Angiopoietin-like proteins 8 knockout reduces intermittent hypoxia-induced vascular remodeling in a murine model of obstructive sleep apnea.

OBJECTIVE: Obstructive sleep apnea (OSA) is a major risk factor for cardiovascular mortality. Apnea-induced chronic intermittent hypoxia (CIH) is a primary pathophysiological manifestation of OSA that promotes various cardiovascular alterations, such as aortic vascular remodeling. In this study, we investigated the association between angiopoietin-like proteins 8 (ANGPTL8) and CIH-induced aortic vascular remodeling in mice. METHODS: C57BL/6J male mice were divided into four groups: Normoxia group, ANGPTL8-/- group, CIH group, CIH + ANGPTL8-/- group. Mice in the normoxia group and ANGPTL8-/- group received no treatment, while mice in the CIH and CIH + ANGPTL8-/- group were subjected to CIH (21%-5% O2, 180 s/cycle, 10 h/day) for 6 weeks. At the end of the experiments, intima-media thickness (IMT), elastin disorganization, and aortic wall collagen abundance were assessed in vivo. Immunohistochemistry and Western-blot were used to detect endoplasmic reticulum stress (ERS) and aortic vascular smooth muscle cell proliferation. ANGPTL8 shRNA and ANGPL8 overexpression were used in aortic vascular smooth muscle cells to investigate the mechanism of ANGPTL8 in CIH. RESULTS: Compared to the control group, CIH exposure significantly increased intima-media thickness (IMT), elastic fibers disorganization, and aortic wall collagen abundance. CIH also significantly increased blood pressure, induced hyperlipidemia, as well as the expression of ERS protein activating transcription factor-6 (ATF6) and aortic vascular smooth muscle cell proliferation. Contrary, ANGPTL8-/- significantly mitigated the CIH-induced vascular remodeling; ANGPTL8-/- decreased CIH-induced hypertension and hyperlipidemia, inhibited the protein expression of ATF6, and aortic vascular smooth muscle cell proliferation. Moreover, our in vitro study suggested that CIH could induce ANGPTL8 expression via hypoxia-inducible factor (HIF-1α); ANGPTL8 induced proliferation of aortic vascular smooth muscle cells via the ERS pathway. CONCLUSION: ANGPTL8-/- can prevent CIH-induced aortic vascular remodeling, probably through the inhibition of the ERS pathway. Therefore, ANGPTL8 might be a potential target in CIH-induced aortic vascular remodeling.

Deciphering the expression dynamics of ANGPTL8 associated regulatory network in insulin resistance using formal modelling approaches.

ANGPTL8 is a recently identified novel hormone which regulates both glucose and lipid metabolism. The increase in ANGPTL8 during compensatory insulin resistance has been recently reported to improve glucose tolerance and a part of cytoprotective metabolic circuit. However, the exact signalling entities and dynamics involved in this process have remained elusive. Therefore, the current study was conducted with a specific aim to model the regulation of ANGPTL8 with emphasis on its role in improving glucose tolerance during insulin resistance. The main contribution of this study is the construction of a discrete model (based on kinetic logic of René Thomas) and its equivalent Stochastic Petri Net model of ANGPTL8 associated Biological Regulatory Network (BRN) which can predict its dynamic behaviours. The predicted results of these models are in-line with the previous experimental observations and provide comprehensive insights into the signalling dynamics of ANGPTL8 associated BRN. The authors' results support the hypothesis that ANGPTL8 plays an important role in supplementing the insulin signalling pathway during insulin resistance and its loss can aggravate the pathogenic process by quickly leading towards Diabetes Mellitus. The results of this study have potential therapeutic implications for treatment of Diabetes Mellitus and are suggestive of its potential as a glucose-lowering agent.

Familial combined hypolipidemia: angiopoietin-like protein-3 deficiency.

PURPOSE OF REVIEW: Angiopoietin-like protein-3 (ANGPTL3) is emerging as a key player in lipoprotein transport with an expanding role on fatty acid and glucose metabolism. Its deficiency is associated with a favorable metabolic profile. The present review will highlight the recent understanding of metabolic and cardiovascular consequences of ANGPTL3 inactivation by considering both genetic and pharmacological investigations. RECENT FINDINGS: Experimental studies have further illustrated the complex interplay between ANGPTL3 and ANGPTL4-8 in orchestrating lipid transport in different nutritional status. Individuals with familial combined hypolipidemia due to homozygous loss-of-function mutations in ANGPTL3 gene showed improved metabolism of triglyceride-rich lipoproteins during fasting and postprandial state and increased fatty acid oxidation and insulin sensitivity. Moreover, mendelian randomizations studies demonstrated that partial ANGPTL3 deficiency associates with reduced risk of atherosclerotic cardiovascular events and, eventually, diabetes mellitus. Finally, inactivation of ANGPTL3, using either a specific mAb or antisense oligonucleotide, was reported to reduce plasma levels of atherogenic lipoprotein in humans and improve hepatic fat infiltration in animal models. SUMMARY: Human and animal studies have further dissected the complex role of ANGPTL3 in the regulation of energy substrate metabolism. Moreover, genetic and pharmacological investigations have convincingly indicated that the inactivation of ANGPTL3 may be a very promising strategy to treat atherogenic metabolic disorders.

Dual functions of angiopoietin-like protein 2 signaling in tumor progression and anti-tumor immunity.

Angiopoietin-like protein 2 (ANGPTL2) is a secreted glycoprotein homologous to angiopoietins. Previous studies suggest that tumor cell-derived ANGPTL2 has tumor-promoting function. Here, we conducted mechanistic analysis comparing ANGPTL2 function in cancer progression in a murine syngeneic model of melanoma and a mouse model of translocation renal cell carcinoma (tRCC). ANGPTL2 deficiency in tumor cells slowed tRCC progression, supporting a tumor-promoting role. However, systemic ablation of ANGPTL2 accelerated tRCC progression, supporting a tumor-suppressing role. The syngeneic model also demonstrated a tumor-suppressing role of ANGPTL2 in host tumor microenvironmental cells. Furthermore, the syngeneic model showed that PDGFRα+ fibroblasts in the tumor microenvironment express abundant ANGPTL2 and contribute to tumor suppression. Moreover, host ANGPTL2 facilitates CD8+ T-cell cross-priming and enhances anti-tumor immune responses. Importantly, ANGPTL2 activates dendritic cells through PIR-B-NOTCH signaling and enhances tumor vaccine efficacy. Our study provides strong evidence that ANGPTL2 can function in either tumor promotion or suppression, depending on what cell type it is expressed in.

Angiopoietin-like-3 knockout protects against glomerulosclerosis in murine adriamycin-induced nephropathy by attenuating podocyte loss.

BACKGROUND: Angiopoietin-like-3 (Angptl3) knockout is known for its protective effects on podocyte injury and proteinuria in the early stage of adriamycin (ADR) nephropathy. The current study re-evaluated the renoprotective effect of Angptl3 knockout in chronic ADR nephropathy and attempted to explore the mechanism underlying the effect associated with Angptl3 knockout in glomerulosclerosis. METHODS: B6; 129S5 mice were injected with ADR to induce nephropathy. Kidney structure and serum and urine parameters were observed during long-term follow-up. Cultured primary mouse podocytes were exposed to ADR and analyzed for the expression of some relative proteins. Podocyte loss was analyzed in both in vivo and in vitro experiments. RESULTS: Angptl3 knockout attenuated proteinuria and hypoproteinemia, protected renal structure and function, and improved the survival of mice over the whole process of ADR nephropathy. Furthermore, Angptl3 knockout reduced the numbers of the detached and apoptotic cells in the renal tissue and alleviated podocyte loss in mice with ADR chronic nephropathy, thereby, delaying the glomerulosclerosis formation. Additional results in vitro showed that Angptl3 knockout attenuated ADR-induced primary podocyte loss, including podocyte detachment and apoptosis. CONCLUSION: In addition to serving a renoprotective role in the early stage of ADR nephropathy, Angptl3 knockout contributed to disease amelioration throughout the ADR nephropathy process. Angptl3 knockout effectively delayed glomerulosclerosis formation by attenuating podocyte loss through rescuing podocytes from detachment and apoptosis. Angptl3 antagonists or inhibitors might have therapeutic potential in the occurrence and progression of nephropathy.

Long non-coding RNA ANGPTL1-3 promotes multiple myeloma bortezomib resistance by sponging miR-30a-3p to activate c-Maf expression.

Multiple Myeloma (MM) is a malignant hematological disease characterized by monoclonal proliferation of plasma cells in the bone marrow. In recent years, the widespread use of new drugs based on bortezomib (Btz) has significantly improved the remission rate of MM patients. However, drug resistance and disease relapse occur within a few years and MM is still considered to be an incurable disease. The amplification of the long arm of chromosome 1 is one of the most common genetic abnormalities in MM patients. Here, we found that long non-coding RNA ANGPTL1-3 which located in 1q region was overexpressed in MM. Lnc-ANGPTL1-3 expression was correlated with MM International Staging System (ISS) and overall survival. Notably, knockdown of lnc-ANGPTL1-3 increased Btz sensitivity of MM cells. Following exploration revealed that lnc-ANGPTL1-3 competitively interacted with miR-30a-3p to c-Maf, a transcription factor which was reported to be associated with Btz resistance. Taken together, our findings demonstrate that lnc-ANGPTL1-3/miR-30a-3p/c-Maf axis plays a critical role in MM Btz resistance.

Metabolomic Signature of Angiopoietin-Like Protein 3 Deficiency in Fasting and Postprandial State.

Objective- Loss-of-function (LOF) variants in the ANGPTL3 (angiopoietin-like protein 3) have been associated with low levels of plasma lipoproteins and decreased coronary artery disease risk. We aimed to determine detailed metabolic effects of genetically induced ANGPTL3 deficiency in fasting and postprandial state. Approach and Results- We studied individuals carrying S17X LOF mutation in ANGPTL3 (6 homozygous and 32 heterozygous carriers) and 38 noncarriers. Nuclear magnetic resonance metabolomics was used to quantify 225 circulating metabolic measures. We compared metabolic differences between LOF carriers and noncarriers in fasting state and after a high-fat meal. In fasting, ANGPTL3 deficiency was characterized by similar extent of reductions in LDL (low-density lipoprotein) cholesterol (0.74 SD units lower concentration per LOF allele [95% CI, 0.42-1.06]) as observed for many TRL (triglyceride-rich lipoprotein) measures, including VLDL (very-low-density lipoprotein) cholesterol (0.75 [95% CI, 0.45-1.05]). Within most lipoprotein subclasses, absolute levels of cholesterol were decreased more than triglycerides, resulting in the relative proportion of cholesterol being reduced within TRLs and their remnants. Further, ß-hydroxybutyrate was elevated (0.55 [95% CI, 0.21-0.89]). Homozygous ANGPTL3 LOF carriers showed essentially no postprandial increase in TRLs and fatty acids, without evidence for adverse compensatory metabolic effects. Conclusions- In addition to overall triglyceride- and LDL cholesterol-lowering effects, ANGPTL3 deficiency results in reduction of cholesterol proportion within TRLs and their remnants. Further, ANGPTL3 LOF carriers had elevated ketone body production, suggesting enhanced hepatic fatty acid ß-oxidation. The detailed metabolic profile in human knockouts of ANGPTL3 reinforces inactivation of ANGPTL3 as a promising therapeutic target for decreasing cardiovascular risk.

ANGPTL2 deletion inhibits osteoclast generation by modulating NF-κB/MAPKs/Cyclin pathways.

Osteoclasts are multinucleated cells essential for bone-resorption. Successful repair of bone defciencies still remains a great challenge worldwide. The signaling factor angiopoietin-like protein 2 (ANGPTL2), one of eight ANGPTL proteins, functions in maintenance of tissue homeostasis partly through regulating inflammation. In the study, ANGPTL2 expression was promoted during osteoclast development and that suppressing ANGPTL2 alleviated osteoclast production regulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). The results suggested that ANGPTL2 knockdown inhibited M-CSF-caused proliferation of osteoclast precursor cells. Further, ANGPTL2 silence reduced nuclear factor of activated T cell c 1 (NFATC1) and NFATC4 expressions in M-CSF-treated cells, along with decreased Runx2, OPN and Colla1. Moreover, silencing ANGPTL2 down-regulated M-CSF-promoted expressions of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, and chemoattractant protein-1 (CCL-2). Consistently, ANGPTL2 knockdown reduced M-CSF-enhanced activation of IKKα, IκBα and nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPKs) (p38 MAPK, ERK1/2 MAPK and JNK MAPK). Additionally, knockdown of ANGPTL2 inhibited the induction of Cyclin D1, Cyclin D2 and Cyclin E1 due to M-CSF exposure. In vivo, we confirmed that ANGPTL2 knockout (KO) mice were protected against osteoporosis induced by ovariectomy (OVX), as proved by the improved bone loss and bone mineral density (BMD). Decreased expression of NFATCs was also observed in OVX-induced mice in the absence of ANGPTL2. Elevated release of pro-inflammatory cytokines was abrogated by ANGPTL2 knockout in femoral heads of mice with OVX operation, accompanied with a significant reduction of phosphorylated NF-κB and MAPKs signaling pathways. And down-regulated expression of Cyclin D1, Cyclin D2 and Cyclin E1 was observed in OVX-operated mice with ANGPTL2 knockout. Therefore, our study indicated that ANGPTL2 played an essential role in osteoclast generation through regulating the proliferation and inflammation of osteoclast lineage cells, providing new insights into the therapeutic strategy to alleviate bone loss.

Angptl2 deficiency attenuates paraquat (PQ)-induced lung injury in mice by altering inflammation, oxidative stress and fibrosis through NF-κB pathway.

Paraquat (PQ) is one of the most extensively used herbicides, possessing high toxicity for humans and animals. The lung is the main target organ by the poisoning of PQ resulting in acute lung injury. Nonetheless, molecular mechanisms underlying PQ-induced lung injury remain unclear. Here, we ask if angiopoietin-like protein 2 (Angptl2), a pro-inflammatory protein, contributes to inflammation that accelerates acute lung injury. The results indicated that abundant Angptl2 expression was observed in lung tissues of PQ-treated mice. Histological analysis revealed that PQ-induced histological changes were alleviated by Angptl2 knockout (Angptl2-/-). Angptl2-/- in PQ-treated mice attenuated acute lung injury progression by reducing the number of total cells, total leukocytes, neutrophils and macrophages in bronchoalveolar lavage fluid (BALF) and reducing inflammatory response through the inactivation of nuclear factor kappa B (NF-κB) pathway. Angptl2-/- reduced oxidative stress in PQ-treated mice, as evidenced by the enhanced superoxide dismutase (SOD) activity and reduced malondialdehyde (MDA) levels in serum or lung tissue samples, which was accompanied with increased expressions of nuclear respiratory factor 2 (Nrf-2), heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO-1). PQ-induced fibrosis was also improved in Angptl2-/- mice by decreasing pulmonary transforming growth factor (TGF)-ß1 expressions. In vitro, we found that Angptl2 knockdown-suppressed inflammation, oxidative stress and fibrosis was restored by increasing NF-κB activation in PQ-incubated A549 cells; however, the results above were significantly reversed by inactivating NF-κB using its inhibitor, Bay 11-7085 or LY2409881. Therefore, Angptl2 could provide therapeutic effects on PQ-induced acute lung injury through inhibiting inflammation, oxidative stress and fibrosis by regulating NF-κB pathway.

Role of angiopoietin-like 3 (ANGPTL3) in regulating plasma level of low-density lipoprotein cholesterol.

BACKGROUND AND AIMS: Angiopoietin-like 3 (ANGPTL3) has emerged as a key regulator of lipoprotein metabolism in humans. Homozygous loss of ANGPTL3 function causes familial combined hypolipidemia characterized by low plasma levels of triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). While known effects of ANGPTL3 in inhibiting lipoprotein lipase and endothelial lipase contribute to the low TG and HDL-C, respectively, the basis of low LDL-C remains unclear. Our aim was to explore the role of ANGPTL3 in modulating plasma LDL-C. METHODS: We performed RNAi-mediated gene silencing of ANGPTL3 in five mouse models and in human hepatoma cells. We validated results by deleting ANGPTL3 gene using the CRISPR/Cas9 genome editing system. RESULTS: RNAi-mediated Angptl3 silencing in mouse livers resulted in very low TG, HDL-C and LDL-C, a pattern similar to the human phenotype. The effect was observed in wild-type and obese mice, while in hCETP/apolipoprotein (Apo) B-100 double transgenic mice, the silencing decreased LDL-C and TG, but not HDL-C. In a humanized mouse model (Apobec1-/- carrying human ApoB-100 transgene) deficient in the LDL receptor (LDLR), Angptl3 silencing had minimum effect on LDL-C, suggesting the effect being linked to LDLR. This observation is supported by an additive effect on LDL-C between ANGPTL3 and PCSK9 siRNAs. ANGPTL3 gene deletion induced cellular long-chain TG and ApoB-100 accumulation with elevated LDLR and LDLR-related protein (LRP) 1 expression. Consistent with this, ANGPTL3 deficiency by gene deletion or silencing reduced nascent ApoB-100 secretion and increased LDL/VLDL uptake. CONCLUSIONS: Reduced secretion and increased uptake of ApoB-containing lipoproteins may contribute to the low LDL-C observed in mice and humans with genetic ANGPTL3 deficiency.

ANGPTL8 requires ANGPTL3 to inhibit lipoprotein lipase and plasma triglyceride clearance.

Angiopoietin-like (ANGPTL)3 and ANGPTL8 are secreted proteins and inhibitors of LPL-mediated plasma triglyceride (TG) clearance. It is unclear how these two ANGPTL proteins interact to regulate LPL activity. ANGPTL3 inhibits LPL activity and increases serum TG independent of ANGPTL8. These effects are reversed with an ANGPTL3 blocking antibody. Here, we show that ANGPTL8, although it possesses a functional inhibitory motif, is inactive by itself and requires ANGPTL3 expression to inhibit LPL and increase plasma TG. Using a mutated form of ANGPTL3 that lacks LPL inhibitory activity, we demonstrate that ANGPTL3 activity is not required for its ability to activate ANGPTL8. Moreover, coexpression of ANGPTL3 and ANGPTL8 leads to a far more efficacious increase in TG in mice than ANGPTL3 alone, suggesting the major inhibitory activity of this complex derives from ANGPTL8. An antibody to the C terminus of ANGPTL8 reversed LPL inhibition by ANGPTL8 in the presence of ANGPTL3. The antibody did not disrupt the ANGPTL8:ANGPTL3 complex, but came in close proximity to the LPL inhibitory motif in the N terminus of ANGPTL8. Collectively, these data show that ANGPTL8 has a functional LPL inhibitory motif, but only inhibits LPL and increases plasma TG levels in mice in the presence of ANGPTL3.

Knockdown of Angiopoietin-Like Protein 2 Inhibits Proliferation and Invasion in Glioma Cells via Suppressing the ERK/MAPK Signaling Pathway.

Angiopoietin-like protein 2 (ANGPTL2), a member of the glycoprotein family, is mainly secreted by adipose tissues under normal conditions. Recently, ANGPTL2 has been found to be upregulated in some types of cancers and is considered to be a tumor promoter. However, the functional significance of ANGPTL2 in glioma has not yet been elucidated. In this study, we investigated the specific role of ANGPTL2 in glioma. The results showed that ANGPTL2 was highly expressed in glioma tissues and cell lines. Knockdown of ANGPTL2 reduced the proliferative and invasive abilities of glioma cells. Moreover, the tumorigenesis assay showed that ANGPTL2 knockdown inhibited glioma tumor growth in vivo. We also found that ANGPTL2 knockdown decreased the protein levels of p-ERK1/2 in glioma cells and thus blocked the activity of the ERK/MAPK signaling pathway. Taken together, our study provided the first evidence that ANGPTL2 played an oncogenic role in glioma development and might be considered as a new therapeutic target for glioma treatment.