Evaluating the susceptibility of AGO2-loaded microRNAs to degradation by nucleases in vitro.

MicroRNAs (miRNAs) comprise a class of small non-coding RNAs that regulate the stability and/or translatability of most protein-coding transcripts. Steady-state levels of mature miRNAs can be controlled through mechanisms that influence their biogenesis and/or decay rates. Pathways that mediate mature miRNA decay are less well understood than those that mediate miRNA biogenesis. We recently described Tudor-staphylococcal/micrococcal-like nuclease (TSN)-mediated miRNA decay (TumiD) as a cellular pathway that promotes the sequence-specific endonucleolytic decay of miRNAs that harbor a CA and/or UA dinucleotide. Here, we describe an in vitro assay for evaluating the susceptibility of AGO2-loaded miRNAs to degradation by different classes of nucleases. This in vitro approach can be used to complement in vivo studies that aim to identify novel miRNA decay factors.

Angiogenin-induced tRNA fragments inhibit translation initiation.

Angiogenin is a stress-activated ribonuclease that cleaves tRNA within anticodon loops to produce tRNA-derived stress-induced fragments (tiRNAs). Transfection of natural or synthetic tiRNAs inhibits protein synthesis and triggers the phospho-eIF2α-independent assembly of stress granules (SGs), essential components of the stress response program. We show that selected tiRNAs inhibit protein synthesis by displacing eIF4G/eIF4A from uncapped > capped RNAs. tiRNAs also displace eIF4F, but not eIF4E:4EBP1, from isolated m(7)G cap. We identify a terminal oligoguanine motif that is required to displace the eIF4F complex, inhibit translation, and induce SG assembly. We show that the tiRNA-associated translational silencer YB-1 contributes to angiogenin-, tiRNA-, and oxidative stress-induced translational repression. Our data reveal some of the mechanisms by which stress-induced tRNA cleavage inhibits protein synthesis and activates a cytoprotective stress response program.

A Novel Extrinsic Pathway for the Unfolded Protein Response in the Kidney.

The ribonuclease angiogenin is a component of the mammalian stress response, and functions in both cell-autonomous and non-cell-autonomous ways to promote tissue adaptation to injury. We recently showed that angiogenin regulates tissue homeostasis during AKI associated with endoplasmic reticulum (ER) stress through the production of transfer RNA fragments that interfere with translation initiation and thereby alleviate ER stress. However, whether the paracrine signaling mediated by angiogenin secretion is a genuine component of the ER stress response to kidney injury is unknown. Here, we explored the molecular mechanisms by which angiogenin is secreted upon ER stress, and determined how it modulates the inflammatory microenvironment. In cultured renal epithelial cells, ER stress specifically induced angiogenin secretion under the selective control of inositol-requiring enzyme 1α, a key activator of the unfolded protein response. The transcription factors spliced X-box-binding protein 1 and p65, which are activated by inositol-requiring enzyme 1α upon ER stress, each bound the angiogenin promoter and controlled the amount of angiogenin secreted. Furthermore, p65 promoted angiogenin transcription in an ER stress-dependent manner. Similar to secretion of the ER stress-induced proinflammatory cytokine IL-6, secretion of angiogenin required the ER-Golgi pathway. Notably, incubation of human macrophages with angiogenin promoted macrophage reprogramming toward an activated and proinflammatory phenotype. In patients, angiogenin expression increased upon renal inflammation, and the urinary concentration of angiogenin correlated with the extent of immune-mediated kidney injury. Collectively, our data identify angiogenin as a mediator of the ER stress-dependent inflammatory response and as a potential noninvasive biomarker of AKI.

Three decades of research on angiogenin: a review and perspective.

As a member of the vertebrate-specific secreted ribonucleases, angiogenin (ANG) was first isolated and identified solely by its ability to induce new blood vessel formation, and now, it has been recognized to play important roles in various physiological and pathological processes through regulating cell proliferation, survival, migration, invasion, and/or differentiation. ANG exhibits very weak ribonucleolytic activity that is critical for its biological functions, and exerts its functions through activating different signaling transduction pathways in different target cells. A series of recent studies have indicated that ANG contributes to cellular nucleic acid metabolism. Here, we comprehensively review the results of studies regarding the structure, mechanism, and function of ANG over the past three decades. Moreover, current problems and future research directions of ANG are discussed. The understanding of the function and mechanism of ANG in a wide context will help to better delineate its roles in diseases, especially in cancer and neurodegenerative diseases.

Bovine brain ribonuclease is the functional homolog of human ribonuclease 1.

Mounting evidence suggests that human pancreatic ribonuclease (RNase 1) plays important roles in vivo, ranging from regulating blood clotting and inflammation to directly counteracting tumorigenic cells. Understanding these putative roles has been pursued with continual comparisons of human RNase 1 to bovine RNase A, an enzyme that appears to function primarily in the ruminant gut. Our results imply a different physiology for human RNase 1. We demonstrate distinct functional differences between human RNase 1 and bovine RNase A. Moreover, we characterize another RNase 1 homolog, bovine brain ribonuclease, and find pronounced similarities between that enzyme and human RNase 1. We report that human RNase 1 and bovine brain ribonuclease share high catalytic activity against double-stranded RNA substrates, a rare quality among ribonucleases. Both human RNase 1 and bovine brain RNase are readily endocytosed by mammalian cells, aided by tight interactions with cell surface glycans. Finally, we show that both human RNase 1 and bovine brain RNase are secreted from endothelial cells in a regulated manner, implying a potential role in vascular homeostasis. Our results suggest that brain ribonuclease, not RNase A, is the true bovine homolog of human RNase 1, and provide fundamental insight into the ancestral roles and functional adaptations of RNase 1 in mammals.

Increased ribonuclease expression reduces inflammation and prolongs survival in TLR7 transgenic mice.

TLR7 activation is implicated in the pathogenesis of systemic lupus erythematosus. Mice that overexpress TLR7 develop a lupus-like disease with autoantibodies and glomerulonephritis and early death. To determine whether degradation of the TLR7 ligand RNA would alter the course of disease, we created RNase A transgenic (Tg) mice. We then crossed the RNase Tg to TLR7 Tg mice to create TLR7 × RNase double Tg (DTg) mice. DTg mice had a significantly increased survival associated with reduced activation of T and B lymphocytes and reduced kidney deposition of IgG and C3. We observed massive hepatic inflammation and cell death in TLR7 Tg mice. In contrast, hepatic inflammation and necrosis were strikingly reduced in DTg mice. These findings indicate that high concentrations of serum RNase protect against immune activation and inflammation associated with TLR7 stimulation and that RNase may be a useful therapeutic strategy in the prevention or treatment of inflammation in systemic lupus erythematosus and, possibly, liver diseases.

Motoneurons secrete angiogenin to induce RNA cleavage in astroglia.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder affecting motoneurons. Mutations in angiogenin, encoding a member of the pancreatic RNase A superfamily, segregate with ALS. We previously demonstrated that angiogenin administration shows promise as a neuroprotective therapeutic in studies using transgenic ALS mice and primary motoneuron cultures. Its mechanism of action and target cells in the spinal cord, however, are largely unknown. Using mixed motoneuron cultures, motoneuron-like NSC34 cells, and primary astroglia cultures as model systems, we here demonstrate that angiogenin is a neuronally secreted factor that is endocytosed by astroglia and mediates neuroprotection in paracrine. We show that wild-type angiogenin acts unidirectionally to induce RNA cleavage in astroglia, while the ALS-associated K40I mutant is also secreted and endocytosed, but fails to induce RNA cleavage. Angiogenin uptake into astroglia requires heparan sulfate proteoglycans, and engages clathrin-mediated endocytosis. We show that this uptake mechanism exists for mouse and human angiogenin, and delivers a functional RNase output. Moreover, we identify syndecan 4 as the angiogenin receptor mediating the selective uptake of angiogenin into astroglia. Our data provide new insights into the paracrine activities of angiogenin in the nervous system, and further highlight the critical role of non-neuronal cells in the pathogenesis of ALS.

The role of angiogenin in pT1-T2 tongue carcinoma neo-angiogenesis and cell proliferation: an exploratory study.

BACKGROUND: Angiogenin (ANG) is a member of the ribonuclease superfamily and of medical interest largely because it supports the growth of primary and metastatic malignancies. This study is the first to investigate the potential role of ANG in tongue carcinoma neo-angiogenesis and cancer cell proliferation. METHODS: Angiogenin expression (in carcinoma cells and endothelial intratumor vessel cells), CD105-assessed micro-vessel density (MVD), and MIB-1 expression were correlated with prognostic parameters in 28 primarily consecutively operated pT1-T2 tongue carcinomas (squamous cell carcinoma [SCC]). Whenever feasible, a computer-based image analysis system was used for the immunohistochemical reaction analysis. RESULTS: No significant correlations emerged between ANG expression in the tongue carcinoma cells or endothelial intratumor vessel cells and tongue SCC recurrence rate or disease-free survival (DFS). ANG expression was also unrelated to CD105-assessed MVD or MIB-1 expression. Conversely, CD105-assessed MVD correlated directly with recurrence rate (P = 0.02) and DFS was significantly shorter in cases with CD105-assessed MVD >167 micro-vessels/mm(2) than in those with CD105-assessed MVD ≤167 micro-vessels/mm(2) (P = 0.042). CONCLUSIONS: Our results support the hypothesis that CD105-assessed MVD would be a valuable parameter for predicting which patients with tongue SCC are at greatest risk of disease recurrence. Despite our study results, the role of ANG in tongue carcinoma warrants further investigation in larger series.

Vitamin D and Parkinson's disease.

Parkinson's disease (PD) is the second most common form of neurodegeneration among the elderly population. PD is clinically characterized by tremors, rigidity, slowness of movement, and postural imbalance. Interestingly, a significant association has been demonstrated between PD and low levels of vitamin D in the serum, and vitamin D supplement appears to have a beneficial clinical effect on PD. Genetic studies have provided the opportunity to determine which proteins link vitamin D to PD pathology, e.g., Nurr1 gene, toll-like receptor, gene related to lipid disorders, vascular endothelial factor, tyrosine hydroxylase, and angiogenin. Vitamin D also exerts its effects on cancer through nongenomic factors, e.g., bacillus Calmette-Guerin vaccination, interleukin-10, Wntß-catenin signaling pathways, mitogen-activated protein kinase pathways, and the reduced form of the nicotinamide adenine dinucleotide phosphate. In conclusion, vitamin D might have a beneficial role in PD. Calcitriol is best used for PD because it is the active form of the vitamin D(3) metabolite and modulates inflammatory cytokine expression. Further investigation with calcitriol in PD is needed.

Role of platelet-derived growth factor-AB in tumour growth and angiogenesis in relation with other angiogenic cytokines in multiple myeloma.

Angiogenesis is a complex process essential for the growth, invasion, and metastasis of various malignant tumours, including multiple myeloma (MM). Various angiogenic cytokines have been implicated in the angiogenic process. Among them, platelet-derived growth factor-AB (PDGF-AB) has been reported to be a potent stimulator of angiogenesis in many solid tumours and haematological malignancies, including MM. The aim of the study was to investigate the relationship between PDGF-AB, microvascular density (MVD), and various angiogenic cytokines, such as basic fibroblast growth factor (b-FGF), angiogenin (ANG), and interleukin-6 (IL-6), in MM patients. Forty-seven MM patients before treatment, 22 of whom were in plateau phase, were studied. We determined the serum levels of the aforementioned cytokines and MVD in bone marrow biopsies before and after treatment. Mean serum values of PDGF-AB, b-FGF, ANG, and MVD were significantly higher in patients compared with controls and with increasing disease stage. Significant positive correlations were observed between serum PDGF-AB, ANG, and IL-6 levels and MVD. Furthermore, we found significant positive correlations between PDGF-AB and b-FGF, IL-6, ANG, and ß2 microglobulin. We also found that patients with high MVD had statistically significantly higher serum levels of PDGF-AB when a median MVD value of 7.7 was used as the cutoff point. Furthermore, a significant difference was found in serum levels of PDGF-AB between pre- and post-treatment patients. Finally, survival time was significantly higher in the low MVD group versus the high MVD group (76 vs 51 months). Our results showed that there is a strong positive correlation between PDGF-AB and the studied angiogenic cytokines and MVD. It seems that PDGF-AB plays a role in the complex network of cytokines inducing bone marrow neovascularization in patients with MM.

Assessment of ANG variants in Parkinson's disease.

Genetic risk factors are occasionally shared between different neurodegenerative diseases. Previous studies have linked ANG, a gene encoding angiogenin, to both Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). Functional studies suggest ANG plays a neuroprotective role in both PD and amyotrophic lateral sclerosis by reducing cell death. We further explored the genetic association between ANG and PD by analyzing genotype data from the International Parkinson's Disease Genomics Consortium (14,671 cases and 17,667 controls) and whole genome sequencing data from the Accelerating Medicines Partnership - Parkinson's disease initiative (AMP-PD, https://amp-pd.org/) (1,647 cases and 1,050 controls). Our analysis did not replicate the findings of previous studies and identified no significant association between ANG variants and PD risk.

Neamine inhibits prostate cancer growth by suppressing angiogenin-mediated rRNA transcription.

PURPOSE: Angiogenin (ANG) undergoes nuclear translocation and stimulates rRNA transcription in both prostate cancer cells and endothelial cells. The purpose of this study is to assess the antitumor activity of neamine, a nontoxic degradation product of neomycin that blocks nuclear translocation of ANG. EXPERIMENTAL DESIGN: The anti-prostate cancer activity of neamine was first evaluated in a xenograft animal model. It was then examined in the murine prostate-restricted AKT transgenic mice that develop prostate intraepithelial neoplasia (PIN) owing to AKT transgene overexpression. RESULTS: Neamine inhibits xenograft growth of PC-3 human prostate cancer cells in athymic mice. It blocks nuclear translocation of ANG and inhibits rRNA transcription, cell proliferation, and angiogenesis. Neamine also prevents AKT-induced PIN formation as well as reverses fully developed PIN in murine prostate-restricted AKT mice, accompanied by a decrease in rRNA synthesis, cell proliferation, and angiogenesis and an increase in prostate epithelial cell apoptosis. CONCLUSION: We confirmed that ANG is a molecular target for cancer drug development and that blocking nuclear translocation of ANG is an effective means to inhibit its activity. Our results also suggested that neamine is a lead compound for further preclinical evaluation.

On the information expressed in enzyme primary structure: lessons from Ribonuclease A.

The information expressed in an enzyme's primary structure is investigated. Brownian computations are directed at Ribonuclease A (RNase A) so as to quantify the information at the atom/covalent bond level. The information content and distribution are crucial because the primary structure underpins the molecule's chemical functions. Brownian computation data are illustrated for the native protein, mutants, and sequence isomers. The results identify signature features of the active protein on new information grounds. The same tools offer rapid screening of proteins and polypeptides whereby several examples are illustrated.

[Biological functions and therapeutic properties of angiogenins].

The review is devoted to an analysis of recently published data on the biological functions of angiogenins with a view to their potential use as an active basis for new pharmaceuticals and cosmetics.

Pericyte Bridges in Homeostasis and Hyperglycemia.

Diabetic retinopathy is a potentially blinding eye disease that threatens the vision of one-ninth of patients with diabetes. Progression of the disease has long been attributed to an initial dropout of pericytes that enwrap the retinal microvasculature. Revealed through retinal vascular digests, a subsequent increase in basement membrane bridges was also observed. Using cell-specific markers, we demonstrate that pericytes rather than endothelial cells colocalize with these bridges. We show that the density of bridges transiently increases with elevation of Ang-2, PDGF-BB, and blood glucose; is rapidly reversed on a timescale of days; and is often associated with a pericyte cell body located off vessel. Cell-specific knockout of KLF4 in pericytes fully replicates this phenotype. In vivo imaging of limbal vessels demonstrates pericyte migration off vessel, with rapid pericyte filopodial-like process formation between adjacent vessels. Accounting for off-vessel and on-vessel pericytes, we observed no pericyte loss relative to nondiabetic control retina. These findings reveal the possibility that pericyte perturbations in location and process formation may play a role in the development of pathological vascular remodeling in diabetic retinopathy.

Ribonuclease inhibitor regulates neovascularization by human angiogenin.

Human angiogenin (ANG) is a homologue of bovine pancreatic ribonuclease (RNase A) that induces neovascularization. ANG is the only human angiogenic factor that possesses ribonucleolytic activity. To stimulate blood vessel growth, ANG must be transported to the nucleus and must retain its catalytic activity. Like other mammalian homologues of RNase A, ANG forms a femtomolar complex with the cytosolic ribonuclease inhibitor protein (RI). To determine whether RI affects ANG-induced angiogenesis, we created G85R/G86R ANG, which possesses 10(6)-fold lower affinity for RI but retains wild-type ribonucleolytic activity. The neovascularization of rabbit corneas by G85R/G86R ANG was more pronounced and more rapid than by wild-type ANG. These findings provide the first direct evidence that RI serves to regulate the biological activity of ANG in vivo.

Blockade of angiogenin by thalidomide inhibits the tumorigenesis of murine hemangioendothelioma.

Thalidomide, a well-known immunomodulatory compound, has an anti-angiogenic activity, which may be utilized for the treatment of angiogenesis-related diseases such as hemangioendothelioma. The aim of the present study was to investigate both the antitumor role of thalidomide on hemangioendothelioma and the underlying mechanism. By using the xenograft mouse model, we found that thalidomide can inhibit the progression of hemangioendothelioma in vivo. Moreover, thalidomide shows no effect on the proliferation of hemangioendothelioma endothelial cell (EOMA), but significantly impairs the pro-angiogenic capacity of the EOMA cells in vitro. By qRT-PCR screening, we observed that the expression of angiogenin was downregulated by thalidomide treatment. We next performed tissue array analysis and found a positive correlation between angiogenin expression level and hemangioendothelioma occurrence in patients. Moreover, we confirmed that the antitumoral role of thalidomide is dependent on angiogenin expression both in vivo and in vitro. Taken together, we concluded that thalidomide can inhibit the progression of hemangioendothelioma by downregulating the expression of pro-angiogenic factor angiogenin and therefore can be used as a potent therapeutic to treat hemangioendothelioma.

Nucleotide substrate binding characterization in human pancreatic-type ribonucleases.

Human genome contains a group of more than a dozen similar genes with diverse biological functions including antiviral, antibacterial and angiogenesis activities. The characterized gene products of this group show significant sequence similarity and a common structural fold associated with binding and cleavage of ribonucleic acid (RNA) substrates. Therefore, these proteins have been categorized as members of human pancreatic-type ribonucleases (hRNases). hRNases differ in cell/tissue localization and display distinct substrate binding preferences and a wide range of ribonucleolytic catalytic efficiencies. Limited information is available about structural and dynamical properties that influence this diversity among these homologous RNases. Here, we use computer simulations to characterize substrate interactions, electrostatics and dynamical properties of hRNases 1-7 associated with binding to two nucleotide substrates (ACAC and AUAU). Results indicate that even with complete conservation of active-site catalytic triad associated with ribonucleolytic activity, these enzymes show significant differences in substrate interactions. Detailed characterization suggests that in addition to binding site electrostatic and van der Waals interactions, dynamics of distal regions may also play a role in binding. Another key insight is that a small difference in temperature of 300 K (used in experimental studies) and 310 K (physiological temperature) shows significant changes in enzyme-substrate interactions.

Ribonucleases with angiogenic and bactericidal activities from the Atlantic salmon.

The importance of fish in vertebrate evolution has been better recognized in recent years after the intense work carried out on fish genomics. The recent discovery that fish genomes comprise homologs of ribonucleases, studied before only in tetrapods, and the isolation of ribonucleases from zebrafish have suggested an experimental model for studying fish and vertebrate evolution. Thus, the cDNAs encoding the RNases from the Atlantic salmon were expressed, and the recombinant RNases (Ss-RNase-1 and Ss-RNase-2) were isolated and characterized as both proteins and for their biological activities. Salmon RNases are less active than RNase A in degrading RNA, but are both sensitive to the action of the human cytosolic RNase inhibitor. The two enzymes possess both angiogenic and bactericidal activities. However, catalytically inactivated Ss-RNases do not exert any angiogenic activity, but preserve their full bactericidal activity, which is surprisingly preserved even when the enzyme proteins are fully denatured. Analyses of the conformational stability of the two RNases has revealed that they are as stable as typical RNases of the superfamily, and Ss-RNase-2, the most active as an enzyme, is also the most resistant to thermal and chemical denaturation. The implications of these findings in terms of the evolution of early RNases, in particular of the physiological significance of the angiogenic and bactericidal activities of fish RNases, are analyzed and discussed.

Therapeutic potential of angiogenin modified mesenchymal stem cells: angiogenin improves mesenchymal stem cells survival under hypoxia and enhances vasculogenesis in myocardial infarction.

BACKGROUND: To examine the effects of angiogenin modified mesenchymal stem cells (MSCs) on ventricular remodeling and cardiac function in a rat model of acute myocardial infarction. METHODS: MSCs were transfected with adenoviral vectors carrying either angiogenin (MSC(AdANG)) or EGFP (MSC(AdEGFP)). Angiogenin gene amplification, protein expression and cell death were assayed after hypoxic treatment. DiI labeled MSC(AdANG) and MSC(AdEGFP) were injected into infracted heart. Six weeks after cell transplantation, echocardiography and histological study were performed. RESULTS: After hypoxia treatment, angiogenin modified MSCs effectively expressed angiogenin for at least 14 days. The death of MSC(AdANG) was one-third of MSCAd(EGFP), and 50% of untreated MSCs. In the infracted myocardium, the number of DiI labeling cells in MSC(AdANG) group with high angiogenin expression was three-fold that in MSC(AdEGFP) group. Echocardiograms suggested that angiogenin modified MSCs significantly improved left ventricular (LV) systolic and diastolic function. Histological study confirmed that ventricular remodeling was attenuated significantly in MSC(AdANG) group. Furthermore, vasculogenesis was enhanced significantly in MSC(AdANG) group as measured by both factor VIII and alpha-SMA staining. CONCLUSION: Angiogenin modified MSCs enhanced the tolerance of engrafted MSCs to hypoxia injury in vitro and improved their viability in infracted hearts, thus helping preserve the LV contractile function and attenuate LV remodeling through vasculogenesis.