Aged fibroblast-derived extracellular vesicles promote angiogenesis in melanoma.

Advancing age is a negative prognostic factor for cutaneous melanoma. However, the role of extracellular vesicles (EVs) within the melanoma tumor microenvironment (TME) has remained unexplored in the context of aging. While the size and morphology of the EVs isolated from young vs. aged fibroblasts remained unaltered, the contents of the protein cargo were changed. Aging reduced the expression of the tetraspanin CD9 in both the dermal fibroblasts and released EVs. CD9 is a crucial regulator of EV cargo sorting. Modulating the CD9 expression in fibroblasts was sufficient to alter its levels in EVs. Mass spectrometry analysis of EVs released by CD9 knockdown (KD) vs. control cells revealed a significant increase in angiopoietin-like protein 2 (ANGPTL2), an angiogenesis promoter. Analysis of primary endothelial cells confirmed increased sprouting under CD9 KD conditions. Together, our data indicate that aged EVs play an important role in promoting a tumor-permissive microenvironment.

Remodeling of tumor microenvironment by extracellular matrix protein 1a differentially regulates ovarian cancer metastasis.

We previously reported that extracellular matrix protein 1 isoform a (ECM1a) promotes epithelial ovarian cancer (EOC) through autocrine signaling by binding to cell surface receptors αXß2. However, the role of ECM1a as a secretory molecule in the tumor microenvironment is rarely reported. In this study, we constructed murine Ecm1-knockout mice and human ECM1a-knockin mice and further generated orthotopic or peritoneal xenograft tumor models to mimic the different metastatic stages of EOC. We show that ECM1a induces oncogenic metastasis of orthotopic xenograft tumors, but inhibits early-metastasis of peritoneal xenograft tumors. ECM1a remodels extracellular matrices (ECM) and promotes remote metastases by recruiting and transforming bone marrow mesenchymal stem cells (BMSCs) into platelet-derived growth factor receptor beta (PDGFRß+) cancer-associated fibroblasts (CAFs) and facilitating the secretion of angiopoietin-like protein 2 (ANGPTL2). Competing with ECM1a, ANGPTL2 also binds to integrin αX through the P1/P2 peptides, resulting in negative effects on BMSC differentiation. Collectively, this study reveals the dual functions of ECM1a in remodeling of TME during tumor progression, emphasizing the complexity of EOC phenotypic heterogeneity and metastasis.

ANGPTL2 knockdown induces autophagy to relieve alveolar macrophage pyroptosis by reducing LILRB2-mediated inhibition of TREM2.

Acute lung injury (ALI) is featured with a robust inflammatory response. Angiopoietin-like protein 2 (ANGPTL2), a pro-inflammatory protein, is complicated with various disorders. However, the role of ANGPTL2 in ALI remains to be further explored. The mice and MH-S cells were administrated with lipopolysaccharide (LPS) to evoke the lung injury in vivo and in vitro. The role and mechanism of ANGPTL was investigated by haematoxylin-eosin, measurement of wet/dry ratio, cell count, terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling, reverse transcription quantitative polymerase chain reaction, immunofluorescence, enzyme-linked immunosorbent assay, detection of autophagic flux and western blot assays. The level of ANGPTL2 was upregulated in lung injury. Knockout of ANGPTL2 alleviated LPS-induced pathological symptoms, reduced pulmonary wet/dry weight ratio, the numbers of total cells and neutrophils in BALF, apoptosis rate and the release of pro-inflammatory mediators, and modulated polarization of alveolar macrophages in mice. Knockdown of ANGPTL2 downregulated the level of pyroptosis indicators, and elevated the level of autophagy in LPS-induced MH-S cells. Besides, downregulation of ANGPTL2 reversed the LPS-induced the expression of leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) and triggering receptor expressed on myeloid cells 2 (TREM2), which was reversed by the overexpression of LILRB2. Importantly, knockdown of TREM2 reversed the levels of autophagy- and pyroptosis-involved proteins, and the contents of pro-inflammatory factors in LPS-induced MH-S cells transfected with si ANGPTL2, which was further inverted with the treatment of rapamycin. Therefore, ANGPTL2 silencing enhanced autophagy to alleviate alveolar macrophage pyroptosis via reducing LILRB2-mediated inhibition of TREM2.

Knockdown of ANGPTL2 promotes left ventricular systolic dysfunction by upregulation of NOX4 in mice.

Background: Angiopoietin-like 2 (ANGPTL2) is a pro-inflammatory and pro-oxidant circulating protein that predicts and promotes chronic inflammatory diseases such as atherosclerosis in humans. Transgenic murine models demonstrated the deleterious role of ANGPTL2 in vascular diseases, while deletion of ANGPTL2 was protective. The nature of its role in cardiac tissues is, however, less clear. Indeed, in adult mice knocked down (KD) for ANGPTL2, we recently reported a mild left ventricular (LV) dysfunction originating from a congenital aortic valve stenosis, demonstrating that ANGPTL2 is essential to cardiac development and function. Hypothesis: Because we originally demonstrated that the KD of ANGPTL2 protected vascular endothelial function via an upregulation of arterial NOX4, promoting the beneficial production of dilatory H2O2, we tested the hypothesis that increased cardiac NOX4 could negatively affect cardiac redox and remodeling and contribute to LV dysfunction observed in adult Angptl2-KD mice. Methods and results: Cardiac expression and activity of NOX4 were higher in KD mice, promoting higher levels of cardiac H2O2 when compared to wild-type (WT) mice. Immunofluorescence showed that ANGPTL2 and NOX4 were co-expressed in cardiac cells from WT mice and both proteins co-immunoprecipitated in HEK293 cells, suggesting that ANGPTL2 and NOX4 physically interact. Pressure overload induced by transverse aortic constriction surgery (TAC) promoted LV systolic dysfunction in WT mice but did not further exacerbate the dysfunction in KD mice. Importantly, the severity of LV systolic dysfunction in KD mice (TAC and control SHAM) correlated with cardiac Nox4 expression. Injection of an adeno-associated virus (AAV9) delivering shRNA targeting cardiac Nox4 expression fully reversed LV systolic dysfunction in KD-SHAM mice, demonstrating the causal role of NOX4 in cardiac dysfunction in KD mice. Targeting cardiac Nox4 expression in KD mice also induced an antioxidant response characterized by increased expression of NRF2/KEAP1 and catalase. Conclusion: Together, these data reveal that the absence of ANGPTL2 induces an upregulation of cardiac NOX4 that contributes to oxidative stress and LV dysfunction. By interacting and repressing cardiac NOX4, ANGPTL2 could play a new beneficial role in the maintenance of cardiac redox homeostasis and function.

Endothelial-derived small extracellular vesicles support B-cell acute lymphoblastic leukemia development.

PURPOSE: The bone marrow niche plays an important role in leukemia development. However, the contributions of different niche components to leukemia development and their underlying mechanisms remain largely unclear. METHOD: Cre/LoxP-based conditional knockout technology was used to delete VPS33B or ANGPTL2 gene in niche cells. Murine B-ALL model was established by overexpressing the N-Myc oncogene in hematopoietic stem progenitor cells. The frequency of leukemia cells and immunophenotypic B220+ CD43+ LICs was detected by flow cytometry. SEVs was isolated by sequential centrifugation and mass spectrometry was performed to analyze the different components of SEVs. Immunoprecipitation and western blot were used to measure the interaction of VPS33B and ANGPTL2. RESULTS: Here, we showed that specific knockout of vascular protein sorting 33b (Vps33b) in endothelial cells (ECs), but not megakaryocytes or mesenchymal stem cells, resulted in a significant decrease in the secretion of small extracellular vesicles (SEVs) and a delay in the development of B-cell lymphoblastic leukemia (B-ALL). Vps33b knockdown endothelial cells contained much lower levels of SEVs that contained angiopoietin-like protein 2 (ANGPTL2) than the control cells. Importantly, conditional knockout of Angptl2 in ECs significantly delayed B-ALL progression. Moreover, C-terminal region of ANGPTL2 (aa247-471) could directly interact with Sec1-like domain 1 of VPS33B (aa1-aa146). We further demonstrated that the point mutations R399H and G402S in ANGPTL2 led to a dramatic decrease in the secretion of ANGPTL2-SEVs. We also showed that wild-type ANGPTL2-containing SEVs, but not mutant ANGPTL2-containing SEVs, significantly enhanced B-ALL development. CONCLUSION: In summary, our findings indicate that the secretion of ANGPTL2-containing SEVs in ECs sustains the leukemogenic activities of B-ALL cells, which is fine-tuned by the direct interaction of VPS33B and ANGPTL2. These findings reveal that niche-specific SEVs play an important role in B-ALL development.

ANGPTL2 aggravates LPS-induced septic cardiomyopathy via NLRP3-mediated inflammasome in a DUSP1-dependent pathway.

Angiopoietin-like protein 2 (ANGPTL2) was implicated in various cardiovascular diseases; however, its role in lipopolysaccharide (LPS)-related septic cardiomyopathy remains unclear. Herein, mice were exposed to LPS to generate septic cardiomyopathy, and adeno-associated viral vector was employed to overexpress ANGPTL2 in the myocardium. Besides, mice were treated with adenoviral vector to knock down ANGPTL2 in hearts. ANGPTL2 expressions in hearts and cardiomyocytes were upregulated by LPS challenge. ANGPTL2 overexpression aggravated, while ANGPTL2 silence ameliorated LPS-associated cardiac impairment and inflammation. Mechanically, we found that ANGPTL2 activated NLRP3 inflammasome via suppressing DUSP1 signaling, and NLRP3 knockdown abrogated the detrimental role of ANGPTL2 in aggravating LPS-induced cardiac inflammation. Furthermore, DUSP1 overexpression significantly inhibited ANGPTL2-mediated NLRP3 activation, and subsequently improved LPS-related cardiac dysfunction. In summary, ANGPTL2 exacerbated septic cardiomyopathy via activating NLRP3-mediated inflammation in a DUSP1-dependent manner, and our study uncovered a promising therapeutic target in preventing septic cardiomyopathy.

ANGPTL2-mediated epigenetic repression of MHC-I in tumor cells accelerates tumor immune evasion.

Loss or downregulation of major histocompatibility complex class I (MHC-I) contributes to tumor immune evasion. We previously demonstrated that angiopoietin-like protein 2 (ANGPTL2) promotes tumor progression using a Xp11.2 translocation renal cell carcinoma (tRCC) mouse model. However, molecular mechanisms underlying ANGPTL2 tumor-promoting activity in the tRCC model remained unclear. Here, we report that ANGPTL2 deficiency in renal tubular epithelial cells slows tumor progression in the tRCC mouse model and promotes activated CD8+ T-cell infiltration of kidney tissues. We also found that Angptl2-deficient tumor cells show enhanced interferon γ-induced expression of MHC-I and increased susceptibility to CD8+ T-cell-mediated anti-tumor immune responses. Moreover, we provide evidence that the ANGPTL2-α5ß1 integrin pathway accelerates polycomb repressive complex 2-mediated repression of MHC-I expression in tumor cells. These findings suggest that ANGPTL2 signaling in tumor cells contributes to tumor immune evasion and that suppressing that signaling in tumor cells could serve as a potential strategy to facilitate tumor elimination by T-cell-mediated anti-tumor immunity.

ANGPTL2 promotes VEGF-A synthesis in human lung cancer and facilitates lymphangiogenesis.

Lung cancer is an extremely common cancer and metastatic lung cancer has a greatly low survival rate. Lymphangiogenesis is essential for the development and metastasis of lung cancer. The adipokine angiopoietin-like protein 2 (ANGPTL2) regulates tumor progression and metastasis, although the functions of ANGPTL2 in lung cancer are unknown. Analysis of data from TCGA genomics program, the GEPIA web server and the Oncomine database revealed that higher levels of ANGPTL2 expression were correlated with progressive disease and lymph node metastasis. ANGPTL2 enhanced VEGF-A-dependent lymphatic endothelial cell (LEC) tube formation and migration. Integrin α5ß1, p38 and nuclear factor (NF)-κB signaling mediated ANGPTL2-regulated lymphangiogenesis. Importantly, overexpression ANGPTL2 facilitated tumor growth and lymphangiogenesis in vivo. Thus, ANGPTL2 is a promising therapeutic object for treating lung cancer.

ANGPTL2 binds MAG to efficiently enhance oligodendrocyte differentiation.

BACKGROUND: Oligodendrocytes have robust regenerative ability and are key players in remyelination during physiological and pathophysiological states. However, the mechanisms of brain microenvironmental cue in regulation of the differentiation of oligodendrocytes still needs to be further investigated. RESULTS: We demonstrated that myelin-associated glycoprotein (MAG) was a novel receptor for angiopoietin-like protein 2 (ANGPTL2). The binding of ANGPTL2 to MAG efficiently promoted the differentiation of oligodendrocytes in vitro, as evaluated in an HCN cell line. Angptl2-null mice had a markedly impaired myelination capacity in the early stage of oligodendrocyte development. These mice had notably decreased remyelination capacities and enhanced motor disability in a cuprizone-induced demyelinating mouse model, which was similar to the Mag-null mice. The loss of remyelination ability in Angptl2-null/Mag-null mice was similar to the Angptl2-WT/Mag-null mice, which indicated that the ANGPTL2-mediated oligodendrocyte differentiation effect depended on the MAG receptor. ANGPTL2 bound MAG to enhance its phosphorylation level and recruit Fyn kinase, which increased Fyn phosphorylation levels, followed by the transactivation of myelin regulatory factor (MYRF). CONCLUSION: Our study demonstrated an unexpected cross-talk between the environmental protein (ANGPTL2) and its surface receptor (MAG) in the regulation of oligodendrocyte differentiation, which may benefit the treatment of many demyelination disorders, including multiple sclerosis.

ANGPTL2 aggravates doxorubicin-induced cardiotoxicity via inhibiting DUSP1 pathway.

Angiopoietin-like protein 2 (ANGPTL2) plays versatile roles in various cardiovascular diseases. Its connection to doxorubicin (DOX)-related cardiomyopathy, however, remains elusive. To determine the role of ANGPTL2, an adeno-associated viral vector was used to overexpress ANGPTL2 in the murine heart 4 weeks before DOX treatment (15 mg/kg). Moreover, mice were injected with adenoviral vectors to knock down ANGPTL2 in the myocardium. Echocardiography and hemodynamics were used to determine the cardiac function. The effect of ANGPTL2 and its downstream target were elucidated by applying molecular and biochemical strategies. We found that ANGPTL2 expression was significantly increased in response to DOX stimulation. Moreover, cardiac-specific ANGPTL2 overexpression exacerbated DOX-related cardiac dysfunction, myocardial apoptosis, and oxidative stress. Mechanistically, ANGPTL2 aggravated DOX-induced cardiac injury via inhibiting the dual specificity phosphatase 1 (DUSP1) pathway and DUSP1 overexpression significantly impeded DOX-induced cardiomyopathy in ANGPTL2-overexpressed mice. Altogether, ANGPTL2 aggravated DOX-related cardiac injury by suppressing the DUSP1 pathway.

Efficient expansion of mouse hematopoietic stem cells ex vivo by membrane anchored Angptl2.

Hematopoietic stem cell (HSC) transplantation represents an important curative therapy for numerous hematological and immune diseases. Many efforts have been applied to achieve attainable ex vivo HSC expansion. We previously showed that angiopoietin-like proteins 2 (Angptl2) binds and activates the immune inhibitory receptor human leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) to support the expansion of HSC. However, soluble Angptl2 is unstable and the downstream signaling would be attenuated by ligand-binding triggered receptor endocytosis, compromising the potential of Angptl2 to expand HSCs. We proposed that membrane anchored Angptl2 will overcome these limitations. In this study, we constructed the C-terminal and N-terminal anchored membrane Angptl2 (Cm-Angptl2 and Nm-Angptl2) by adding a transmembrane domain at the C-terminal or an anchor sequence at the N-terminal respectively. Both forms of Angptl2 showed efficient expression on the surface of feeder cells. Nm-Angptl2, but not Cm-Angptl2, induces a potent activation of LILRB2 reporter, indicating the fibronectin (FBN) domain at the C-terminus of Angptl2 is essential to stimulate LILRB2 signaling. Compared to soluble Angptl2, Nm-Angptl2 displays higher activities to activate LILRB2 reporter, and to promote the expansion of mouse HSCs as determined by transplantation and limiting dilution assay. Our study revealed the importance of FBN domain for Angptl2 to activate LILRB2 and demonstrated that Nm-Angptl2 have enhanced activities than the soluble protein in LILRB2 activation and HSC expansion, providing a strategy to explore the mode of ligand induced receptor signaling, and an optimized approach to expand HSCs ex vivo.

Knockdown of hypoxia-inducible factor 1-alpha (HIF1α) interferes with angiopoietin-like protein 2 (ANGPTL2) to attenuate high glucose-triggered hypoxia/reoxygenation injury in cardiomyocytes.

To investigate the role of hypoxia-inducible factor 1-alpha (HIF1A) in hypoxia/reoxygenation (H/R) injury of cardiomyocytes induced by high glucose (HG). The in vitro model of coronary heart disease with diabetes was that H9c2 cells were stimulated by H/R and HG. Quantitative reverse transcription PCR (RT-qPCR) and Western blot analysis were used to detect the expression of HIF1A and angiopoietin-like protein 2 (ANGPTL2) in H9c2 cells. Cell viability and apoptosis were, respectively, estimated by Cell Counting Kit 8 (CCK-8) and TUNEL assays. Lactate dehydrogenase (LDH) activity, inflammation and oxidative stress were in turn detected by their commercial assay kits. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were used to confirm the association between HIF1A and ANGPTL2 promoter. The expression of nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway-related proteins and apoptosis-related proteins were also detected by Western blot analysis. As a result, ANGPTL2 expression was upregulated in H9c2 cells induced by HG or/and H/R. ANGPTL2 positively modulated HIF1A expression in H9c2 cells. HG or/and H/R suppressed the cell viability and promoted apoptosis, inflammatory response and oxidative stress levels in H9c2 cells. However, the knockdown of ANGPTL2 could reverse the above phenomena in H/R-stimulated-H9c2 cells through activation of Nrf2/HO-1 pathway. HIF1A transcriptionally activated ANGPTL2 expression. The effect of knockdown of ANGPTL2 on H/R triggered-H9c2 cells was weakened by HIF1A overexpression. In conclusion, knockdown of HIF1A downregulated ANGPTL2 to alleviate H/R injury in HG-induced H9c2 cells by activating the Nrf2/HO-1 pathway.

Evaluation of serum Angiopoietin-like protein 2 (ANGPTL-2), Angiopoietin-like protein 8 (ANGPTL-8), and high-sensitivity C-reactive protein (hs-CRP) levels in patients with gestational diabetes mellitus and normoglycemic pregnant women.

OBJECTIVE: In the present study, we aimed to investigate the role of the fasting serum levels of Anjiopoetin 2 - like protein (ANGPTL2), Anjiopoetin 8-like protein (ANGPTL8), and high-sensitivity C-reactive protein (hs-CRP) in the etiopathogenesis of gestational diabetes mellitus (GDM), and analyze the relationships between insulin resistance parameters. MATERIAL AND METHOD: The 90 individuals admitted to Izmir Katip Celebi University Hospital Internal Medicine, Endocrinology and Obstetrics, and gynecology outpatient clinic were included in the study of similar ages and similar demographic characteristics. Forty-five women with diet-controlled GDM and 45 women with normoglycemic pregnancy were enrolled. ANGPTL-2, ANGPTL-8, hs-CRP, creatinine, ALT, GGT, lipid profile, HBA1c(%), and serum insülin, c-peptide levels were studied in the fasting serum samples of research groups. All individuals had 75-g OGTT testing. GDM screening was performed at 24-28 weeks' gestation. Exclusion criteria were as follows: Age <18 years or >40 years, pregestational diabetes (type 1 or 2), drug or alcohol abuse, thyroid dysfunction, Hepatitis B, and other infectious diseases (Herpes virus, Streptococcus B carriers, Chlamydia and Candida), Thalassemia carriers or other significant medical conditions, the use of any medication that interferes with lipid or glucose metabolism that would affect glucose regulation. RESULT: Forty-five women with GDM and for the control group, 45 women with normoglycemic pregnant women were identified. The mean gestational age was 30.7 (18-38) for GDM and 29.6 (24-39) for the control group. Serum ANGPTL-8 (GDM =19.5 ± 93 Control = 0.73 ± 3.78 p = <.001). There was a statistically significant difference between the case and control groups for serum ANGPTL-8 levels. Serum ANGPTL-2 (GDM =19.9 ± 23.1 Control = 26.0 ± 23.4 p = .105) and serum hs-CRP(GDM =106 ± 65.1 Control =98.2 ± 87.3 p = .768). There was no statistically significant difference between the case and control groups for serum ANGPTL-2 and hsCRP levels. Serum ANGPTL8 levels were positively correlated with FPG (r = 0.391, p = <.001), FPI (r = 0.212, p = .045), 1-h PPG (r = 0.514, p = <.001), 2-h PPG (r = 0.502, p = <.001), HOMA-IR) score (r = 0.310, p = .003), TG (r = 0.245, p = .020); they were not except for BMI, hs-CRP levels and ANGPTL2 levels. CONCLUSIONS: ANGPTL8 levels were significantly higher in GDM than in healthy control group. ANGPTL2 levels and hs-CRP levels were similar to the healthy control group. Elevated serum ANGPTL8 levels were correlated significantly with insulin resistance parameters, the main component of GDM pathophysiology. Our data showed that ANGPTL8 could be a new biomarker for diagnosing GDM.

Angiopoietin-like protein 2 deficiency promotes periodontal inflammation and alveolar bone loss.

BACKGROUND: Human periodontitis is a highly prevalent inflammatory disease that leads to connective tissue degradation, alveolar bone resorption, and tooth loss. Angiopoietin-like 2 (ANGPTL2) regulates chronic inflammation in various diseases and is functionally involved in maintaining tissue homeostasis and promoting tissue regeneration, but there is limited information about its function in periodontitis. Here we investigated the expression and explicit role of ANGPTL2 in periodontitis. METHODS: Immunohistochemistry and quantitative real-time PCR (qRT-PCR) were used to detect the ANGPTL2 expression in periodontal tissues and periodontal ligament cells (PDLCs). A ligature-induced periodontitis model was generated in wild-type and ANGPTL2 knockout mice. qRT-PCR and enzyme-linked immunosorbent assay were used to assess the production of inflammatory cytokines and matrix metalloproteinases (MMPs) in cultured PDLCs. Western blot was performed to detect proteins in relevant signaling pathways. RESULTS: Increased ANGPTL2 expression was observed in inflamed periodontal tissues and PDLCs. ANGPTL2 deficiency promoted alveolar bone loss with enhanced osteoclastogenesis and inflammatory reactions in ligature-induced periodontitis. Downregulation of ANGPTL2 remarkably enhanced expression levels of interleukin (IL)-6, IL-8, MMP1, and MMP13 in Porphyromonas gingivalis lipopolysaccharide-induced PDLCs, whereas ANGPTL2-overexpressing PDLCs showed opposite trends. ANGPTL2 downregulation activated STAT3 and nuclear factor-κB pathways and blocked Akt signaling under inflammatory environment. Treatment with a STAT3 inhibitor partially suppressed the inflammatory reaction of PDLCs mediated by ANGPTL2 knockdown. CONCLUSIONS: Our study provides the first evidence of an anti-inflammatory effect of ANGPTL2 in murine periodontitis. The findings demonstrate the critical and protective role of ANGPTL2 in alveolar bone loss and periodontal inflammation.

Blocking angiotensin 2 receptor attenuates diabetic nephropathy via mitigating ANGPTL2/TL4/NF-κB expression.

BACKGROUND: Diabetic nephropathy (DN) is a consequence of diabetes mellitus (DM) and is associated with early changes in renal angiotensin II (ANG II). These changes were evaluated using ANG II blocker valsartan early from week two of diabetes (experiment I, renoprotective) and late from week nine of diabetes (experiment II, renotherapeutic) to the end of both experiments at week twelve. METHODS AND RESULTS: In both experiments, adult male Wister rats were divided into (i) vehicle group; (ii) valsartan received oral 30 mg/Kg/day; (iii) diabetic received single 50 mg/Kg intraperitoneal streptozotocin injection; (iv) renoprotection, diabetic rats received valsartan treated in experiments I and II. DM effects on urine albumin excretion, blood pressure, and renal ANG II were measured. Urinary nephrin, kidney injury molecule-1 (KIM-1), renal angiopoietin-like protein 2 (ANGPTL2), and toll-like receptor 4 (TLR 4) mRNA expression were tested. DM-initiated fibrotic markers integrin, α-smooth muscle actin expression, and collagen IV and apoptotic protein caspase 3 were tested. DM induced early changes starting from week four in the tested variables. At week twelve, in both experiments, valsartan intervention showed a significant reduction in ANG II, ANGPTL2, TLR 4 and integrin expression and improvement in albuminuria, blood pressure, urinary biomarkers, fibrotic and apoptotic markers. CONCLUSIONS: Changes leading to DN starts early in the disease course and ANG II reduction decreased the expression of ANGPTL2 and integrin which preserve the glomerular barrier. Blocking ANG II was able to decrease TLR 4 and inflammatory cytokines leading to decreasing DN.

Angiopoietin-like protein 2 decreases peritoneal metastasis of ovarian cancer cells by suppressing anoikis resistance.

Peritoneal metastasis is a common mode of spread of ovarian cancer. Despite therapeutic advances, some patients have intractable peritoneal metastasis. Therefore, in-depth characterization of the molecular mechanism of peritoneal metastasis is a key imperative. Angiopoietin-like protein 2 (ANGPTL2) is an inflammatory factor which activates NF-κB signaling and plays an important role in the pathogenesis of various inflammatory diseases including cancers, such as lung and breast cancer. In this study, we examined the role of ANGPTL2 in ovarian cancer peritoneal metastasis. We observed no difference of cell proliferation between ANGPTL2-expressing and control cells. In the mouse intraperitoneal xenograft model, formation of peritoneal metastasis by ANGPTL2-expressing cells was significantly decreased compared to control. In the in vitro analysis, the expressions of integrin α5ß1, α6, and ß4, but not those of αvß3, α3, α4, and ß1, were significantly decreased in ANGPTL2-expressing cells compared to control cells. ANGPTL2-expressing cells showed significantly inhibited adherence to laminin compared to control. In addition, we observed upregulation of anoikis (a form of programmed cell death occurring under an anchorage-independent condition) and significant decrease in the expression of Bcl-2 in ANGPTL2-expressing cells as compared to control cells. These results suggest that ANGPTL2 expression in ovarian cancer cells represses peritoneal metastasis by suppressing anoikis resistance.

CD146 is a Novel ANGPTL2 Receptor that Promotes Obesity by Manipulating Lipid Metabolism and Energy Expenditure.

Obesity and its related complications pose an increasing threat to human health; however, targetable obesity-related membrane receptors are not yet elucidated. Here, the membrane receptor CD146 is demonstrated to play an essential role in obesity. In particular, CD146 acts as a new adipose receptor for angiopoietin-like protein 2 (ANGPTL2), which is thought to act on endothelial cells to activate adipose inflammation. ANGPTL2 binds to CD146 to activate cAMP response element-binding protein (CREB), which then upregulates CD146 during adipogenesis and adipose inflammation. CD146 is present in preadipocytes and mature adipocytes, where it is mediated by its ligands ANGPTL2 and galectin-1. In preadipocytes, CD146 ablation suppresses adipogenesis, whereas the loss of CD146 in mature adipocytes suppresses lipid accumulation and enhances energy expenditure. Moreover, anti-CD146 antibodies inhibit obesity by disrupting the interactions between CD146 and its ligands. Together, these findings demonstrate that ANGPTL2 directly affects adipocytes via CD146 to promote obesity, suggesting that CD146 can be a potential target for treating obesity.

ANGPTL2 Induces Synovial Inflammation via LILRB2.

Angiopoietin-like proteins (ANGPTLs) are circulating proteins that are expressed in various cells and tissues and are thought to be involved in the repair and remodeling of damaged tissues; however, ANGPTL2 hyperfunction has been shown to cause chronic inflammation, leading to the progression of various diseases. ANGPTL2 is known to exert cellular effects via receptors such as integrin α5ß1 and leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2); however, their roles in ANGPTL2-induced inflammation remain unclear. In this study, we investigated the mechanisms underlying ANGPTL2-induced inflammation involving LILRB2 and various signaling pathways in human fibroblast-like synoviocytes (HFLS). The effects of ANGPTL2 and an anti-LILRB2 antibody on the gene expression of various inflammation-related factors were examined using real-time RT-PCR, while their effects on MAPK, NF-κB, and Akt phosphorylation were analyzed by western blotting. We found that the addition of ANGPTL2 enhanced the gene expression of inflammatory factors, whereas pretreatment with the anti-LILRB2 antibody for 12 h decreased the expression of these factors. Similarly, ANGPTL2 addition activated the phosphorylation of ERK, p38, JNK, NF-κB, and Akt in HFLS; however, this effect was significantly inhibited by pretreatment with the anti-LILRB2 antibody. Together, the findings of this study demonstrate that ANGPTL2 induces the expression of inflammatory factors via LILRB2 in synovial cells. Therefore, LILRB2 could be a potential therapeutic agent for treating matrix degradation in osteoarthritis.

CSN5 Promotes Carcinogenesis of Thyroid Carcinoma Cells Through ANGPTL2.

COP9 signalosome subunit 5 (CSN5) plays a key role in carcinogenesis of multiple cancers and contributes to the stabilization of target proteins through deubiquitylation. However, the underlying role of CSN5 in thyroid carcinoma has not been reported. In this research, our data showed that CSN5 was overexpressed in thyroid carcinoma tissues compared with paracancerous tissues. Furthermore, a series of gain/loss functional assays were performed to demonstrate the role of CSN5 in facilitating thyroid carcinoma cell proliferation and metastasis. Additionally, we found there was a positive correlation between CSN5 and angiopoietin-like protein 2 (ANGPTL2) protein levels in thyroid carcinoma tissues and that CSN5 promoted thyroid carcinoma cell proliferation and metastasis through ANGPTL2. We also identified the underlying mechanism that CSN5 elevated ANGPTL2 protein level by directly binding it, decreasing its ubiquitination and degradation. Overall, our results highlight the significance of CSN5 in promoting thyroid carcinoma carcinogenesis and implicate CSN5 as a promising candidate for thyroid carcinoma treatment.

Relationship between miR-204 and ANGPTL2 expression and diagnosis, pathological stage, and prognosis in patients with colon cancer.

BACKGROUND: Angiopoietin-like protein 2 (ANGPTL2) is linked to various tumors. MicroRNA-204 (miR-204) is associated with colorectal cancer (CRC). Bioinformatic analysis has demonstrated a targeting relationship between miR-204 and ANGPTL2. The present study aimed to investigate the role of miR-204 in the proliferation and apoptosis of colorectal tumor cells. METHODS: Colorectal tumor tissues were collected. Normal colon mucosa was used as a control. The relationship between miR-204 and ANGPTL2 expression and tumor stage and prognosis was analyzed. The dual-luciferase reporter assay confirmed targeted regulation between miR-204 and ANGPTL2. SW480 cells were allocated to the miR-NC group and the miR-204 mimic group, followed by apoptotic analysis using flow cytometry and cellular proliferation analysis using EdU staining. RESULTS: Compared with normal colonic mucosa, miR-204 expression was decreased in colorectal tumor tissues and ANGPTL2 expression was increased, which correlated with TNM staging. The prognosis of patients with low miR-204 expression and high ANGPTL2 expression was worse than for patients with high miR-204 expression and low ANGPTL2 expression. The dual-luciferase reporter assay confirmed a targeting regulation relationship between miR-204 and ANGPTL2. Transfection of miR-204 mimic significantly inhibited the expression of ANGPTL2 and cell proliferation in SW480 cells and promoted apoptosis. CONCLUSIONS: Downregulating miR-204 expression plays a vital role in upregulating ANGPTL2 expression and promoting the pathogenesis of CRC. MiR-204 is able to hinder the proliferation of colorectal tumor cells and encourage apoptosis by targeting the inhibition of ANGPTL2 expression.