Presence Of Angiogenin Research Articles

IL-1β knockout increases the intestinal abundancy of Akkermansia muciniphila.

The proinflammatory cytokine interleukin-1β (IL-1β) is known to be upregulated in patients suffering from metabolic syndrome. IL-1β contributes to insulin resistance in obesity and type 2 diabetes, yet its influence on the intestinal microbiome is incompletely understood. The data presented here demonstrate that mice genetically deficient in IL-1β show a specific alteration of intestinal colonisation of a small group of bacteria. Especially Akkermansia muciniphila, a bacterium reported to be inversely associated with obesity, diabetes, cardiometabolic diseases and low-grade inflammation, showed increased colonisation in IL-1β knockout mice. In comparative microarray analysis from mucus scrapings of the colon mucosa of IL-1β knockout and wildtype mice, angiogenin 4 mRNA was strongly reduced in IL-1β knockout animals. Since the presence of angiogenin 4 in the culture medium showed a significant growth inhibition on A. muciniphila which was not detectable for other bacteria tested, IL-1β induced expression of angiogenin 4 is a strong candidate to be responsible for the IL-1β induced suppression of A. muciniphila colonisation. Thus, the data presented here indicate that IL-1β might be the lacking link between inflammation and suppression of A. muciniphila abundance as observed in a variety of chronic inflammatory disorders.

Cytosine-5 RNA Methylation Regulates Neural Stem Cell Differentiation andMotility.

SummaryLoss-of-function mutations in the cytosine-5 RNA methylase NSUN2 cause neurodevelopmental disorders in humans, yet the underlying cellular processes leading to the symptoms that include microcephaly remain unclear. Here, we show that NSUN2 is expressed in early neuroepithelial progenitors of the developing human brain, and its expression is gradually reduced during differentiation of human neuroepithelial stem (NES) cells in vitro. In the developing Nsun2−/− mouse cerebral cortex, intermediate progenitors accumulate and upper-layer neurons decrease. Loss of NSUN2-mediated methylation of tRNA increases their endonucleolytic cleavage by angiogenin, and 5′ tRNA fragments accumulate in Nsun2−/− brains. Neural differentiation of NES cells is impaired by both NSUN2 depletion and the presence of angiogenin. Since repression of NSUN2 also inhibited neural cell migration toward the chemoattractant fibroblast growth factor 2, we conclude that the impaired differentiation capacity in the absence of NSUN2 may be driven by the inability to efficiently respond to growth factors.

Angiogenin interacts with the plasminogen activation system at the cell surface of breast cancer cells to regulate plasmin formation and cell migration

Angiogenin (ANG), a 14-kDa pro-angiogenic secreted protein, has been shown to play a role in cell migration and tumor invasion, which involve proteolytic cleavage of plasminogen to generate plasmin. However, the mechanism by which ANG regulates plasmin formation and cell migration was not known. Our studies here detected elevated levels of secreted and cell surface-bound ANG in highly invasive metastatic breast cancer cells. ANG was also detected at very high levels in the tumor cells in infiltrating ductal carcinomas. By immunofluorescence and immunoprecipitation analysis, ANG was detected at the leading edges of the cell surfaces where it colocalized and interacted with members of the plasminogen activation system (PAS) such as annexin A2 (A2), calpactin (S100-A10) and urokinase plasminogen activator receptor (uPAR). Analysis of lipid raft (LR) and non-lipid raft (NLR) regions of the cell membranes showed the predominance of ANG, A2 and S100-A10 in the LR regions. In contrast, uPAR was detected predominantly in the NLR fractions, suggesting that ANG interacts with uPAR at the junctions of LR and NLR regions. ANG knockdown in T47D and MDA-MB-231 breast cancer cell lines did not affect the cellular expression of A2, S100-A10 and uPAR but decreased cell migration and plasmin formation. Neutralization of ANG with monoclonal antibodies similarly decreased the migration of MDA-MB-231 cells. In the presence of ANG, uPAR was observed to interact with uPA, which is necessary for plasmin formation. Conversely, in the absence of ANG, uPAR did not interact with uPA and FAK and Src kinases were observed to be dephosphorylated. Exogenous addition of recombinant ANG to ANG knocked down MDA-MB-231 cells restored FAK phosphorylation, uPAR interactions with uPA, plasmin formation as well as migration of these cells. Taken together, our results identified a novel role for ANG as a member of the uPAR interactome that facilitates the interaction of uPAR with uPA, leading to plasmin formation and cell migration necessary for tumor invasion and metastasis of breast cancer cells.

Abstract A087: Angiogenin (ANG) localized at the leading edges of breast cancer cells regulates plasmin formation by plasminogen activation complex (PAC) and cell migration

Abstract Angiogenin (ANG), a 14-kDa pro-angiogenic protein, had been shown to play a role in cell migration and tumor invasion processes, which involve proteolytic cleavage of plasminogen to generate plasmin. However, the mechanism by which ANG regulates plasmin formation and cell migration was not known. Our studies detected elevated levels of secreted ANG and cell surface-bound ANG in highly invasive metastatic breast cancer cells. By immunofluorescence and immunoprecipitation analysis, ANG was detected at leading edges of the cell surfaces where it colocalized and interacted with members of the plasminogen activation complex (PAC) such as Annexin A2 (A2), Calpactin (S100A10) and Urokinase plasminogen receptor (uPAR). Analysis of lipid raft (LR) and non-lipid raft (NLR) regions of the cell membranes showed the predominance of ANG, A2 and S100A10 in the LR regions. In contrast, uPAR was detected only in the NLR fractions, suggesting that ANG interacts with uPAR at junctions of LR and NLR regions. ANG knockdown in T47D and MDA-MB-231 breast cancer cell lines did not affect the cellular expression of A2, S100A10 and uPAR but decreased cell migration and plasmin formation. Neutralization of ANG with monoclonal antibodies similarly decreased the migration of MDA-MB-231 cells. In the presence of ANG on cell surfaces, uPAR was observed to interact with UPA, which is known to be necessary for proteolytic cleavage of plasminogen by UPA to form plasmin. However, in the absence of ANG, uPAR interacted exclusively with PAI, which is known to inhibit plasmin formation by blocking UPA mediated proteolytic cleavage of plasminogen. Assessment of phosphorylation states of cellular FAK and Src kinases under these conditions further showed that both FAK and Src are dephosphorylated in the absence of ANG. Taken together, our results identified a novel regulatory role of ANG as a bridging protein that facilitates the interaction of uPAR with UPA, which leads to plasmin formation and cell migration necessary for tumor invasion and metastasis of breast cancer cells. These studies also suggest that ANG could serve as a bio-marker for identifying the leading edges of metastatic breast cancer cells and targeting ANG could be a novel method to block breast cancer invasiveness. Citation Format: Sujoy Dutta, Chirosree Bandyopadhyay, Lydia Wilson, Case Warshall, Virginie Bottero, Karen E. Johnson, Bala Chandran. Angiogenin (ANG) localized at the leading edges of breast cancer cells regulates plasmin formation by plasminogen activation complex (PAC) and cell migration. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A087.

ACTIBIND, an actin‐binding fungal T2‐RNase with antiangiogenic and anticarcinogenic characteristics

ACTIBIND is an Aspergillus niger extracellular ribonuclease (T2-ribonuclease [RNase]) that possesses actin-binding activity. In plants, ACTIBIND inhibits the elongation and alters the orientation of pollen tubes by interfering with the intracellular actin network. The question rose whether ACTIBIND can also affect mammalian cancer development. Cell colony formation was performed in human colon (HT-29, Caco-2, RSB), breast (ZR-75-1), and ovarian (2780) cancer cells in the presence or absence of 1 muM ACTIBIND. In HT-29 and ZR-75-1 cells, the effect of ACTIBIND on cell migration was studied by microscopic observations and by invasion assay through Matrigel. Tube formation was assessed in human umbilical vein endothelial cells (HUVEC) in the presence of angiogenin or basic fibroblast growth factor (bFGF) (1 microg/mL each) following overnight incubation with 1 or 10 microM ACTIBIND. In an athymic mouse xenograft model, HT-29 cells were injected subcutaneously, followed by subcutaneous (0.4-8 mg/mouse/injection) or intraperitoneal (0.001-1 mg/mouse/injection) injections of ACTIBIND. In a rat dimethylhydrazine (DMH)-colorectal carcinogenesis model, ACTIBIND was released directly into the colon via osmotic micropumps (250 microg/rat/day) or given orally via microcapsules (1.6 mg/rat/day). Aberrant crypt foci, tumors in the distal colon, and tumor blood vessels were examined. ACTIBIND had an anticlonogenic effect unrelated to its ribonuclease activity. It also inhibited angiogenin-induced HUVEC tube formation in a dose-responsive manner. ACTIBIND was found to bind actin in vitro. It also bound to cancer cell surfaces, leading to disruption of the internal actin network and inhibiting cell motility and invasiveness through Matrigel-coated filters. In mice, ACTIBIND inhibited HT-29 xenograft tumor development, given either as a subcutaneous or intraperitoneal treatment. In rats, ACTIBIND exerted preventive and therapeutic effects on developing colonic tumors induced by DMH. It also reduced the degree of tumor observation. This study indicated that ACTIBIND is an effective antiangiogenic and anticarcinogenic factor.

Hypoxic conditions stimulate the production of angiogenin and vascular endothelial growth factor by human renal proximal tubular epithelial cells in culture

Chronic low oxygen in the tubulointerstitial area is a crucial cause of renal degradation and tubulointerstitial damage. Previous reports have suggested that the maintenance of renal blood flow plays a role in the suppression of progressive renal damage. Neovascularization is important for the maintenance of blood flow. We studied the production of angiogenic factors by culturing renal proximal tubular epithelial cells (PTEC) under hypoxic conditions. Cultured PTEC were exposed to normal and low-oxygen conditions. The levels of angiogenin (ANG) and vascular endothelial growth factor (VEGF) in the cell supernatants were measured by enzyme-linked immunosorbent assay. The messenger RNAs (mRNAs) of ANG and VEGF in the PTEC were examined by real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR). The presence of ANG, VEGF and hypoxia-inducible factor-1 (HIF-1) was studied by immunofluorescence techniques. The effect of cobalt chloride (CoCl(2)), which is an HIF-1 inducer, on the production of ANG and VEGF was also examined in order to elucidate the contribution of the HIF-1 pathway to the production of these cytokines. ANG and VEGF were demonstrated to exist in the cell supernatants, and ANG and VEGF mRNAs were detected in the PTEC. Hypoxic conditions stimulated the secretion of ANG (2.5-fold vs normoxia, P<0.001) and VEGF (3.2-fold vs normoxia, P<0.001) by PTEC. Hypoxic conditions increased the mRNA expression of ANG for 6 h (1.38-fold vs normoxia, P<0.05) and VEGF for 24 h (2.04-fold vs normoxia, P<0.01). Hypoxic conditions also enhanced ANG, VEGF and HIF-1 protein expression in PTEC. The CoCl(2) increased the secretion of ANG (5.2-fold vs control, P<0.0001) and VEGF (2.3-fold vs control, P<0.0001) by PTEC. Under hypoxic conditions, the ANG and VEGF secreted by PTEC may modulate angiogenesis and vascular remodeling in the renal interstitium via an increase in the production of HIF-1.

Expression and localization of angiogenin in placenta: enhanced levels at term over first trimester villi.

Human angiogenin, a 14-kDa non-glycosylated polypeptide with both angiogenic and ribonucleolytic activities, is implicated in angiogenesis, a complex process of proliferation and formation of new capillary blood vessels from existing blood vessels. Placental growth requires extensive angiogenesis, which develops its vascular structure in both fetal chorionic villi and maternal deciduas. In this study, we investigated the expression profiles of angiogenin in placental villi from early and late gestation at both mRNA and protein levels using explant cultures in vitro followed by RT-PCR, immunoblot, and immunohistochemical analyses. From functionally active placental explants, angiogenin was detected in conditioned media of all the samples from first trimester and term group. The mean levels of angiogenin produced by term villi were found to be 2.6-, 2.1-, and 2.2-fold higher (P < 0.01) than first trimester villi at 24, 48, and 72 hr of culture, respectively. Expression profiles of angiogenin from term and first trimester villi seem to agree with its mRNA levels and immunoblot analysis; the expression in term villi was twice that in first trimester villi. The presence of angiogenin in placental villi and upregulation of its production towards term indicate that angiogenin production by the placenta is specific to the developmental stage. In conclusion, the observed changes in the localization and mRNA expression of angiogenin during placental development raise the possibility that it is involved in morphological and angiogenic changes in this endocrine organ vital to the successful fetal outcome during pregnancy.

Angiogenin promotes invasiveness of cultured endothelial cells by stimulation of cell-associated proteolytic activities.

Angiogenin, a potent inducer of neovascularization in the chicken chorioallantoic membrane and rabbit cornea, promotes endothelial cell invasion of Matrigel basement membrane. A transformed bovine aortic endothelial cell line, GM 7373, is 5 times more invasive when cultured in the presence of 1 microgram of bovine angiogenin per ml than in its absence. A polyclonal anti-angiogenin antibody and alpha 2-antiplasmin neutralize the effect of angiogenin, but an angiogenin-binding protein (actin) does not. Further, this concentration of angiogenin induces a 14-fold increase in the cell-associated proteolytic activity of cultured endothelial cells, determined with a tissue-type plasminogen activator-specific peptide as the substrate. In addition, cells cultured on a three-dimensional fibrin gel in the presence of angiogenin are 3 times more capable of dissolving the gel and forming focal defects in the underlying matrix. The results indicate that angiogenin can enhance the ability of endothelial cells to digest extracellular matrix components and degrade basement membrane, thereby facilitating cell invasion and migration. Binding of angiogenin to its cell-surface binding protein (actin) followed by dissociation of the angiogenin-actin complex from the cell surface and subsequent activation of tissue-type plasminogen activator/plasmin are likely steps involved in the processes of endothelial cell invasion and angiogenesis.