Angiogenin Research Articles

Characterization of oral biomarkers during early healing at augmented dental implant sites.

The aim of this study is to assess early wound healing expression of local angiogenic biomarkers following connective tissue graft (CTG) at dental implant sites. Twenty-eight subjects with single dental implants exhibiting a soft tissue dehiscence were included and randomly treated with CTG, either with coronally advanced flap (CAF) or with tunnel technique (TUN). Peri-implant crevicular fluid (PICF) was collected at the midfacial and midlingual aspect of the implant sites at baseline and at 3, 7, 14, 30, and 90 days after the surgical intervention. The expression of angiogenin (ANG), fibroblast growth factor-2 (FGF-2), platelet-derived growth factor (PDGF), tissue inhibitor of metalloproteinases-2 (TIMP-2), and vascular endothelial growth factor (VEGF) was investigated over a period of 3 months. Patient-reported outcomes, clinical measurements, and ultrasonography scans at multiple time points were also evaluated. The longitudinal regression revealed a significant difference in the expression of VEGF and TIMP-2 between CAF- and TUN-treated sites over 3 months (p = .033 and p = .004, respectively), whereas no significant differences were observed for ANG, FGF-2 and PDGF between the two groups. At 7 days, a direct correlation was observed between ANG levels and ultrasonographic color velocity in the CAF group (p < .001) and between ANG levels and ultrasonographic color power in the TUN group (p = .028). VEGF levels and ultrasonographic mean perfused area of the CTG were significantly correlated at the 7-day time point (p < .001 for both CAF and TUN). The expression of VEGF at 7 days was directly associated with mucosal thickness gain at 1 year (p < .001 for both groups). Early TIMP-2 expression showed an inverse correlation with time to recovery (p = .002). TIMP-2 levels at 3 months exhibited inverse correlations with mean dehiscence coverage (p = .004) and the rate of complete dehiscence coverage (p = .012). PICF biomarkers can be used to monitor early wound healing events following soft tissue grafting at implant sites. VEGF and TIMP-2 showed correlations with the 1-year clinical and volumetric outcomes, as well as with post-operative patient-reported outcomes and Doppler Ultrasonographic tissue perfusion-related parameters.

Secretome from iPSC-derived MSCs exerts proangiogenic and immunosuppressive effects to alleviate radiation-induced vascular endothelial cell damage

BackgroundRadiation therapy is the standard of care for central nervous system tumours. Despite the success of radiation therapy in reducing tumour mass, irradiation (IR)-induced vasculopathies and neuroinflammation contribute to late-delayed complications, neurodegeneration, and premature ageing in long-term cancer survivors. Mesenchymal stromal cells (MSCs) are adult stem cells that facilitate tissue integrity, homeostasis, and repair. Here, we investigated the potential of the iPSC-derived MSC (iMSC) secretome in immunomodulation and vasculature repair in response to radiation injury utilizing human cell lines.MethodsWe generated iPSC-derived iMSC lines and evaluated the potential of their conditioned media (iMSC CM) to treat IR-induced injuries in human monocytes (THP1) and brain vascular endothelial cells (hCMEC/D3). We further assessed factors in the iMSC secretome, their modulation, and the molecular pathways they elicit.ResultsIncreasing doses of IR disturbed endothelial tube and spheroid formation in hCMEC/D3. When IR-injured hCMEC/D3 (IR ≤ 5 Gy) were treated with iMSC CM, endothelial cell viability, adherence, spheroid compactness, and proangiogenic sprout formation were significantly ameliorated, and IR-induced ROS levels were reduced. iMSC CM augmented tube formation in cocultures of hCMEC/D3 and iMSCs. Consistently, iMSC CM facilitated angiogenesis in a zebrafish model in vivo. Furthermore, iMSC CM suppressed IR-induced NFκB activation, TNF-α release, and ROS production in THP1 cells. Additionally, iMSC CM diminished NF-kB activation in THP1 cells cocultured with irradiated hCMEC/D3, iMSCs, or HMC3 microglial lines. The cytokine array revealed that iMSC CM contains the proangiogenic and immunosuppressive factors MCP1/CCL2, IL6, IL8/CXCL8, ANG (Angiogenin), GROα/CXCL1, and RANTES/CCL5. Common promoter regulatory elements were enriched in TF-binding motifs such as androgen receptor (ANDR) and GATA2. hCMEC/D3 phosphokinome profiling revealed increased expression of pro-survival factors, the PI3K/AKT/mTOR modulator PRAS40 and β-catenin in response to CM. The transcriptome analysis revealed increased expression of GATA2 in iMSCs and the enrichment of pathways involved in RNA metabolism, translation, mitochondrial respiration, DNA damage repair, and neurodevelopment.ConclusionsThe iMSC secretome is a comodulated composite of proangiogenic and immunosuppressive factors that has the potential to alleviate radiation-induced vascular endothelial cell damage and immune activation.

Effects of Pathogenic Mutants of the Neuroprotective RNase 5-Angiogenin in Amyotrophic Lateral Sclerosis (ALS).

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that affects the motoneurons. More than 40 genes are related with ALS, and amyloidogenic proteins like SOD1 and/or TDP-43 mutants are directly involved in the onset of ALS through the formation of polymorphic fibrillogenic aggregates. However, efficacious therapeutic approaches are still lacking. Notably, heterozygous missense mutations affecting the gene coding for RNase 5, an enzyme also called angiogenin (ANG), were found to favor ALS onset. This is also true for the less-studied but angiogenic RNase 4. This review reports the substrate targets and illustrates the neuroprotective role of native ANG in the neo-vascularization of motoneurons. Then, it discusses the molecular determinants of many pathogenic ANG mutants, which almost always cause loss of function related to ALS, resulting in failures in angiogenesis and motoneuron protection. In addition, ANG mutations are sometimes combined with variants of other factors, thereby potentiating ALS effects. However, the activity of the native ANG enzyme should be finely balanced, and not excessive, to avoid possible harmful effects. Considering the interplay of these angiogenic RNases in many cellular processes, this review aims to stimulate further investigations to better elucidate the consequences of mutations in ANG and/or RNase 4 genes, in order to achieve early diagnosis and, possibly, successful therapies against ALS.

Abstract A006: Bladder cancer risk stratification with the Oncuria 10 plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms

Abstract Introduction and Objectives Oncuria® is a bead-based multiplex fluorescence immunoassay that coordinately measures 10 protein biomarkers in urine samples. The current study compared assay performance and output when urine samples were evaluated with the Oncuria assay using three different fluorescence-analyzing instruments commonly used in diagnostic laboratories worldwide. Methods We compared the performance of the clinically validated Oncuria bladder cancer (BC) multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96 well microtiter plates, and consecutively evaluated on the LED/image based MagPix, and laser/flow based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX). The BC assay uses magnetic bead based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. Results All three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically &amp;lt;5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥1 analyte above the highest standard concentration, i.e., A1AT (n=7/18), IL-8 (n=5), and/or ANG (n=2). In Controls, A1AT was higher in one sample. Conclusions In conclusion, the Oncuria BC assay performed similarly well across three different flow analysis platforms for all 10 analytes simultaneously evaluated in urine samples. This agreement across instruments indicates that the test is amenable to standardized performance in laboratories using existing xMAP, without requiring costly outlays for new equipment. Citation Format: Toru Sakatani, Sunao Tanaka, Kaoru Murakami, Richard Waldron, Wayne Hogrefe, Charles Rosser, Hideki Furuya. Bladder cancer risk stratification with the Oncuria 10 plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A006.

Long-timescale atomistic simulations uncover loss-of-function mechanisms of uncharacterized Angiogenin mutants associated with ALS

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive degeneration of motor neurons, resulting in respiratory failure and mortality within 3–5 years. Mutations in the Angiogenin (ANG) cause loss of ribonucleolytic and nuclear translocation activities, contributing to ALS pathogenesis. This study focused on investigating two uncharacterized ANG mutations, T11S and R122H, newly identified in the Project Mine consortium. Using extensive computational analysis, including structural modeling and microsecond-timescale molecular dynamics (MD) simulations, we observed conformational changes in the catalytic residue His114 of ANG induced by T11S and R122H mutations. These alterations impaired ribonucleolytic activity, as inferred through molecular docking and binding free energy calculations. Gibbs free energy landscape and residue-residue interaction network analysis further supported our findings, revealing the energetic states and allosteric pathway from the mutated site to His114. Additionally, we assessed the binding of NCI-65828, an inhibitor of ribonucleolytic activity of ANG, and found reduced effectiveness in binding to T11S and R122H mutants when His114 assumed a non-native conformation. This highlights the crucial role of His114 and its association with ALS. Elucidating the relationship between physical structure and functional dynamics of frequently mutated ANG mutants is essential for understanding ALS pathogenesis and developing more effective therapeutic interventions.

Activation of angiogenin expression in macrophages by lipopolysaccharide via the TLR4/NF-κB pathway in colitis.

Inflammatory bowel disease (IBD) is a debilitating condition that can lead to life-threatening complications. Macrophages are crucial in IBD management because they secrete various cytokines and regulate tissue repair. Macrophage-derived angiogenin (ANG) has been shown to be essential for limiting colonic inflammation, but its upstream regulatory pathway and role in macrophages remain unclear. Here we show that ANG expression is up-regulated in macrophages during colitis treatment or upon lipopolysaccharides (LPS) treatment. Mechanistically, LPS activates Toll-like receptor 4 (TLR4) to initiate NF-κB translocation from the cytoplasm to the nucleus, where it binds to the ANG promoter and enhances its transcriptional activity, leading to increased ANG expression. Interestingly, our data also reveal that the deletion of ANG in macrophages has no adverse effect on key macrophage functions, such as phagocytosis, chemotaxis, and cell survival. Our findings establish a "LPS-TLR4-NF-κB-ANG" regulatory axis in inflammatory disorders and confirm that ANG controls inflammation in a paracrine manner, highlighting the importance of ANG as a key mediator in the complex network of inflammatory processes.

The dynamics of microRNAs level associated with pathological venous angiogenesis in experimental toxic liver fibrosis in rats

BACKGROUND: It is known that miRNAs are important in liver fibrogenesis. However, their use as targets for early diagnosis and treatment of fibrosis is far from use in clinical practice. Angiogenesis and sinusoid capillarization are important histological features of the process. Studies regarding the role of miRNAs in pathological angiogenesis and sinusoid capillarization are insufficient.&#x0D; AIM: To study the molecular targets (miRNAs and mRNAs) dynamics of associated with pathological angiogenesis in toxic fibrosis of the liver; to evaluate the relationship of the selected molecular factors to the processes of restructuring the intrahepatic vascular system.&#x0D; METHODS: Fibrosis and subsequent cirrhosis of the liver in rats of the Wistar line (males) were induced for 17 weeks by a freshly prepared solution of thioacetamide. The level of miRNA-19а-3р, miRNA-29b-3р, miRNA-29b-1-5p, miRNA-34b-5р, miRNA-125b-5р, miRNA-130a-5p, miRNA-195-5р, miRNA-449а-5р, miRNA-449с-5р, miRNA-466d, miRNA-489-3р, miRNA-495, miRNA-664-3р, miRNA-3085, miRNA-3558-3р in fresh frozen liver samples, was determined by Two-tailed RT-qPCR.&#x0D; RESULTS: In this study, we found that throughout the experiment, the relative level of microRNAs varied in a wide range of values (10–3–104 rel. units). In most cases, it decreased at the point of transition from fibrosis to cirrhosis, while growth was observed only for microRNA-29b-3p. Statistically significant correlation relationships were established between microRNAs and the number of interlobular veins, interlobular arteries, sinusoids, and the area of connective tissue (p 0.05).&#x0D; CONCLUSION: A joint analysis of morphological and molecular-genetic parameters allowed us to suggest that within the framework of the current experimental model of liver fibrosis and cirrhosis, the restructuring of the intrahepatic vascular bed and the progression of fibrosis are associated with the dynamics of the level of a number of microRNAs that we studied and Ang mRNA level.

Abstract 3773: Bladder cancer risk stratification with the Oncuria 10 plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms

Abstract Introduction and Objectives: Oncuria® is a bead-based multiplex fluorescence immunoassay that coordinately measures 10 protein biomarkers in urine samples. The current study compared assay performance and output when urine samples were evaluated with the Oncuria assay using three different fluorescence-analyzing instruments commonly used in diagnostic laboratories worldwide. Methods: We compared the performance of the clinically validated Oncuria bladder cancer (BC) multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96 well microtiter plates, and consecutively evaluated on the LED/image based MagPix, and laser/flow based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX). The BC assay uses magnetic bead based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. Results: All three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically &amp;lt;5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥1 analyte above the highest standard concentration, i.e., A1AT (n=7/18), IL-8 (n=5), and/or ANG (n=2). In Controls, A1AT was higher in one sample. Conclusions: In conclusion, the Oncuria BC assay performed similarly well across three different flow analysis platforms for all 10 analytes simultaneously evaluated in urine samples. This agreement across instruments indicates that the test is amenable to standardized performance in laboratories using existing xMAP, without requiring costly outlays for new equipment. Citation Format: Sunao Tanaka, Toru Sakatani, Kaoru Murakami, Richard T. Waldron, Walter Morales, Wayne Hogrefe, Charles J. Rosser, Hideki Furuya. Bladder cancer risk stratification with the Oncuria 10 plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3773.

Herpes Simplex Virus 1 Glycoprotein B from a Hyperfusogenic Virus Mediates Enhanced Cell-Cell Fusion.

Herpes simplex virus 1 (HSV-1) causes significant morbidity and death in humans worldwide. Herpes simplex virus 1 has a complex fusion mechanism that is incompletely understood. The HSV-1 strain ANG has notable fusion and entry activities that distinguish it from wild type. HSV-1 ANG virions fused with the Vero cell surface at 4 °C and also entered cells more efficiently at 15 °C, relative to wild type HSV-1 strain KOS virions, consistent with a hyperfusogenic phenotype. Understanding the molecular basis for the unique entry and fusion activities of HSV-1 strain ANG will help decipher the HSV fusion reaction and entry process. Sequencing of HSV-1 ANG genes revealed multiple changes in gB, gC, gD, gH, and gL proteins relative to wild type HSV-1 strains. The ANG UL45 gene sequence, which codes for a non-essential envelope protein, was identical to wild type KOS. HSV-1 ANG gB, gD, and gH/gL were necessary and sufficient to mediate cell-cell fusion in a virus-free reporter assay. ANG gB, when expressed with wild type KOS gD and gH/gL, increased membrane fusion, suggesting that ANG gB has hyperfusogenic cell-cell fusion activity. Replacing the KOS gD, gH, or gL with the corresponding ANG alleles did not enhance cell-cell fusion. The novel mutations in the ANG fusion and entry glycoproteins provide a platform for dissecting the cascade of interactions that culminate in HSV fusion and entry.

Neural stem cell homeostasis is affected in cortical organoids carrying a mutation in Angiogenin.

Mutations in Angiogenin (ANG) and TARDBP encoding the 43 kDa transactive response DNA binding protein (TDP-43) are associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). ANG is neuroprotective and plays a role in stem cell dynamics in the haematopoietic system. We obtained skin fibroblasts from members of an ALS-FTD family, one with mutation in ANG, one with mutation in both TARDBP and ANG, and one with neither mutation. We reprogrammed these fibroblasts to induced pluripotent stem cells (iPSCs) and generated cortical organoids as well as induced stage-wise differentiation of the iPSCs to neurons. Using these two approaches we investigated the effects of FTD-associated mutations in ANG and TARDBP on neural precursor cells, neural differentiation, and response to stress. We observed striking neurodevelopmental defects such as abnormal and persistent rosettes in the organoids accompanied by increased self-renewal of neural precursor cells. There was also a propensity for differentiation to later-born neurons. In addition, cortical neurons showed increased susceptibility to stress, which is exacerbated in neurons carrying mutations in both ANG and TARDBP. The cortical organoids and neurons generated from patient-derived iPSCs carrying ANG and TARDBP gene variants recapitulate dysfunctions characteristic of frontotemporal lobar degeneration observed in FTD patients. These dysfunctions were ameliorated upon treatment with wild type ANG. In addition to its well-established role during the stress response of mature neurons, ANG also appears to play a role in neural progenitor dynamics. This has implications for neurogenesis and may indicate that subtle developmental defects play a role in disease susceptibility or onset. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Bladder cancer risk stratification with the Oncuria 10-plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms

BackgroundNo single marker of bladder cancer (BC) exists in urine samples with sufficient accuracy for disease diagnosis and treatment monitoring. The multiplex Oncuria BC assay noninvasively quantifies the concentration of 10 protein analytes in voided urine samples to quickly generate a unique molecular profile with proven BC diagnostic and treatment-tracking utility. Test adoption by diagnostic and research laboratories mandates reliably reproducible assay performance across a variety of instrumentation platforms used in different laboratories.MethodsWe compared the performance of the clinically validated Oncuria BC multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96-well microtiter plates on Day 1, and consecutively evaluated on the LED/image-based MagPix, and laser/flow-based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX) on Day 2. The BC assay uses magnetic bead-based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. All three analyzers quantify fluorescence signals generated by the Oncuria assay.ResultsAll three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically < 5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥ 1 analyte above the highest standard concentration, i.e., A1AT (n = 7/18), IL-8 (n = 5), and/or ANG (n = 2), while only one control sample registered an analyte (A1AT) above the highest standard concentration.ConclusionMultiplex BC assays generate detailed molecular signatures useful for identifying BC, predicting treatment responsiveness, and tracking disease progression and recurrence. The similar performance of the Oncuria assay across three different analyzer systems supports test adaptation by clinical and research laboratories using existing xMAP platforms.Trial Registration: This study was registered at ClinicalTrials.gov as NCT04564781, NCT03193528, NCT03193541, and NCT03193515.

A Neglected Gene: The Role of the ANG Gene in the Pathogenesis of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease with a poor prognosis. To date, more than 40 ALS-related genes have been identified. However, there is still a lack of targeted therapeutic drugs for the treatment of ALS, especially for patients with acute onset and severe disease. A series of studies reported missense heterozygous mutations with loss of function in the coding region of the ANG gene in ALS patients. ANG deficiency is related to the pathogenesis of ALS, but the underlying mechanism has not been determined. This article aimed to synthesize and consolidate the knowledge of the pathological mechanism of ALS induced by ANG mutation and provide a theoretical basis for ALS diagnosis and targeted therapy. This article further delves into the mechanisms underlying the current understanding of the structure and function of the ANG gene, the association between ANG and ALS, and its pathogenesis. Mutations in ANG may lead to the development of ALS through the loss of neuroprotective function, induction of oxidative stress, or inhibition of rRNA synthesis. ANG mutations and genetic and environmental factors may cause disease heterogeneity and more severe disease than in ALS patients with the wild-type gene. Exploring this mechanism is expected to provide a new approach for ALS treatment through increasing ANG expression or angiogenin activity. However, the related study is still in its infancy; therefore, this article also highlights the need for further exploration of the application of ANG gene mutations in clinical trials and animal experiments is needed to achieve improved early diagnosis and treatment of ALS.

CompCorona: A web application for comparative transcriptome analyses of coronaviruses reveals SARS-CoV-2-specific host response.

Understanding the mechanism of host transcriptomic response to infection by the SARS-CoV-2 virus is crucial, especially for patients suffering from long-term effects of COVID-19, such as long COVID or pericarditis inflammation, potentially linked to side effects of the SARS-CoV-2 spike proteins. We conducted comprehensive transcriptome and enrichment analyses on lung and peripheral blood mononuclear cells (PBMCs) infected with SARS-CoV-2, as well as on SARS-CoV and MERS-CoV, to uncover shared pathways and elucidate their common disease progression and viral replication mechanisms. We developed CompCorona, the first interactive online tool for visualizing gene response variance among the family Coronaviridae through 2D and 3D principal component analysis (PCA) and exploring systems biology variance using pathway plots. We also made preprocessed datasets of lungs and PBMCs infected by SARS-CoV-2, SARS-CoV, and MERS-CoV publicly available through CompCorona. One remarkable finding from the lung and PBMC datasets for infections by SARS-CoV-2, but not infections by other coronaviruses (CoVs), was the significant downregulation of the angiogenin (ANG) and vascular endothelial growth factor A (VEGFA) genes, both directly involved in epithelial and vascular endothelial cell dysfunction. Suppression of the TNF signaling pathway was also observed in cells infected by SARS-CoV-2, along with simultaneous activation of complement and coagulation cascades and pertussis pathways. The ribosome pathway was found to be universally suppressed across all three viruses. The CompCorona online tool enabled the comparative analysis of 9 preprocessed host transcriptome datasets of cells infected by CoVs, revealing the specific host response differences in cases of SARS-CoV-2 infection. This included identifying markers of epithelial dysfunction via interactive 2D and 3D PCA, Venn diagrams, and pathway plots. Our findings suggest that infection by SARS-CoV-2 might induce pulmonary epithelial dysfunction, a phenomenon not observed in cells infected by other CoVs. The publicly available CompCorona tool, along with the preprocessed datasets of cells infected by various CoVs, constitutes a valuable resource for further research into CoV-associated syndromes.

Generation of Porcine Angiogenin 4-Expressing Pichia pastoris and Its Protection against Intestinal Inflammatory Injury.

Antimicrobial peptides have been extensively studied as potential alternatives to antibiotics. Porcine angiogenin 4 (pANG4) is a novel antimicrobial peptide in the angiogenin (ANG) family, which may have a regulatory effect on intestinal microflora. The object of present study is obtained pANG4 protein by heterologous expression, so as to explore the biological function of recombinant pANG4 (rpANG4). The pANG4 was expressed in Pichia pastoris (P. pastoris) and anti-inflammatory effects were investigated in intestinal porcine epithelial cell line-J2 (IPEC-J2) and mice. Purified rpANG4 had bacteriostatic activity and did not cause hemolysis or cytotoxicity at concentrations below 128 μg/mL. Purified rpANG4 increased the activity of IPEC-J2 and reduced apoptosis in vitro. rpANG4 reduced the pro-inflammatory gene expression and upregulated tight junction protein gene expression during inflammation. rpANG4 alleviated lipopolysaccharide (LPS)-induced liver and spleen damage, intestinal inflammation, jejunal apoptosis genes' expression, and improved immune function in an in vivo mice model. rpANG4 increased tight junction protein gene expression in jejunum, thereby improving the jejunum intestinal barrier function. In conclusion, rpANG4 had antibacterial activity, inhibited intestinal inflammation, improved intestinal barrier function, and alleviated liver and spleen damage. The current study contributes to the development of antibiotic substitutes and the improvement of animal health.

Advanced breast cancer diagnosis: Multiplex RT-qPCR for precise typing and angiogenesis profiling

Breast cancer (BC) remains the foremost cause of cancer-related mortality, with an estimated 2.3 million new cases anticipated globally. The timely diagnosis of BC is pivotal for effective treatment. Currently, BC diagnosis predominantly relies on Immunohistochemistry (IHC), a method known for its sluggishness, expense, and dependence on proficient pathologists for confident cancer typing. In this study, we introduce a novel approach to enhance the accuracy, speed, and cost-effectiveness of BC diagnosis. We employ multiplex Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) with touch-down methods, which consistently yield significantly lower Cycle Threshold (CT) values. The study evaluates gene expression profiles of HER2, PGR, ESR, and Ki67 genes across 61 samples representing four BC subtypes, using RPL13A as the endogenous control gene. The results demonstrate that our method offers remarkable precision, nearly equivalent to IHC, in detecting gene expressions vital for BC diagnosis and subtyping. Moreover, we explore the gene expression of Hif1A, ANG, and VEGFR genes involved in angiogenesis, shedding light on the metastatic potential of the tested BC tumours. Notably, numerous samples exhibit elevated levels of Hif1A and VEGFR, indicating their potential as valuable biomarkers for assessing metastatic status. Collectively, our RT-qPCR methodology emerges as a powerful diagnostic tool for swiftly identifying BC subtypes and can be complemented with other essential tumorigenic biomarker assessments, such as angiogenesis, to further refine cancer characterisation and inform personalised therapeutic strategies for BC patients. This innovation holds the promise of revolutionising BC diagnosis and treatment, offering expedited and reliable insights for improved patient care.

Abstract C116: Impact of human iPSC-derived MSC secretome in modulating radiation induced inflammation and brain vascular endothelial cell damage

Abstract Introduction: Radiation therapy (RT) is the standard of care for central nervous system (CNS) cancers. Despite the success of RT in reducing tumor mass, RT-induced side effects such as ECM remodeling, vasculopathies and neuroinflammation are well documented that cause reduced neurogenecity and premature aging like complications during post-cancer survivorship. Since mesenchymal stromal cells (MSCs) are multipotent adult stem cells that support tissue integrity, homeostasis and repair through factors they release, we here investigated the potential of iPSC-derived MSC (iMSC)-secretome in immunomodulation and vasculature repair in response to radiation-induced alterations utilizing models of human monocytic and microglial cells (THP1, HMC-3) and human brain endothelial cells (HCMEC/D3). Approaches: (a) Investigate the impact of iMSC secretome on vasculature damage caused by radiation-induced cell injury and RT-induced inflammation, by evaluating its effects on (i) endothelial cell survival and morphogenesis (ii) activation of peripheral monocytes stimulated by X-ray radiation therapy (RT), RT-induced microglial injury, and vasculature injury mediated inflammation (b) Identify factors and pathways that may contribute to the observed phenotype. Results: Decline in intact angiogenic tube formation and less compact spheroid formation was observed for HCMEC/D3 cells after irradiation at doses (Gy): 2.5, 5, 10, 15, and 30, with maximal effect observed at 30Gy. Evaluating the effects of iMSC secretome on radiation induced endothelial cell damage and inflammation revealed increased cell viability with reduced apoptosis, higher cell adherance and better spheroid compactness in 5Gy irradiated HCMEC/D3 cells after iMSC-conditioned media (iMSC-CM) treatment. Reduction in NFκB promoter activity was observed in irradiated THP1- NFκB-Luc2 reporter line, with reduced levels of TNF-α release per unit cell viability in THP1 after iMSC-CM treatment. NFκB promoter activity was also reduced in effect of iMSC treatment in THP1- NFκB-Luc2 reporter cells subjected to irradiated vasculature cells (D3), with or without iMSC coculture and, irradiated microglial cells (HMC3), mimicking vasculature injury, microglial injury, and immune cell crosstalk, respectively. iMSC secretome facilitated a more networked tube formation in HCMEC/D3 cells in coculture of D3+iMSC cells that was longer retained. Cytokine arrays revealed iMSC-CM to have proangiogenic, immunomodulatory (immune suppressive) and chemotactic factors: MCP1, ANG (angiogenin), IL6, IL8, GRO, GROα, RANTES, and SDF1. Our initial pathway analysis using human phospho-proteome profiling showed iMSC secretome elevates levels of beta-catenin, and PRAS40 (AKT/mTOR modulator) and lowers level of pRSK1/2/3 in HCMEC/D3. Conclusions: Taken together, we demonstrated that iMSC-secretome complements the proangiogenic and immunosuppressive effects of iMSC during radiation induced injury. Further studies are required to validate the in vivo potential of iMSC-secretome as safe biological therapy in long-term cancer survivors. Citation Format: Kshama Gupta, Ralph B. Perkerson III, Tammee M Parsons, Prasanna Vibhute, Abba C. Zubair, Alfredo Quinones-Hinojosa, Takahisa Kanekiyo. Impact of human iPSC-derived MSC secretome in modulating radiation induced inflammation and brain vascular endothelial cell damage [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C116.

Molecular and clinical characterization of ANG expression in gliomas and its association with tumor-related immune response.

Angiogenin (ANG) has been widely reported as a crucial molecular regulator in multiple malignancies. However, its role in gliomagenesis remains unclear. This study aimed to investigate the molecular and clinical characterization of ANG expression at transcriptome level and the association with glioma-related immune response. A total of 301 glioma samples with mRNA microarray data (CGGA301) was obtained from the official website of CGGA project for yielding preliminary results, followed by validation in two independent RNAseq datasets, including TCGA with 697 samples and CGGA325 with 325 patients. Moreover, CGGA single-cell RNAseq (scRNAseq) data were analyzed to identify differential and dynamic ANG expression in different cells. Immunohistochemistry was performed to evaluate ANG protein expression across different WHO grades in a tissue microarray (TMA). Figure generation and statistical analysis were conducted using R software. ANG expression was associated with clinical features, malignant phenotypes, and genomic alterations. Based on significantly correlated genes of ANG, subsequent gene ontology (GO) and gene set enrichment analysis (GSEA) concordantly pointed to the significant association of ANG in immune-related biological processes. Moreover, ANG showed robust correlations with canonical immune checkpoint molecules, including PD1 signaling, CTLA4, TIM3, and B7H3. Gene sets variation analysis (GSVA) found that ANG was particularly associated with activities of macrophages and antigen presentation cells (APCs) in both LGG and GBM across different datasets. Furthermore, the higher-ANG milieu seemed to recruit monocyte-macrophage lineage and dendritic cells into the glioma microenvironment. According to scRNAseq analysis, ANG was mainly expressed by neoplastic cells and tumor-associated macrophages (TAMs) and was correlated with the initiation and progression of tumor cells and the polarization of TAMs. Finally, Kaplan-Meier plots demonstrated that higher expression of ANG was significantly correlated with shorter survival in gliomas. Cox regression analysis further confirmed ANG as an independent predictor of prognosis for gliomas of all three datasets. ANG is significantly correlated with a range of malignant and aggressive characteristics in gliomas and reveals considerable prognostic value for glioma patients. ANG seems to be primarily associated with immune activities of macrophages and APCs in gliomas. Furthermore, ANG is mainly expressed in neoplastic cells and TAMs and is involved in the initiation and progression of neoplastic cells as well as macrophage polarization.

The effect of growth hormone on ovarian function recovery in a mouse model of ovarian insufficiency.

To evaluate the effects and mechanisms of action of growth hormone (GH) in the recovery of ovarian function in ovarian insufficiency induced by cyclophosphamide (CP) in a mouse model. After inducing ovarian insufficiency by administering 400 mg/kg of CP intraperitoneally to 6-week-old ICR mice, the mice were divided into four groups (control, CP, 1 mg/kg GH, and 2 mg/kg GH) with 10 mice in each group. GH was administered a week later for 7 days. Five mice from each group were sacrificed the next day, and their ovaries were collected for histological examination. The remaining mice were superovulated for in vitro fertilization (IVF). The terminal deoxynucleotidyl transferase dUTP-nick end labeling assay was performed to detect apoptosis. Masson's trichrome staining was used to analyze the degree of fibrosis. To quantify angiogenesis, CD31 immunohistochemistry was performed. Angiogenesis-related gene expression profiles were assessed using quantitative reverse transcription polymerase chain reaction. CP induced the loss of non-growing (primordial and primary) follicles while GH significantly protected primordial follicles and increased follicular quality. The CP group showed a decrease in fertilization and blastocyst formation rates in IVF. In contrast, the GH treatment group showed dose-dependent enhanced IVF outcomes. Furthermore, GH treatment decreased apoptosis and stromal fibrosis and increased angiogenesis. Many genes involved in angiogenesis, especially Leptin (Lep), platelet endothelial cell adhesion molecule 1 (Pecam-1), and angiogenin (Ang) were up-regulated in the GH treatment groups. GH treatment may promote the recovery of ovarian function in ovarian insufficiency induced by the administration of CP via decreasing apoptosis and stromal fibrosis and upregulating Lep, Pecam-1, and Ang genes.

Expression analysis, clinical significance and potential function of PLXNB2 in acute myeloid leukaemia.

The overall survival (OS) rate of adult patients suffering from acute myeloid leukaemia (AML) remains unsatisfactory at less than 40%. Current risk stratification systems fail to provide accurate guidelines for precise treatment. Novel biomarkers for predicting prognosis are urgently needed. Plexin B2 (PLXNB2), a functional receptor of angiogenin (ANG), has been found to be aberrantly expressed in multitudinous tumours. We detected overexpression of PLXNB2 mRNA in AML via transcriptome microarray analysis. This study aims to explore the potential role of PLXNB2 as a biomarker of prognosis and a prospective target of AML. qRT‒PCR was conducted to verify the expression of PLXNB2 mRNA in bone marrow mononuclear cells from AML patients. Immunohistochemical and immunofluorescence staining were performed and confirmed increased expression of PLXNB2 protein in AML bone marrow tissues. Data on PLXNB2 expression, prognosis and clinical features were accessed from multiple bioinformatic databases, including The Cancer Genome Atlas (TCGA). Genes coexpressed and correlated with PLXNB2 were identified and analysed in the TCGA AML cohort. Metascape was applied for functional and pathway enrichment analysis of genes related to PLXNB2. Small molecular agents and traditional Chinese medicines potentially targeting genes related to PLXNB2 were screened via the Connectivity Map, TCMSP and HIT databases. PLXNB2 mRNA and protein levels are higher in AML samples than in normal controls. Overexpression of PLXNB2 is associated with worse OS in AML. Patients with high PLXNB2 expression might benefit more from haematopoietic stem cell transplantation (HSCT) (indicated by prolonged OS) than those with only chemotherapy treatment. Differentially expressed genes between the high and low PLXNB2 expression groups were overlapped with PLXNB2-coexpressed genes, and genes that overlapped were enriched in immune functions, endothelial cell regulation and cell interaction gene sets, indicating the potential function of PLXNB2 in AML. A total of 36 hub genes were identified from the differentially expressed genes, and MRC1, IL10, CD163 and CCL22 had significant prognostic value for AML. Analysis of the connectivity map and traditional agents revealed that honokiol, morphines, triptolide and paeoniflorin could be potential treatment regimens. The overexpression of PLXNB2 is an adverse prognostic factor in adult AML patients and could be used as a potential biomarker. PLXNB2 might exert an oncogenic role by modulating immune functions, endothelial cell functions and cell interactions. AML patients with high PLXNB2 expression could benefit more from HSCT.

Comparative Study of the Cytokine Profiles of Serum and Tissues from Patients with the Ossification of the Posterior Longitudinal Ligament.

The ossification of the posterior longitudinal ligament (OPLL) is one of the contributing factors leading to severe cervical spondylotic myelopathy (CSM). The mechanism causing ossification is still unclear. The current study was designed to analyze the specimens of patients with or without OPLL. The study collected 51 patients with cervical spondylosis. There were six serum samples in both the non-OPLL (NOPLL) and OPLL groups. For tissue analysis, there were seven samples in the NOPLL group and five samples in the OPLL group. The specimens of serum and tissue were analyzed by using Human Cytokine Antibody Arrays to differentiate biomarkers between the OPLL and NOPLL groups, as well as between serum and OPLL tissue. Immunohistochemical staining of the ligament tissue was undertaken for both groups. For OPLL vs. NOPLL, the serum leptin levels are higher in the OPLL group, corroborating others' observations that it may serve as a disease marker. In the tissue, angiogenin (ANG), osteopontin (OPN), and osteopro-tegerin (OPG) are higher than they are in the OPLL group (p < 0.05). For serum vs. OPLL tissue, many chemotactic cytokines demonstrated elevated levels of MIP1 delta, MCP-1, and RANTES in the serum, while many cytokines promoting or regulating bone genesis were up-regulated in tissue (oncostatin M, FGF-9, LIF, osteopontin, osteoprotegerin, TGF-beta2), as well as the factor that inhibits osteoclastogenesis (IL-10), with very few cytokines responsible for osteoclastogenesis. Molecules promoting angiogenesis, including angiotensin, vEGF, and osteoprotegerin, are abundant in the OPLL tissue, which paves the way for robust bone growth.