarely reported. In this study, we observed that ANGPTL3 expression was distinctly downregulated in both RCC specimens from TCGA datasets and cell lines. Survival assays also revealed that patients with low ANGPTL3 expression exhibited a shorter overall survival and disease-free survival than those with high ANGPTL3 expression. Cell counting kit-8 (CCK-8) assay, Colony formation assay, and flow cytometry showed that overexpression of ANGPTL3 distinctly suppressed the proliferation of RCC cells, and promoted apoptosis. Transwell assays and Wound healing assays revealed that ANGPTL3 upregulation suppressed the migration and invasion of RCC cells. Then, we explored whether ANGPTL3 dysregulation influenced the alteration of Wnt/ß-catenin signaling using TOP/FOP flash reporter assays and western blot. The results showed that overexpression of ANGPTL3 distinctly suppressed the activity of Wnt/ß-catenin signaling. Overall, our results confirmed that overexpression of ANGPTL3 was related to the malignancy and good prognosis of RCC patients, and ANGPTL3 upregulation inhibited the tumor proliferation and metastasis via the Wnt/ß-catenin pathway. ANGPTL3 may be a novel therapeutic target and a prognostic biomarker for RCC patients.

Antisense oligonucleotide-mediated inhibition of angiopoietin-like protein 3 increases reverse cholesterol transport in mice.

Supported by an abundance of experimental and genetic evidence, angiopoietin-like protein 3 (ANGPTL3) has emerged as a promising therapeutic target for cardiovascular disease. ANGPTL3 is primarily produced by the liver and is a potent modulator of plasma lipids and lipoproteins. Experimental models and subjects with loss-of-function Angptl3 mutations typically present with lower levels of HDL-C than noncarriers. The effect of ANGPTL3 on HDL-C is typically attributed to its function as an inhibitor of the enzyme endothelial lipase. The ability to facilitate reverse cholesterol transport (RCT), the transport of cholesterol from peripheral tissues back to the liver, is a proposed antiatherogenic property of HDL. However, the effect of ANGPTL3 inhibition on RCT remains unclear. Here, we performed a series of dose-response and RCT studies using an Angptl3 antisense oligonucleotide (ASO) in mouse models with varying plasma lipid profiles ranging from moderately to severely hyperlipidemic. Angptl3 ASO-mediated reduction in HDL-C was limited to the model with moderate lipidemia, where the majority of plasma cholesterol was associated with HDL. Surprisingly, regardless of the effect on HDL-C, treatment with the Angptl3 ASO enhanced RCT in all models tested. The observations from the RCT assays were confirmed in HDL clearance studies, where mice treated with the Angptl3 ASO displayed increased plasma clearance and hepatic uptake of labeled HDL. The results from our studies suggest that inhibition of ANGPTL3 not only reduces levels of proatherogenic lipids but also improves HDL-mediated RCT.

Angiopoietin-like 3 inhibition of endothelial lipase is not modulated by angiopoietin-like 8.

High plasma triglyceride (TG) levels and low HDL-C levels are risk factors for atherosclerosis and cardiovascular disease. Both plasma TG and HDL-C levels are regulated in part by the circulating inhibitor, angiopoietin-like 3 (ANGPTL3). ANGPTL3 inhibits the phospholipase, endothelial lipase (EL), which hydrolyzes the phospholipids of HDL, thus decreasing plasma HDL levels. ANGPTL3 also inhibits LPL, the lipase primarily responsible for the clearance of TGs from the circulation. Previous studies have shown that ANGPTL3 requires complex formation with the related ANGPTL protein, angiopoietin-like 8 (ANGPTL8), to efficiently inhibit LPL, but the role of ANGPTL8 in EL inhibition is not known. In this study, we characterized inhibition and binding of EL by ANGPTL3 and investigated the role of ANGPTL8 in EL inhibition. We found that inhibition of EL by ANGPTL3 was dose dependent and temperature dependent. Interestingly, this inhibition was diminished when EL was bound to endothelial cells or in the presence of heparin. Unlike previous findings with LPL, we found that ANGPTL8 did not significantly alter the binding or the inhibition of EL by ANGPTL3. In addition, we found that a common ANGPTL8 variant, which encodes an R59W mutation, altered the ability of ANGPTL3 to bind and inhibit LPL but not EL. Together, our data indicate that ANGPTL8 is not necessary for EL inhibition. We conclude that ANGPTL8 is specific for the regulation of TG-rich lipoproteins through the LPL pathway and that therapeutically targeting ANGPTL8 for the treatment of hypertriglyceridemia or cardiovascular disease may have different outcomes than targeting ANGPTL3.

Efficacy and Safety of Evinacumab for the Treatment of Hypercholesterolemia: A Meta-Analysis.

ABSTRACT: Angiopoietin-like protein 3 is essential in lipid metabolism regulation. However, the efficacy and safety of evinacumab (angiopoietin-like protein 3 inhibition drug) for hypercholesterolemia treatment is unknown. In this study, a meta-analysis of randomized controlled trials (RCTs) was conducted to assess the efficacy and safety of evinacumab. RCTs published between January 1, 2000, and November 1, 2020, were obtained from PubMed, Embase, and Cochrane Library. All RCTs evaluating the efficacy and safety of evinacumab were included without language restrictions. Our primary end points included the percent change of low-density lipoprotein cholesterol (LDL-C) from baseline and the incidence of at least one treatment emergent adverse events including nasopharyngitis, influenza-like illness, headache, dizziness, injection-site reaction, increased aspartate aminotransferase, increased alanine aminotransferase, and any other discomfort during treatments. Percentage changes of triglycerides and high-density lipoprotein cholesterol (HDL-C) from baseline indicated secondary end points. A random-effects model was used to assess pooled data if there was moderate to high heterogeneity between studies. Four studies with 5 RCTs (568 participants) were identified. Evinacumab significantly reduced LDL-C [mean difference (MD) -33.123%, 95% confidence interval (CI), -48.639% to -17.606%, P < 0.0001], triglycerides (MD -50.959%, 95% CI, -56.555% to -45.362%, P < 0.0001), and HDL-C (MD -12.773%, 95% CI, -16.359% to -9.186%, P < 0.0001) compared with placebo. The incidence of at least 1 treatment emergent adverse events was not significantly different between evinacumab and placebo groups (relative risk 1.080, 95% CI, 0.901-1.296, P = 0.405). Evinacumab decreased triglycerides, LDL-C, and HDL-C without significant adverse effects, indicating that it can be a therapeutic strategy for hypercholesterolemia.