Angiogenin regulates epithelial-mesenchymal transition of hepatocellular carcinoma through upregulation of HMGA2.

Angiogenin (ANG) is known to alter multiple cell behaviors by directly targeting downstream targets, but its role in hepatocellular carcinoma (HCC) remains to be elucidated. The expression of ANG in HCC cell lines was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The effects of ANG expression on cell proliferation, cell migration, and hallmarks of epithelial-mesenchymal transition (EMT) process were also investigated. The relationship between ANG and high mobility group AT-hook 2 (HMGA2) was evaluated. ANG expression was increased in HCC cell lines. Downregulating of ANG inhibits proliferation, migration, and EMT of HCC cells. The direct regulation of ANG on HMGA2 was verified by luciferase activity reporter assay and western blot assay. Furthermore, overexpression of HMGA2 reversed the inhibitory effects of ANG downregulation on HCC cell behaviors. Our results illustrated the mechanism that ANG promote the EMT of HCC through targeting HMGA2.

A multifunctional bioactive material that stimulates osteogenesis and promotes the vascularization bone marrow stem cells and their resistance to bacterial infection.

The main limitation of tissue engineering lies in the inability to stimulate osteogenesis, angiogenesis of stem cells and broad-spectrum antimicrobial activity. However, the development of multifunctional bioactive materials with these capabilities remains a great challenge. In this study, we prepared mesoporous silica nanoparticles encapsulated with silver nanocrystals (AG-MSN) with uniform sphere size and mesopores. Platelet-derived growth factor BB (PDGF-BB) was effectively loaded in the AG-MSN mesopores (P-AG-MSN). The silicon ions (Si) released by P-AG-MSN stimulate osteogenic differentiation of bone marrow stromal cells (BMSC) by activating the alkaline phosphatase (ALP) activity of bone-related genes and increasing protein (OCN, RUNX2 and OPN) expression. Ag+ ions could be slowly released from the interior of the shell, highlighting their durable antibacterial activity. The sustained release of PDGF-BB from P-AG-MSN stimulated the angiogenic differentiation of BMSC, as indicated by the enhanced secretion of vascular endothelial growth factor (VEGF), HIF-1α, HGF and ANG-1 and protein expression. Our results show that P-AG-MSN can clearly promote BMSC osteostimulation and vascularization. This research serves as a preliminary study of the utilization of this multifunctional mixture to fabricate a new active biological scaffold that integrates BMSC osteostimulation, vascularization and bactericidal effects by 3D printing technology.

Angiogenin elevates the invasive potential of squamous cell lung carcinoma cells through epithelial­mesenchymal transition.

Squamous cell carcinoma of the lung is one of the most aggressive cancers, and its aggressiveness is in part due to its intrinsic high rate of metastasis. Moreover, the process of epithelial-mesenchymal transition (EMT) appears to be involved in these neoplastic processes. Furthermore, EMT-type cells share many biological characteristics with the function of angiogenin (ANG) in squamous cell lung carcinoma. We conducted immunohistochemical analysis to detect the expression of ANG, E-cadherin, vimentin, N-cadherin, ß-catenin and TGF-ß1 in 60 cases of squamous cell lung carcinoma tissues. Western blot analysis was adopted to detect the protein expression levels of ANG and EMT markers. The effects of ANG on proliferation, migration and invasion of squamous cell lung carcinoma cells was analyzed by Cell Counting Kit-8, scratch assay and Transwell invasion chamber in order to reveal the role of ANG in the process of EMT in squamous cell lung carcinoma. The results revealed that ANG was aberrantly expressed in the squamous cell lung carcinoma specimens and was closely correlated with the differentiation of the cell lines. The expression of ANG was also significantly associated with metastasis and the stage of the squamous cell lung carcinoma cases. In addition, we validated that ANG influenced the expression of vimentin, E-cadherin, N-cadherin, ß-catenin and TGF-ß1 in SK-MES-1 cells. Most importantly, overexpression of ANG enhanced the migration and invasion of SK-MES-1 cells, while knockdown resulted in opposite effects. In the present study, we found that ANG plays an important role in EMT in squamous cell lung carcinoma and may be a valuable therapeutic target for squamous cell lung carcinoma.

Human Th17 Cells Lack HIV-Inhibitory RNases and Are Highly Permissive to Productive HIV Infection.

UNLABELLED: Human immunodeficiency virus (HIV) infects and depletes CD4(+) T cells, but subsets of CD4(+) T cells vary in their susceptibility and permissiveness to infection. For example, HIV preferentially depletes interleukin-17 (IL-17)-producing T helper 17 (Th17) cells and T follicular helper (Tfh) cells. The preferential loss of Th17 cells during the acute phase of infection impairs the integrity of the gut mucosal barrier, which drives chronic immune activation-a key determinant of disease progression. The preferential loss of Th17 cells has been attributed to high CD4, CCR5, and CXCR4 expression. Here, we show that Th17 cells also exhibit heightened permissiveness to productive HIV infection. Primary human CD4(+) T cells were sorted, activated under Th17- or Th0-polarizing conditions and infected, and then analyzed by flow cytometry. Th17-polarizing cytokines increased HIV infection, and HIV infection was disproportionately higher among Th17 cells than among IL-17(-) or gamma interferon-positive (IFN-γ(+)) cells, even upon infection with a replication-defective HIV vector with a pseudotype envelope. Further, Th17-polarized cells produced more viral capsid protein. Our data also reveal that Th17-polarized cells have diminished expression of RNase A superfamily proteins, and we report for the first time that RNase 6 inhibits HIV. Thus, our findings link Th17 polarization to increased HIV replication. IMPORTANCE: Our study compares the intracellular replicative capacities of several different HIV isolates among different T cell subsets, providing a link between the differentiation of Th17 cells and HIV replication. Th17 cells are of key importance in mucosal integrity and in the immune response to certain pathogens. Based on our findings and the work of others, we propose a model in which HIV replication is favored by the intracellular environment of two CD4(+) T cell subsets that share several requirements for their differentiation: Th17 and Tfh cells. Characterizing cells that support high levels of viral replication (rather than becoming latently infected or undergoing cell death) informs the search for new therapeutics aimed at manipulating intracellular signaling pathways and/or transcriptional factors that affect HIV replication.

Synthetic Terrein Inhibits Progression of Head and Neck Cancer by Suppressing Angiogenin Production.

BACKGROUND/AIM: Head and neck cancers are the fifth most common cancer type worldwide, affecting more than half a million patients annually. Development of effective therapeutic drugs is, therefore, required for this type of disease. This study assessed the effects of synthetic terrein on head and neck cancer. MATERIALS AND METHODS: Synthetic terrein was prepared by using the modified Altenhach's procedure. The effect of synthetic terrein on cell proliferation of head and neck cancer cells and HUVECs was assessed. Angiogenin secretion and ribosome biogenesis were examined by ELISA and silver staining of the nucleolar organizer region. A mouse xenograft model was prepared by inoculating mice with suspensions of cells of the human head and neck cancer cell line OSC-19 subcutaneously into the dorsal region of each mouse. Ki-67, CD31 and angiogenin expression in xenografted tumors was examined by immunohistochemistry. RESULTS: Synthetic terrein inhibited the growth of various head and neck cancer cells. In addition, an in vivo experiment revealed that synthetic terrein inhibited a xenograft tumor growth in athymic mice. Immunohistochemical analysis revealed that expression of Ki-67, CD31 and ANG was down-regulated in synthetic terrein-treated tumors, compared to controls. Synthetic terrein suppressed the ANG secretion and ribosome biogenesis in cancer cells, and cell proliferation in vascular endothelial cells. CONCLUSION: The mechanism underlying the anti-tumor effects of synthetic terrein against head and neck cancer consists of the inhibition of both tumor cell proliferation and angiogenesis via the suppression of ANG production.

[Experimental approach to the gene therapy of motor neuron disease with the use of genes hypoxia-inducible factors].

Motor neuron disease (MND), or amyotrophic lateral sclerosis, is a fatal neurodegenerative disorder characterized by a progressive loss of motor neurons in the spinal cord and the brain. Several angiogenic and neurogenic growth factors, such as the vascular endothelial growth factor (VEGF), angiogenin (ANG), insulin-like growth factor (IGF) and others, have been shown to promote survival of the spinal motor neurons during ischemia. We constructed recombinant vectors using human adenovirus 5 (Ad5) carrying the VEGF, ANG or IGF genes under the control of the cytomegalovirus promoter. As a model for MND, we employed a transgenic mice strain, B6SJL-Tg (SOD1*G93A)d11 Gur/J that develops a progressive degeneration of the spinal motor neurons caused by the expression of a mutated Cu/Zn superoxide dismutase gene SOD1. Delivery of the therapeutic genes to the spinal motor neurons was done using the effect of the retrograde axonal transport after multiple injections of the Ad5-VEGF, Ad5-ANG and Ad5-IGF vectors and their combinations into the limbs and back muscles of the SOD1(G93A) mice. Viral transgene expression in the spinal cord motor neurons was confirmed by immunocytochemistry and RT-RCR. We assessed the neurological status, motor activity and lifespan of experimental and control animal groups. We discovered that SOD1(G93A) mice injected with the Ad5-VEGF + Ad5-ANG combination showed a 2-3 week delay in manifestation of the disease, higher motor activity at the advanced stages of the disease, and at least a 10% increase in the lifespan compared to the control and other experimental groups. These results support the safety and therapeutic efficacy of the tested recombinant treatment. We propose that the developed experimental MND treatment based on viral delivery of VEGF + ANG can be used as a basis for gene therapy drug development and testing in the preclinical and clinical trials of the MND.

Overexpression of angiogenin in pterygium body fibroblasts and its association with proliferative potency.

PURPOSE: Angiogenin (ANG) originally was identified as an angiogenic tumor factor, and recently its biologic activity is extended to stimulating cell proliferation. With viewing pterygium as a tumorigenic mimicry, we investigated ANG profiles within pterygia. METHODS: Expression levels of ANG were assessed using immunohistochemistry, RT-PCR, and Western blotting through examination of excised specimens and cultured fibroblasts from pterygium and conjunctiva tissues. The phenotypes of pterygia were classified by four grading indices, including recurrence, growth activity, pterygium body translucency (T), and vascularity (V). Then, ANG levels in pterygia were differentiated according to phenotypes of pterygia, and were compared to levels in normal conjunctiva. Furthermore, to investigate ANG-related acquisition of proliferative potency in fibroblasts, the correlation between ANG and α-smooth muscle actin (α-SMA) levels was evaluated. RESULTS: In immunohistochemistry, ANG was expressed strongly in pterygium stroma with all four severe phenotypes (with recurrence, active growth, thick body [T3], and marked vascularization [V3]), especially at the perivascular areas. There was a trend toward higher ANG expression in cultured fibroblasts of pterygia with severe phenotype, compared to those without and with normal conjunctiva. However, pterygium body V had a weak association with ANG expression. Additionally, Western blotting revealed a significant positive correlation between the expression levels of α-SMA and ANG. CONCLUSIONS: Overexpression of ANG in pterygium body fibroblasts might be involved in active pterygium growth with thick pterygium body formation and increased risk of recurrence. A possible mechanism for this finding includes ANG-related transition of pterygium fibroblasts to the proliferative state.

Tumor angiogenesis is caused by single melanoma cells in a manner dependent on reactive oxygen species and NF-κB.

Melanomas have a high angiogenic potential, but respond poorly to medical treatment and metastasize very early. To understand the early events in tumor angiogenesis, animal models with high tumor resolution and blood vessel resolution are required, which provide the opportunity to test the ability of small molecule inhibitors to modulate the angiogenic tumor program. We have established a transgenic melanoma angiogenesis model in the small laboratory fish species Japanese medaka. Here, pigment cells are transformed by an oncogenic receptor tyrosine kinase in fish expressing GFP throughout their vasculature. We show that angiogenesis occurs in a reactive oxygen species (ROS)- and NF-κB-dependent, but hypoxia-independent manner. Intriguingly, we observed that blood vessel sprouting is induced even by single transformed pigment cells. The oncogenic receptor as well as human melanoma cells harboring other oncogenes caused the production of pro-angiogenic factors, most prominently angiogenin, through NF-κB signaling. Inhibiting NF-κB prevented tumor angiogenesis and led to the regression of existing tumor blood vessels. In conclusion, our high-resolution medaka melanoma model discloses that ROS and NF-κB signaling from single tumor cells causes hypoxia-independent angiogenesis, thus, demonstrating that the intrinsic malignant tumor cell features are sufficient to initiate and maintain a pro-angiogenic signaling threshold.

Antiangiogenic effects of ganetespib in colorectal cancer mediated through inhibition of HIF-1α and STAT-3.

Hypoxia-inducible factors (HIFs) and STAT-3 play essential roles in angiogenesis. HIF-1α and STAT-3 are clients of the heat shock protein 90 (HSP90). We hypothesized that ganetespib, a potent HSP90 inhibitor, would disrupt angiogenesis in colorectal cancer (CRC) through inhibition of HIF-1α and STAT-3. CRC cell lines (HCT116 and HT29) were used in all the experiments. Egg CAM and HUVEC assays revealed decreased angiogenesis in ganetespib treated cell lines. Ganetespib inhibited matrigel plug vascularization and tumor growth of xenografts. Significant inhibition of PDGFA, FGF2, Ang-1, Ang-2, TGFß1, VEGF, HIF-1α and STAT-3 expression was observed in both cell lines treated ganetespib. HIF-1α overexpression resulted in the increase VEGF and STAT-3 expression and this was inhibited by ganetespib. HIF-1α knockdown inhibited VEGF and STAT-3 expression. STAT-3 knockdown inhibited VEGF but not HIF-1α expression. HSP90, STAT-3 and VEGF expression was significantly higher in CRC compared to adjacent normal tissue. Significant downregulation of PDGFA, FGF2, Ang-1, Ang-2, TGFß1, VEGF, STAT-3 and HIF-1α mRNA was observed in the post ganetespib treatment tumor samples from patients with rectal cancer. These results collectively suggest that inhibition of HSP90 is a promising antiangiogenic strategy in CRC. HSP90 angiogenic effects are mediated through HIF-1α and STAT-3.

Angiogenin in middle-aged type 1 diabetes patients.

BACKGROUND: Angiogenin levels are increased in children and adolescent patients with type 1 diabetes, regardless of the extent of diabetic microangiopathy. However, little is known about the angiogenin concentrations in adults with type 1 diabetes. Thus we studied its level in middle aged subjects with the presence of diabetic nephro-, retino and neuropathy. METHODS: We investigated the data of 57 (age 39±6.6 years, 45.6% of males) patients with type 1 diabetes and 38 age-matched control subjects without diabetes (age 37.1±5.9 years, 42.1% of males), including medical histories, evidences of microangiopathy and serum angiogenin concentrations. RESULTS: Serum angiogenin level was lower in patients with type 1 diabetes [384.2(190.4-999.8) ng/ml] compared to controls [460.4(230.6-708.2) ng/ml], p=0.04. In patients with overt diabetic nephropathy the angiogenin level was higher when compared to patients without nephropathy [568.2(269.6-999.8) vs 369.4(190.4-999.8) ng/ml, p=0.01]. There were no differences between angiogenin levels in subgroups of patients distinguished by the presence of other microvascular complications or other concomitant vascular risk factors despite cigarette smoking [smokers: 516.2(294.4-999.8) vs. non-smokers: 372.1(190.4-924.8) ng/ml, p=0.01]. CONCLUSIONS: Regardless of the presence of diabetic microangiopathy, angiogenin level in middle-aged type 1 diabetes patients is lower than in controls. The presence of overt nephropathy and smoking habit in middle-aged patients with type 1 diabetes are associated with higher angiogenin level.

Generation of new cytotoxic human ribonuclease variants directed to the nucleus.

Ribonucleases are promising agents for use in anticancer therapy. Engineering a nuclear localization signal into the sequence of the human pancreatic ribonuclease has been revealed as a new strategy to endow this enzyme with cytotoxic activity against tumor cells. We previously described a cytotoxic human pancreatic ribonuclease variant, named PE5, which is able to cleave nuclear RNA, inducing the apoptosis of cancer cells and reducing the amount of P-glycoprotein in different multidrug-resistant cell lines. These results open the opportunity to use this ribonuclease in combination with other chemotherapeutics. In this work, we have investigated how to improve the properties of PE5 as an antitumor drug candidate. When attempting to develop a recombinant protein as a drug, two of the main desirable attributes are minimum immunogenicity and maximum potency. The improvements of PE5 have been designed in both senses. First, in order to reduce the potential immunogenicity of the protein, we have studied which residues mutated on PE5 can be reverted to those of the wild-type human pancreatic ribonuclease sequence without affecting its cytotoxicity. Second, we have investigated the effect of introducing an additional nuclear localization signal at different sites of PE5 in an effort to obtain a more cytotoxic enzyme. We show that the nuclear localization signal location is critical for the cytotoxicity. One of these variants, named NLSPE5, presents about a 10-fold increase in cytotoxicity respective to PE5. This variant induces apoptosis and kills the cells using the same mechanism as PE5.

Expression, purification and characterization of recombinant human angiogenin in Pichia pastoris.

The potential of angiogenin (Ang) for clinical use has been highlighted in view of its important roles in inducing angiogenesis, facilitating cell proliferation, and inhibiting cell apoptosis. To produce soluble, correctly folded recombinant protein with a high yield, a DNA fragment encoding human Ang was inserted into eukaryotic expression vector pPIC9 and transformed into Pichia pastoris. The expression of recombinant human Ang (rhAng) accounted for about 70% of total secreted proteins. Purifying the Ang from the culture supernatant yielded 30 mg/L at 90% purity by chromatography with a SP Sepharose FF column. Biological assays indicated that rhAng can induce new blood-vessel formation, promote HeLa cell proliferation, increase Erk1/2 phosphorylation, and upregulate c-myc expression. Preparation of bioactive rhAng might lay the basis for further functional study, and might provide an effective strategy for large-scale production of soluble human Ang.

Inflammation-driven dermal lymphangiogenesis in atopic dermatitis is associated with CD11b+ macrophage recruitment and VEGF-C up-regulation in the IL-4-transgenic mouse model.

OBJECTIVE: To investigate the presence and extent of inflammatory lymphangiogenesis in AD and determine the role of IL-4 in lymphatic proliferation in both K14-IL-4 Tg mouse model of AD and cultured human epidermal cells. METHODS: Skin tissues from Tg mice were collected for immunostaining against PDPN, LYVE-1, CD11b and VEGF-C. The regulation of specific lymphatic biomarkers and growth factors were determined using qPCR and Western Blot analyses. Dermal lymphatic uptake and drainage were assessed using intradermal EB dye micro-injections. Total RNA from IL-4-stimulated HaCaT cells was analyzed in a PCR array to evaluate the regulation of lymphangiogenic-related genes. RESULTS: Prominent dermal microvascular lymphangiogenesis occurs in the Tg mice, characterized by a significant increase in number and caliber of the vasculature. The extent of both lymphatic proliferation and drainage parallels the progression of lesion severity, as does the up-regulation of pro-lymphangiogenic factors VEGF-C, VEGFR-3, ANG-1, and ANG-2. IL-4-stimulated HaCaT cells express high levels of MCP-1, a strong macrophage chemo-attractant. Additionally, Tg mice show significantly increased number of dermal CD11b+ macrophages expressing VEGF-C in the skin. CONCLUSIONS: Our results provide the first demonstration of inflammation-mediated lymphangiogenesis in AD and that IL-4 triggered macrophage recruitment may be closely linked to this phenomenon.

Multifunction protein staphylococcal nuclease domain containing 1 (SND1) promotes tumor angiogenesis in human hepatocellular carcinoma through novel pathway that involves nuclear factor κB and miR-221.

Staphylococcal nuclease domain-containing 1 (SND1) is a multifunctional protein that is overexpressed in multiple cancers, including hepatocellular carcinoma (HCC). Stable overexpression of SND1 in Hep3B cells expressing a low level of SND1 augments, whereas stable knockdown of SND1 in QGY-7703 cells expressing a high level of SND1 inhibits establishment of xenografts in nude mice, indicating that SND1 promotes an aggressive tumorigenic phenotype. In this study we analyzed the role of SND1 in regulating tumor angiogenesis, a hallmark of cancer. Conditioned medium from Hep3B-SND1 cells stably overexpressing SND1 augmented, whereas that from QGY-SND1si cells stably overexpressing SND1 siRNA significantly inhibited angiogenesis, as analyzed by a chicken chorioallantoic membrane assay and a human umbilical vein endothelial cell differentiation assay. We unraveled a linear pathway in which SND1-induced activation of NF-κB resulted in induction of miR-221 and subsequent induction of angiogenic factors Angiogenin and CXCL16. Inhibition of either of these components resulted in significant inhibition of SND1-induced angiogenesis, thus highlighting the importance of this molecular cascade in regulating SND1 function. Because SND1 regulates NF-κB and miR-221, two important determinants of HCC controlling the aggressive phenotype, SND1 inhibition might be an effective strategy to counteract this fatal malady.

Laryngeal carcinoma prognosis after postoperative radiotherapy correlates with CD105 expression, but not with angiogenin or EGFR expression.

Patients with head and neck squamous cell carcinoma (SCC) respond very differently to radiotherapy (RT). Since clinical factors cannot accurately predict its effects, biological parameters have been investigated, including tumor hypoxia. CD105 is a hypoxia-inducible glycoprotein emerging as a potential prognostic indicator for several solid malignancies. Angiogenin is upregulated under hypoxic conditions and supports primary and metastatic tumor growth. Epidermal growth factor receptor (EGFR) activation stimulates tumor proliferation and angiogenesis. The aim of the present study was to ascertain the prognostic importance of hypoxia-inducible factors (CD105, angiogenin) and EGFR in a series of patients who underwent primary surgery followed by RT for laryngeal SCC. 25 consecutive patients with laryngeal SCC given postoperative RT have been investigated. CD105, angiogenin, and EGFR immunohistochemical expressions in primary laryngeal SCCs have been evaluated also with image analysis. The recurrence rate was significantly higher in SCC patients with a CD105 expression >10.0% (P = 0.012) and their disease-free survival (DFS) was shorter (P = 0.044). Neither angiogenin (in the carcinoma cells or endothelial cells) nor EGFR expression were associated with the prognosis in our patients after primary surgery followed by RT for laryngeal SCC. CD105 should be studied as a potentially predictive biomarker for identifying laryngeal SCCs at higher risk of early recurrence after postoperative RT. Targeted anti-CD105 therapy associated with RT should also be investigated in patients with laryngeal SCCs characterized by high CD105 expression.

[Construction, expression and refolding of recombinant luteinizing hormone releasing hormone-angiogenin toxin].

OBJECTIVE: To express, purify and refold recombinant luteinizing hormone releasing hormone-angiogenin (LHRH-Ang) toxin using E. coli. expression system. METHODS: Recombinant LHRH-Ang expression vector was constructed by replacing of EGF fragment in plasmid pET28a/EGF-Ang with LHRH-PII fragment amplified from plasmid pET28/MSH-PE40. DNA sequencing would be used to verify the correction of fused LHRH-PII-Ang gene. Then, E. coli strain BL21 (DE3) was transformed by pET28a/LHRH-Ang vector. Expression of recombinant LHRH-Ang toxin was induced by Isopropyl-ß-D-Thiogalactoside (IPTG). Refolding effects of gradient dialysis was evaluated by SDS-PAGE. RESULTS: Prokaryotic expression vector pET28a/LHRH-Ang, containing LHRH-PII-Ang fusion gene, was constructed by PCR amplification, restriction enzyme digestion and ligation method. Sequence correction of fusion gene was confirmed by DNA sequencing. After IPGT induction, recombinant LHRH-Ang protein was expressed in BL21 (DE3) as inclusion body, it took 18.43% of total protein. Inclusion body was resolved in 8 mol/L urea and purified by DEAE-Sepharose FF column, the purity was 85%. Recombinant LHRH-Ang toxin was refolded and concentrated by gradient dialysis and PEG 20000, respectively. CONCLUSIONS: Recombinant LHRH-Ang protein was expressed in E. coli and refolded successfully.

Neoangiogenesis in laryngeal carcinoma: angiogenin and CD105 expression is related to carcinoma recurrence rate and disease-free survival.

AIMS: Angiogenin regulates angiogenesis supporting primary and metastatic tumour growth. CD105 is a proliferation-associated protein expressed in angiogenic endothelial cells. The aim of this study was to investigate for the first time angiogenin expression in laryngeal squamous cell carcinoma (LSCC) and to evaluate the relationship between angiogenin and CD105 expression, clinicopathological and prognostic parameters. METHODS AND RESULTS: A total of 108 consecutive operable LSCCs were studied. Angiogenin expression was determined immunohistochemically in both carcinoma cells and intratumoral vessels. CD105 expression was evaluated in endothelial cells of LSCC and calculated by a computer-based image analysis system. The percentage of the fields occupied by CD105-assessed microvessels was determined. Angiogenin expression in carcinoma cells was higher in LSCC patients who experienced loco-regional recurrence of disease (P=0.032). Disease-free survival (DFS) was shorter in cases with carcinoma cells showing angiogenin expression >21.0% (P=0.035). Angiogenin expression in carcinoma cells was correlated strongly with the angiogenin score in endothelial cells of intratumoral vessels (P=0.0000). Higher loco-regional carcinoma recurrence rate and shorter DFS in patients with high CD105 expression were found (P=0.0004, P=0.0001). CONCLUSIONS: Angiogenin expression in laryngeal carcinoma cells and CD105 expression can be considered as potentially useful to detect LSCC patients with higher risk of disease recurrence who might benefit from more aggressive therapy.

Human mast cells synthesize and release angiogenin, a member of the ribonuclease A (RNase A) superfamily.

ANG is a plasma protein with angiogenic and ribonucleolytic activity implicated in tumor growth, heart failure, wound healing, asthma, and the composition of the adult gut microflora. Human mast cells (HuMC) are similarly associated with modulation of vascular permeability, angiogenic processes, wound healing, and asthma. We hypothesized that HuMC express and secrete ANG in response to divergent stimuli. ANG expression was evaluated in the LAD2 HMC, the HMC-1, and CD34+-derived HuMC, following exposure to live Escherichia coli, TLR ligands, or neuropeptides and following FcepsilonRI aggregation. Expression and production of ANG were determined by microarray analysis, qRT-PCR, confocal microscopy, and ELISA. Microarray analysis showed that ANG is up-regulated by LAD2 cells exposed to live E. coli. qRT-PCR analysis revealed that LAD2, HMC-1, and HuMC constitutively expressed ANG mRNA and that it was up-regulated by exposure to E. coli. Activation of HuMC by FcepsilonRI aggregation resulted in release of small amounts of ANG (<100 pg/mL), whereas compound 48/80, NGF, LPS, PGN, and flagellin activated HuMC to secrete >160 pg/mL ANG. These observations demonstrate that HuMC store and secrete ANG to a variety of stimuli and suggest that MC-derived ANG is available in the subsequent inflammatory response.

Increases in serum TARC/CCL17 levels are associated with progression-free survival in advanced melanoma patients in response to dendritic cell-based immunotherapy.

INTRODUCTION: Changes in the levels of serum cytokines and growth factors are associated with response to therapy. We examined cytokine, chemokine, and growth factor levels in serum collected from normal volunteers or metastatic melanoma patients receiving dendritic cell-based immunotherapy. MATERIALS AND METHODS: Using an array for 42 cytokines, chemokines, or growth factors, sera from normal controls and metastatic melanoma patients at baseline and week 4 were analyzed for qualitative changes. Quantitative determination of the levels of the chemokine thymus and activation-regulated chemokine (TARC/CCL17) was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Significant qualitative differences were noted in serum cytokine, chemokine, and growth factor levels of metastatic melanoma patients versus the normal controls at baseline. The results also demonstrated a significant decrease in the level of angiogenin (P = 0.026) and a significant increase in TARC/CCLl7 (P = 0.008) from week 0 to week 4 which was associated with improved overall survival (P = 0.059). Higher TARC/CCL17 levels were observed by ELISA at week 4 and a log-rank comparison revealed a significant association between high serum TARC/CCL17 levels at week 4 and progression-free survival (P = 0.005). Receiver-operator characteristic analysis revealed that week 4 serum TARC/CCL17 levels were predictive of progression-free and overall survival, indicating that serum TARC/CCL17 might be of predictive value of response to dendritic cell-based anti-melanoma immunotherapy.

Angiogenin-stimulated rRNA transcription is essential for initiation and survival of AKT-induced prostate intraepithelial neoplasia.

Angiogenin (ANG), originally identified as an angiogenic ribonuclease, has recently been shown to play a direct role in prostate cancer cell proliferation by mediating rRNA transcription. ANG is up-regulated in human prostate cancer and is the most significantly up-regulated gene in AKT-driven prostate intraepithelial neoplasia (PIN) in mice. Enhanced cell proliferation in the PIN lesions requires increased ribosome biogenesis, a multistep process involving an orchestrated production of ribosomal proteins and rRNA. AKT is known to enhance ribosomal protein production through the mammalian target of rapamycin pathway. However, it was unknown how rRNA is proportionally increased. Here, we report that ANG is essential for AKT-driven PIN formation and survival. We showed that up-regulation of ANG in the AKT-overexpressing mouse prostates is an early and lasting event. It occurs before PIN initiation and lasts beyond PIN is fully developed. Knocking down ANG expression by intraprostate injection of lentivirus-mediated ANG-specific small interfering RNA prevents AKT-induced PIN formation without affecting AKT expression and its signaling through the mammalian target of rapamycin pathway. Neomycin, an aminoglycoside that blocks nuclear translocation of ANG, and N65828, a small-molecule enzymatic inhibitor of the ribonucleolytic activity of ANG, both prevent AKT-induced PIN formation and reverse established PIN. They also decrease nucleolar organizer region, restore cell size, and normalize luminal architectures of the prostate despite continuous activation of AKT. All three types of the ANG inhibitor suppress rRNA transcription of the prostate luminal epithelial cells and inhibit AKT-induced PIN, indicating an essential role of ANG in AKT-mediated cell proliferation and survival.