scholarly journals VEGF Regulation of Angiogenic Factors via Inflammatory Signaling in Myeloproliferative Neoplasms

2021 ◽  
Vol 22 (13) ◽  
pp. 6671
Author(s):  
Tijana Subotički ◽  
Olivera Mitrović Ajtić ◽  
Emilija Živković ◽  
Miloš Diklić ◽  
Dragoslava Đikić ◽  
...  
Keyword(s):  
Protein Expression ◽  
Chronic Inflammation ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Angiogenic Factors ◽  
Mtor Signaling ◽  
Angiogenic Factor ◽  
Inflammatory Signaling ◽  
Gene And Protein Expression ◽  
Hel Cells

Background: Chronic inflammation has been recognized in neoplastic disorders, including myeloproliferative neoplasm (MPN), as an important regulator of angiogenesis. Aims: We investigated the influence of vascular endothelial growth factor (VEGF) and pro-inflammatory interleukin-6 (IL-6) on the expression of angiogenic factors, as well as inflammation-related signaling in mononuclear cells (MNC) of patients with MPN and JAK2V617F positive human erythroleukemic (HEL) cells. Results: We found that IL-6 did not change the expression of angiogenic factors in the MNC of patients with MPN and HEL cells. However, IL-6 and the JAK1/2 inhibitor Ruxolitinib significantly increased angiogenic factors—endothelial nitric oxide synthase (eNOS), VEGF, and hypoxia-inducible factor-1 alpha (HIF-1α)—in patients with polycythemia vera (PV). Furthermore, VEGF significantly increased the expression of HIF-1α and eNOS genes, the latter inversely regulated by PI3K and mTOR signaling in the MNC of primary myelofibrosis (PMF). VEGF and inhibitors of inflammatory JAK1/2, PI3K, and mTOR signaling reduced the eNOS protein expression in HEL cells. VEGF also decreased the expression of eNOS and HIF-1α proteins in the MNC of PMF. In contrast, VEGF increased eNOS and HIF-1α protein expression in the MNC of patients with PV, which was mediated by the inflammatory signaling. VEGF increased the level of IL-6 immunopositive MNC of MPN. In summary, VEGF conversely regulated gene and protein expression of angiogenic factors in the MNC of PMF, while VEGF increased angiogenic factor expression in PV mediated by the inflammation-related signaling. Conclusion: The angiogenic VEGF induction of IL-6 supports chronic inflammation that, through positive feedback, further promotes angiogenesis with concomitant JAK1/2 inhibition.

Abstract 151: A Novel Role for DDiT4L in Regulation of mTOR and Autophagy in the Heart

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Bridget Simonson ◽  
Hannabeth Franchino ◽  
Ashley Knight ◽  
Anthony Rosenzweig ◽  
Saumya Das
Keyword(s):  
Heart Failure ◽  
Protein Expression ◽  
Glucose Deprivation ◽  
Mtor Signaling ◽  
Confocal Imaging ◽  
Negative Regulator ◽  
Neonatal Rat ◽  
Pathological Hypertrophy ◽  
Gene And Protein Expression ◽  
Physiological Hypertrophy

Introduction: DDiT4L is a known negative regulator of mTOR signaling in skeletal muscle; however its role in the heart is unknown. We have recently showed increased DDiT4L mRNA in a murine transgenic model of pathological but not physiological hypertrophy. Here we test the hypothesis that DDiT4L is a regulator of mTOR signaling in the heart and may play a role in pathological hypertrophy and heart failure. Methods: We investigated the regulation of DDiT4L in murine models of hypertrophy and in cultured neonatal rat ventricular cardiomyocytes (NRVMs). Loss and gain of function of DDiT4L in mTOR regulation and autophagy was investigated using confocal imaging, immunoblotting, and qRT-PCR in NRVMs. Results: DDiT4L gene and protein expression was increased four-fold in pressure overload hypertrophy (n = 4-6, p<0.001), but not in a swim model of physiological hypertrophy. DDiT4L gene expression also significantly increased in a genetic model of dilated cardiomyopathy model (n = 4, p<0.001). In NRVMs, DDiT4L was induced by cardiac stressors such as pathological stretch, hypoxia, and glucose deprivation (n = 3-5 in duplicate, p<0.05-0.01). Increased DDiT4L expression correlated with inhibition of mTOR signaling, and an increase in autophagy markers. siRNA ablation of DDiT4L revealed that inhibition of mTOR signaling by DDiT4L was necessary for glucose deprivation induced autophagy, as determined by imaging of GFP-LC3 autophagosomes (n = 3 in duplicate, p<0.01), and immunoblotting of autophagy markers. Conversely, adenoviral-driven overexpression of DDiT4L inhibited mTOR signaling and significantly increased basal autophagy (n = 3 in duplicate, p<0.05). In TAC mice, the increase in DDiT4L protein expression correlated to inhibition of mTOR signaling, increases in autophagy markers (p<0.01), and preceded the transition to LV dilation and HF. Conclusion: Our data suggests that DDiT4L expression is altered in diverse models of pathological hypertrophy and precedes the development of LV dilatation and overt heart failure. DDiT4L inhibition of mTOR and modulation of autophagy may play a role in the progression to heart failure. DDiT4L may represent a novel therapeutic target to prevent this transition.

scholarly journals Synergic Crosstalk between Inflammation, Oxidative Stress, and Genomic Alterations in BCR–ABL-Negative Myeloproliferative Neoplasm

Antioxidants ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1037
Author(s):  
Alessandro Allegra ◽  
Giovanni Pioggia ◽  
Alessandro Tonacci ◽  
Marco Casciaro ◽  
Caterina Musolino ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stem Cell ◽  
Chronic Inflammation ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Tumor Stage ◽  
Driver Mutations ◽  
Hematopoietic Stem ◽  
Chronic Myeloproliferative Neoplasms ◽  
Inflammatory State

Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) have recently been revealed to be related to chronic inflammation, oxidative stress, and the accumulation of reactive oxygen species. It has been proposed that MPNs represent a human inflammation model for tumor advancement, in which long-lasting inflammation serves as the driving element from early tumor stage (over polycythemia vera) to the later myelofibrotic cancer stage. It has been theorized that the starting event for acquired stem cell alteration may occur after a chronic inflammation stimulus with consequent myelopoietic drive, producing a genetic stem cell insult. When this occurs, the clone itself constantly produces inflammatory components in the bone marrow; these elements further cause clonal expansion. In BCR–ABL1-negative MPNs, the driver mutations include JAK 2, MPL, and CALR. Transcriptomic studies of hematopoietic stem cells from subjects with driver mutations have demonstrated the upregulation of inflammation-related genes capable of provoking the development of an inflammatory state. The possibility of acting on the inflammatory state as a therapeutic approach in MPNs appears promising, in which an intervention operating on the pathways that control the synthesis of cytokines and oxidative stress could be effective in reducing the possibility of leukemic progression and onset of complications.

scholarly journals CD47 but Not Calreticulin Is over Expressed in Patients with Myeloproliferative Neoplasms

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5492-5492
Author(s):  
Kristian Boasman ◽  
Matthew J Simmonds ◽  
Ciro R Rinaldi
Keyword(s):  
Cell Surface ◽  
Protein Expression ◽  
Mononuclear Cells ◽  
Immune Escape ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Response To Therapy ◽  
Solid Tumours ◽  
Polycythaemia Vera ◽  
Gene And Protein Expression

Abstract Myeloproliferative neoplasms (MPN) are chronic myeloid cancers characterized by the overproduction of mature blood cells, and the tendency to evolve into acute leukaemia. In solid tumours, calreticulin (CALR) overexpression on the cell surface, produces a pro-phagocytic signal and is counteracted by concomitant expression of anti- phagocytic CD47, reflecting an apoptosis vs survival mechanism. In this study, we investigated the expression of CALR and CD47 in patients with MPN and potential changes induced by treatment. CALR and CD47 gene and protein expression were measured by Real Time PCR and western blotting, in K562 and in mononuclear cells obtained by FICOLL separation, from peripheral blood of 13 MPN patients [4 Polycythaemia Vera, 8 Essential Thrombocythemia, and 1 myelofibrosis; 7 treated (IFN or Hydroxyurea) and 6 untreated] and compared with 4 healthy controls. Cells were also fractionised into 4 compartments: membrane, cytoplasm, cytosol and nucleus and protein fractions analysed. We found a significant increase in CD47 protein expression into the membrane of MPN patients comparing with controls (91.9 % vs 19.7%). Interestingly in treated MPN the CD47 expression increases even further in comparison with untreated (92.6% vs 91.9%). No significant differences were found in total CALR expression comparing with controls (3.23, vs 2.95-fold), however in treated patients we observed a reduction in the expression in the membrane comparing with untreated (26.4 % vs 39.5 %) and an increase into cytoplasm (69.4 % vs 54.6 %). These findings suggest CD47 exposure as a mechanism of defence in MPN cells during treatment. To better understand the potential effects of therapy on CALR and CD47 expression, we incubated K562 with 0.05μM/ml of Ruxolitinib (RUXO), re-dosed at 24 hours and harvested at 48 hours. RUXO induced CALR internalization, reducing its expression into the membrane (RUXO 8.2% vs Untreated: 13.3%), but increasing its expression in cytosol (RUXO 51% vs Untreated 48.4%) and in the cytoplasm (RUXO 13.5% vs Untreated 2.9%). In contrast, CD47 expression remains relatively unmodified, with only slight increases on the membrane (RUXO 43.1% vs Untreated 40.4%) and in the cytoplasm (RUXO 34.5% vs UT 32.5%). In this study we demonstrated that CD47 but not CALR, is overexpressed on the membrane of patients with MPN. This opposes previous studies in solid tumours, which show significant increases of both CALR and CD47. This suggests a role for CD47 as a strong anti-phagocytic signal responsible for immune survival in MPN. We have also shown a potential mechanism adopted by MPN cells in response to therapy, with the internalization of CALR and the enhanced membrane expression of CD47 which remains strongly expressed on cell surface. The addition of Anti-CD47 compounds in combination with conventional therapies might represent a future therapeutically strategy to counteract MPN cells immune-escape mechanism. Disclosures No relevant conflicts of interest to declare.

Therapeutic Potential of Melatonin in the Regulation of MiR-148a-3p and Angiogenic Factors in Breast Cancer

MicroRNA ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 237-247 ◽  
Author(s):  
Jéssica Zani Lacerda ◽  
Lívia Carvalho Ferreira ◽  
Beatriz Camargo Lopes ◽  
Andrés Felipe Aristizábal-Pachón ◽  
Marcio Chaim Bajgelman ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Protein Expression ◽  
Real Time ◽  
Tumor Cells ◽  
Therapeutic Potential ◽  
Xenograft Model ◽  
Angiogenic Factors ◽  
Migration And Invasion ◽  
Gene And Protein Expression

Background: The high mortality rate of breast cancer is related to the occurrence of metastasis, a process that is promoted by tumor angiogenesis. MicroRNAs are small molecules of noncoding mRNA that play a key role in gene regulation and are directly involved in the progression and angiogenesis of various tumor types, including breast cancer. Several miRNAs have been described as promoters or suppressors angiogenesis and may be associated with tumor growth and metastasis. Melatonin is an oncostatic agent with a capacity of modifying the expression of innumerable genes and miRNAs related to cancer. Objective: The aim of this study was to evaluate the role of melatonin and the tumor suppressor miR- 148a-3p on angiogenesis of breast cancer. Method: MDA-MB-231 cells were treated with melatonin and modified with the overexpression of miR-148a-3p. The relative quantification in real-time of miR-148a-3p, IGF-IR and VEGF was performed by real-time PCR. The protein expression of these targets was performed by immunocytochemistry and immunohistochemistry. Survival, migration and invasion rates of tumor cells were evaluated. Finally, the xenograft model of breast cancer was performed to confirm the role of melatonin in the tumor. Results: The melatonin was able to increase the gene level of miR-148a-3p and decreased the gene and protein expression of IGF-1R and VEGF, both in vitro and in vivo. In addition, it also had an inhibitory effect on the survival, migration and invasion of breast tumor cells. Conclusion: Our results confirm the role of melatonin in the regulation of miR-148a-3p and decrease of angiogenic factors.

Increased Dkk3 protein expression in platelets and megakaryocytes of patients with myeloproliferative neoplasms

2011 ◽  
Vol 105 (01) ◽  
pp. 72-80 ◽  
Author(s):  
Alexandar Tzankov ◽  
Johann Kern ◽  
Andreas Pircher ◽  
Martin Hermann ◽  
Helmut-Werner Ott ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Blood Cell ◽  
Protein Expression ◽  
Myeloproliferative Neoplasms ◽  
Protein A ◽  
Primary Myelofibrosis ◽  
Tumour Suppressor Gene ◽  
Cell Populations ◽  
Bone Marrow Biopsies ◽  
Gene And Protein Expression

SummaryDickkopf-3 (Dkk3) has been proposed as tumour suppressor gene and a marker for tumour blood vessels. We analysed the expression and function of Dkk3 in platelets and megakaryocytes from healthy controls and patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN). Dkk3 protein and gene expression in platelets was compared with endothelial and other blood cell populations by ELISA, real-time PCR, and immunofluorescence. Moreover, megakaryocytes were isolated from bone marrow aspirates by CD61 microbeads. Immunohisto-chemical studies of Dkk3 expression were performed in essential thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis (PMF) and control reactive bone marrow cases (each n=10). Compared to all other blood cell populations platelets showed the highest concentration of Dkk3 protein (150 ± 19 ng/mg total protein). A strong DKK3 gene and protein expression was also observed in isolated megakaryocytes. Dkk3 co-localised with VEGF in α-granules of platelets and was released similar to VEGF upon stimulation. Addition of recombinant Dkk3 had no influence on blood coagulation (aPTT, INR) and platelet aggregation. Significantly more Dkk3+ megakaryocytes/mm2 could be found in bone marrow biopsies from patients with MPN (ET 40 ± 10, PV 31 ± 4, PMF 22 ± 3) than in controls (15 ± 3). The mean proportion of Dkk3+ megakaryocytes was increased in MPN as well (ET 83% ± 15%; PV 84% ± 12%; PMF 77% ± 8%) compared to controls (53% ± 11%). Dkk3+ megakaryocytes correlated with microvessel density in PV and PMF. We conclude that Dkk3 might be involved in the pathogenesis of MPN.

scholarly journals Inhibition of USP9X Downregulates JAK2-V617F and Induces Apoptosis Synergistically with BH3 Mimetics Preferentially in Ruxolitinib-Persistent JAK2-V617F-Positive Leukemic Cells

Cancers ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 406
Author(s):  
Hiroki Akiyama ◽  
Yoshihiro Umezawa ◽  
Daisuke Watanabe ◽  
Keigo Okada ◽  
Shinya Ishida ◽  
...  
Keyword(s):  
Map Kinases ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Leukemic Cells ◽  
Bh3 Mimetics ◽  
Downstream Signaling ◽  
Hel Cells ◽  
Induced Apoptosis ◽  
Novel Therapeutic Strategies

JAK2-V617F plays a key role in the pathogenesis of myeloproliferative neoplasm. However, its inhibitor ruxolitinib has shown limited clinical efficacies because of the ruxolitinib-persistent proliferation of JAK2-V617F-positive cells. We here demonstrate that the USP9X inhibitor WP1130 or EOAI3402143 (G9) inhibited proliferation and induced apoptosis more efficiently in cells dependent on JAK2-V617F than on cytokine-activated JAK2. WP1130 preferentially downregulated activated and autophosphorylated JAK2-V617F by enhancing its K63-linked polyubiquitination and inducing its aggresomal translocation to block downstream signaling. Furthermore, JAK2-V617F associated physically with USP9X in leukemic HEL cells. Induction of apoptosis by inhibition of USP9X was mediated through the intrinsic mitochondria-mediated pathway, synergistically enhanced by BH3 mimetics, prevented by overexpression of Bcl-xL, and required oxidative stress to activate stress-related MAP kinases p38 and JNK as well as DNA damage responses in HEL cells. Although autophosphorylated JAK2-V617F was resistant to WP1130 in the ruxolitinib-persistent HEL-R cells, these cells expressed Bcl-2 and Bcl-xL at lower levels and showed an increased sensitivity to WP1130 as well as BH3 mimetics as compared with ruxolitinib-naive HEL cells. Thus, USP9X represents a promising target along with anti-apoptotic Bcl-2 family members for novel therapeutic strategies against JAK2-V617F-positive myeloproliferative neoplasms, particularly under the ruxolitinib persistence conditions.

scholarly journals Novel Insights into the Effect of Hyperforin and Photodynamic Therapy with Hypericin on Chosen Angiogenic Factors in Colorectal Micro-Tumors Created on Chorioallantoic Membrane

2019 ◽  
Vol 20 (12) ◽  
pp. 3004 ◽  
Author(s):  
Martin Majerník ◽  
Rastislav Jendželovský ◽  
Marián Babinčák ◽  
Ján Košuth ◽  
Juraj Ševc ◽  
...  
Keyword(s):  
Photodynamic Therapy ◽  
Experimental Model ◽  
Metabolic Activity ◽  
Chorioallantoic Membrane ◽  
Angiogenic Factors ◽  
Angiogenic Factor ◽  
Treatment Modalities ◽  
Cell Models ◽  
Gene And Protein Expression ◽  
Factor Expression

Photodynamic therapy with hypericin (HY-PDT) and hyperforin (HP) could be treatment modalities for colorectal cancer (CRC), but evidence of their effect on angiogenic factors in CRC is missing. Convenient experimental model utilization is essential for angiogenesis research. Therefore, not only 2D cell models, but also 3D cell models and micro-tumors were used and compared. The micro-tumor extent and interconnection with the chorioallantoic membrane (CAM) was determined by histological analyses. The presence of proliferating cells and HY penetration into the tumor mass were detected by fluorescence microscopy. The metabolic activity status was assessed by an colorimetric assay for assessing cell metabolic activity (MTT assay) and HY accumulation was determined by flow cytometry. Pro-angiogenic factor expression was determined by Western blot and quantitative real-time polymerase chain reaction (RT-qPCR). We confirmed the cytotoxic effect of HY-PDT and HP and showed that their effect is influenced by structural characteristics of the experimental model. We have pioneered a method for analyzing the effect of HP and cellular targeted HY-PDT on pro-angiogenic factor expression in CRC micro-tumors. Despite the inhibitory effect of HY-PDT and HP on CRC, the increased expression of some pro-angiogenic factors was observed. We also showed that CRC experimental micro-tumors created on quail CAM could be utilized for analyses of gene and protein expression.

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