Angiogenic factors are increased in circulating granulocytes and CD34⁺ cells of myeloproliferative neoplasms

Abstract

It has been shown that angiogenesis and inflammation play an important role in development of most hematological malignancies including the myeloproliferative neoplasm (MPN). The aim of this study was to investigate and correlate the levels of key angiogenic molecules such as hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in peripheral blood and bone marrow cells of MPN patients, along with JAK2V617F mutation allele burden and effects of therapy. HIF-1α and VEGF gene expression were decreased, while eNOS mRNA levels were increased in granulocytes of MPN patients. Furthermore, positively correlated and increased VEGF and eNOS protein levels were in negative correlation with HIF-1α levels in granulocytes of MPN patients. According to immunoblotting, the generally augmented angiogenic factors demonstrated JAK2V617F allele burden dependence only in granulocytes of PMF. The angiogenic factors were largely reduced after hydroxyurea therapy in granulocytes of MPN patients. Levels of eNOS protein expression were stimulated by Calreticulin mutations in granulocytes of essential thrombocythemia. Immunocytochemical analyses of CD34 cells showed a more pronounced enhancement of angiogenic factors than in granulocytes. Increased gene expression linked to the proinflammatory TGFβ and MAPK signaling pathways were detected in CD34 cells of MPN patients. In conclusion, the angiogenesis is increased in several cell types of MPN patients supported by the transcriptional activation of inflammation-related target genes, and is not limited to bone marrow stroma cells. It also appears that some of the benefit of hydroxyurea therapy of the MPN is mediated by effects on angiogenic factors. © 2016 Wiley Periodicals, Inc.

IL6 inhibition of inflammatory S100A8/9 proteins is NF‐κB mediated in essential thrombocythemia

“…After these treatments, the MNC were washed once in PBS, incubated in RIPA lysis buffer at 4°C for 45 minutes and centrifuged at 10000× g at 4°C for 15 minutes. In turn, genomic DNA extraction and subsequent JAK2 V617F mutation analyses were performed as previously reported . JAK2 V617F positivity had the parallel confirmation in granulocytes and MNC of MPN.…”

“…Proteins from MPN‐derived MNC were isolated and processed as previously reported . Equal amounts of protein (30 μg) were run on polyacrylamide gels and transferred to polyvinylidene difluoride membranes.…”

“…For the assessment of mRNA expression levels in PB‐derived CD34 + cells biological replicates of nine healthy donors, seven PV patients, nine ET patients, and four PMF patients were analysed. The human oligo probe set used was the Operon Human Genome Array‐Ready Oligo Set Version 4.0 (Eurofins MWG Operon, Huntsville, Alabama) and the RNA samples were processed and analysed as reported before using total human universal RNA (HuURNA, BD Biosciences, Palo Alto, California) as a reference in the competitive hybridizations . The microarray data obtained are available at the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo; accession no.…”

Can Novel Insights into the Pathogenesis of Myeloproliferative Neoplasm-Related Thrombosis Inform Novel Treatment Approaches?

“…Markers of endothelial activation are elevated in the plasma of patients with MPNs and increased levels of vWF, soluble thrombomodulin and soluble P-selectin, E-selectin and L-selectin were previously demonstrated in the plasma of patients with ET and PV [69,102,103]. Markers of angiogenic activity such as vascular endothelial growth factor (VEGF) were found to be increased in patients with MPNs [104][105][106] as were higher levels of circulating endothelial cells (ECs).…”

“…P-selectin-mediated expression of TF and plateletinduced cytokine expression are characteristic of monocytes in MPN [97][98][99]. Endothelial-Markers of endothelial activation are increased [69,102,103] as are markers of angiogenic activity [104][105][106]. The activated endothelium interacts with platelets via vWF-mediated platelet expression of CD40 ligand with endothelial CD40 as well as cross-talk with RBC's (via expression of Lu/BCAM) and leukocytes (via expression of β1 and β2 integrins).…”