In the WHO diagnostic classification, prefibrotic myelofibrosis (pre-MF) is included in the category of primary myelofibrosis (PMF). However, strong evidence for this position is lacking.
We ...investigated whether pre-MF may be aligned along a clinical and biological continuum in 683 consecutive patients who received a WHO diagnosis of PMF.
As compared with PMF-fibrotic type, pre-MF (132 cases) showed female dominance, younger age, higher hemoglobin, higher platelet count, lower white blood cell count, smaller spleen index and higher incidence of splanchnic vein thrombosis. Female to male ratio and hemoglobin steadily decreased, while age increased from pre-MF to PMF- fibrotic type with early and to advanced bone marrow (BM) fibrosis. Likely, circulating CD34+ cells, LDH levels, and frequency of chromosomal abnormalities increased, while CXCR4 expression on CD34+ cells and serum cholesterol decreased along the continuum of BM fibrosis. Median survival of the entire cohort of PMF cases was 21 years. Ninety-eight, eighty-one and fifty-six percent of patients with pre-MF, PMF-fibrotic type with early and with advanced BM fibrosis, respectively, were alive at 10 years from diagnosis.
Pre-MF is a presentation mode of PMF with a very indolent phenotype. The major consequences of this contention is a new clinical vision of PMF, and the need to improve prognosis prediction of the disease.
Endothelial colony-forming cells (ECFCs) are the only endothelial progenitor cells (EPCs) that are capable of acquiring a mature endothelial phenotype. ECFCs are mainly mobilized from bone marrow to ...promote vascularization and represent a promising tool for cell-based therapy of severe ischemic diseases. Vascular endothelial growth factor (VEGF) stimulates the proliferation of peripheral blood-derived ECFCs (PB-ECFCs) through oscillations in intracellular Ca(2+) concentration (Ca(2+)i). VEGF-induced Ca(2+) spikes are driven by the interplay between inositol-1,4,5-trisphosphate (InsP3)-dependent Ca(2+) release and store-operated Ca(2+) entry (SOCE). The therapeutic potential of umbilical cord blood-derived ECFCs (UCB-ECFCs) has also been shown in recent studies. However, VEGF-induced proliferation of UCB-ECFCs is faster compared with their peripheral counterpart. Unlike PB-ECFCs, UCB-ECFCs express canonical transient receptor potential channel 3 (TRPC3) that mediates diacylglycerol-dependent Ca(2+) entry. The present study aimed at investigating whether the higher proliferative potential of UCB-ECFCs was associated to any difference in the molecular underpinnings of their Ca(2+) response to VEGF. We found that VEGF induces oscillations in Ca(2+)i that are patterned by the interaction between InsP3-dependent Ca(2+) release and SOCE. Unlike PB-ECFCs, VEGF-evoked Ca(2+) oscillations do not arise in the absence of extracellular Ca(2+) entry and after pharmacological (with Pyr3 and flufenamic acid) and genetic (by employing selective small interference RNA) suppression of TRPC3. VEGF-induced UCB-ECFC proliferation is abrogated on inhibition of the intracellular Ca(2+) spikes. Therefore, the Ca(2+) response to VEGF in UCB-ECFCs is shaped by a different Ca(2+) machinery as compared with PB-ECFCs, and TRPC3 stands out as a promising target in EPC-based treatment of ischemic pathologies.
Endothelial progenitor cell (EPC) mobilization has been reported following tissue damage, whereas no data are available regarding the mobilization of hematopoietic progenitor cells (HPCs). We ...performed the phenotypic and functional analysis of circulating CD34+ progenitor cells in patients with acute myocardial infarction (AMI), assessed from admission up to 60 days, in patients with stable angina pectoris (SA), and in healthy controls (CTRLs). In patients with AMI at admission (T0), the number of circulating CD34+ cells was higher (P < .001) than in CTRLs and became comparable with CTRLs within 60 days. Both the number of CD34+ cells coexpressing CD33, CD38, or CD117 and the number of HPCs was higher (P < .02 for all) in patients with AMI at T0 than in CTRLs, as was the number of hematopoietic colonies (P < .03). Patients with AMI (T0) had a significantly increased number of CD34+ vascular endothelial growth factor receptor 2–positive (VEGFR-2+) cells (P < .002) with respect to CTRLs, including CD34+ CD133+VEGFR-2+ and CD34+ CD117+VEGFR-2+ EPCs. The number of endothelial colonies was higher in patients with AMI (T0) than in CTRLs (P < .05). No significant difference was documented between patients with SA and CTRLs. Spontaneous mobilization of both HPCs and EPCs occurs within a few hours from the onset of AMI and is detectable until 2 months.
Anemia is a common complication of inflammatory bowel disease, but its epidemiology may be changing due to earlier diagnosis and improved treatments. We investigated the prevalence and pathogenesis ...of anemia in patients with inflammatory bowel disease.
In a cross-sectional study 263 out-patients with inflammatory bowel disease (165 with Crohn's disease, 98 with ulcerative colitis) were investigated. The influence of time from diagnosis, disease activity, inflammation and the status of iron and hematinic vitamins on the level of hemoglobin and prevalence of anemia were evaluated. In a second group of 27 patients with Crohn's disease, undergoing anti-tumor necrosis factor-alpha treatment with infliximab because of refractory or fistulizing disease, we determined the effects of infliximab on disease activity, hemoglobin, serum erythropoietin levels, iron status and inflammation.
In all, 104 of the 263 patients with inflammatory bowel disease were anemic. Age, gender and azathioprine treatment had no influence on anemia. The prevalence of anemia was highest at diagnosis (65%), decreased during the first 4 years after disease onset, and was stable thereafter. Active disease was associated with higher rates of anemia. At diagnosis most anemic patients had anemia of chronic disease; during follow-up iron deficiency and multifactorial forms of anemia became more prevalent. Eighteen of 27 patients undergoing treatment with infliximab were anemic; most of them had anemia of chronic disease. Infliximab reduced disease activity and improved anemia in 12 patients. This was mediated by an increased production of erythropoietin for the degree of anemia. In vitro infliximab increased the growth of erythroid progenitors from the peripheral blood of patients with active disease. Conclusions Anemia is a common problem in out-patients with inflammatory bowel disease; the prevalence and severity of anemia are related to the activity of the bowel disorder. The pathogenesis of anemia changes during the course of the disease, with anemia of chronic disease having a major role at diagnosis and iron deficiency and multifactorial forms of anemia during follow-up. In patients requiring anti-tumor necrosis factor-alpha treatment, response to therapy improves erythropoiesis.
Few investigators have evaluated the usefulness of the JAK2 V617F mutation for explaining the phenotypic variations and for predicting the risk of major clinical events in primary myelofibrosis ...(PMF). In a transversal survey we assayed by allele-specific polymerase chain reaction (PCR) the JAK2 V617F mutational status in 304 patients with PMF. Multiple DNA samples were collected prospectively from 64 patients, and a highly sensitive quantitative PCR was used as a confirmatory test. In a longitudinal prospective study we determined the progression rate to clinically relevant outcomes in 174 patients who had JAK2 mutation determined at diagnosis. JAK2 V617F was identified in 63.4% of patients. None of the V617F-negative patients who were sequentially genotyped progressed to become V617F positive, whereas progression rate from heterozygous to homozygous mutation was 10 per 100 patient-years. JAK2 V617F mutation contributed to hemoglobin, aquagenic pruritus, and platelet count variability, whereas homozygous mutation was independently associated with higher white blood cell count, larger spleen size, and greater need for cytoreductive therapies. Adjusting for conventional risk factors, V617F mutation independently predicted the evolution toward large splenomegaly, need of splenectomy, and leukemic transformation. We conclude that JAK2 V617F genotype should be considered in any future risk stratification of patients with PMF.
Primary myelofibrosis is characterized by the development of fibrosis in the bone marrow that contributes to ineffective hematopoiesis. Bone marrow fibrosis is the result of a complex and not yet ...fully understood interaction among megakaryocytes, myeloid cells, fibroblasts, and endothelial cells. Here, we report that >30% of the endothelial cells in the small vessels of the bone marrow and spleen of patients with primary myelofibrosis have a mesenchymal phenotype, which is suggestive of the process known as endothelial-to-mesenchymal transition (EndMT). EndMT can be reproduced in vitro by incubation of cultured endothelial progenitor cells or spleen-derived endothelial cells with inflammatory cytokines. Megakaryocytes appear to be implicated in this process, because EndMT mainly occurs in the microvessels close to these cells, and because megakaryocyte-derived supernatant fluid can reproduce the EndMT switch in vitro. Furthermore, EndMT is an early event in a JAK2-V617F knock-in mouse model of primary myelofibrosis. Overall, these data show for the first time that microvascular endothelial cells in the bone marrow and spleen of patients with primary myelofibrosis show functional and morphologic changes that are associated to the mesenchymal phenotype.
Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently discovered Ca2+‐releasing messenger that increases the intracellular Ca2+ concentration by mobilizing the lysosomal Ca2+ ...store through two‐pore channels 1 (TPC1) and 2 (TPC2). NAADP‐induced lysosomal Ca2+ release regulates multiple endothelial functions, including nitric oxide release and proliferation. A sizeable acidic Ca2+ pool endowed with TPC1 is also present in human endothelial colony‐forming cells (ECFCs), which represent the only known truly endothelial precursors. Herein, we sought to explore the role of the lysosomal Ca2+ store and TPC1 in circulating ECFCs by harnessing Ca2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly–Phe β‐naphthylamide, and nigericin, which dissipates the proton gradient which drives Ca2+ sequestration by acidic organelles, caused endogenous Ca2+ release in the presence of a replete inositol‐1,4,5‐trisphosphate (InsP3)‐sensitive endoplasmic reticulum (ER) Ca2+ pool. Likewise, the amount of ER releasable Ca2+ was reduced by disrupting lysosomal Ca2+ content. Liposomal delivery of NAADP induced a transient Ca2+ signal that was abolished by disrupting the lysosomal Ca2+ store and by pharmacological and genetic blockade of TPC1. Pharmacological manipulation revealed that NAADP‐induced Ca2+ release also required ER‐embedded InsP3 receptors. Finally, NAADP‐induced lysosomal Ca2+ release was found to trigger vascular endothelial growth factor‐induced intracellular Ca2+ oscillations and proliferation, while it did not contribute to adenosine‐5′‐trisphosphate‐induced Ca2+ signaling. These findings demonstrated that NAADP‐induced TPC1‐mediated Ca2+ release can selectively be recruited to induce the Ca2+ response to specific cues in circulating ECFCs.
Nicotinic acid adenine dinucleotide phosphate (NAADP) stimulated to the endolysosomal two‐pore channel 1 to mediate intracellular Ca2+ signals in endothelial colony‐forming cells. NAADP‐induced endolysosomal (EL) Ca2+ release was then amplified by inositol‐1,4,5‐trisphosphate receptors through the Ca2+‐induced Ca2+‐release process. NAADP‐induced EL Ca2+ release triggered vascular endothelial growth factor‐induced proangiogenic Ca2+ oscillations.
•Unlike liver and lung fibrosis and ET BM, BM from PMF expresses a signature of noncanonical TGF-β signaling activation.•This signature identifies TGF-β as potential target for micro-environment ...therapy in PMF.
Megakaryocytes have been implicated in the micro-environmental abnormalities associated with fibrosis and hematopoietic failure in the bone marrow (BM) of primary myelofibrosis (PMF) patients, the Philadelphia-negative myeloproliferative neoplasm (MPN) associated with the poorest prognosis. To identify possible therapeutic targets for restoring BM functions in PMF, we compared the expression profiling of PMF BM with that of BM from essential thrombocytopenia (ET), a fibrosis-free MPN also associated with BM megakaryocyte hyperplasia. The signature of PMF BM was also compared with published signatures associated with liver and lung fibrosis. Gene set enrichment analysis (GSEA) identified distinctive differences between the expression profiles of PMF and ET. Notch, K-Ras, IL-8, and apoptosis pathways were altered the most in PMF as compared with controls. By contrast, cholesterol homeostasis, unfolded protein response, and hypoxia were the pathways found altered to the greatest degree in ET compared with control specimens. BM from PMF expressed a noncanonical transforming growth factor β (TGF-β) signature, which included activation of ID1, JUN, GADD45b, and genes with binding motifs for the JUN transcriptional complex AP1. By contrast, the expression of ID1 and GADD45b was not altered and there was a modest signal for JUN activation in ET. The similarities among PMF, liver fibrosis, and lung fibrosis were modest and included activation of integrin-α9 and tropomyosin-α1 between PMF and liver fibrosis, and of ectoderm–neural cortex protein 1 and FRAS1-related extracellular matrix protein 1 between PMF and lung fibrosis, but not TGF-β. These data identify TGF-β as a potential target for micro-environmental therapy in PMF.
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Numerous studies have documented ultrastructural abnormalities in malignant megakaryocytes from bone marrow (BM) of myelofibrosis patients but the morphology of these cells in spleen, an important ...extramedullary site in this disease, was not investigated as yet. By transmission-electron microscopy, we compared the ultrastructural features of megakaryocytes from BM and spleen of myelofibrosis patients and healthy controls. The number of megakaryocytes was markedly increased in both BM and spleen. However, while most of BM megakaryocytes are immature, those from spleen appear mature with well-developed demarcation membrane systems (DMS) and platelet territories and are surrounded by platelets. In BM megakaryocytes, paucity of DMS is associated with plasma (thick with protrusions) and nuclear (dilated with large pores) membrane abnormalities and presence of numerous glycosomes, suggesting a skewed metabolism toward insoluble polyglucosan accumulation. By contrast, the membranes of the megakaryocytes from the spleen were normal but these cells show mitochondria with reduced crests, suggesting deficient aerobic energy-metabolism. These distinctive morphological features suggest that malignant megakaryocytes from BM and spleen express distinctive metabolic impairments that may play different roles in the pathogenesis of myelofibrosis.
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