Spleen endothelial cells from patients with myelofibrosis harbor the JAK2V617F mutation.

Increased microvessel density contributes to abnormal BM and spleen microenvironment in myelofibrosis (MF). Taking advantage of the JAK2V617F mutation as a marker of malignancy, in the present study, we investigated whether splenic endothelial cells (ECs) obtained from capillaries by laser microdissection or from fresh spleen tissue by cell culture or cell sorting harbored such mutation in patients bearing the mutation in their granulocytes and undergoing splenectomy for therapeutical reasons. To extend the analysis to the ECs of large vessels, endothelial tissue from the splenic vein was also studied. We found JAK2V617F(+) ECs in 12 of 18 patients also bearing the mutation in their granulocytes. In 3 patients, the mutation was found in at least 2 different EC samples obtained by laser microdissection, cell culture, or cell sorting. The mutation was detected in the splenic vein ECs of 1 of 6 patients investigated. In conclusion, we provide evidence that some ECs from the spleen and splenic veins of patients with MF bear the JAK2V617F mutation. We suggest that splenic ECs are involved in the process of malignant transformation in MF.

Kinetic and Angiogenic Activity of Circulating Endothelial Colony Forming Cells in Patients with Infantile Haemangioma Receiving Propranolol.

Endothelial progenitor cells (EPCs) have been suggested to contribute to the neovascularization of infantile haemangioma (IH). There is strong evidence of the efficacy of propranolol in the treatment of IH, possibly by inhibiting both vasculogenesis and angiogenesis in the tumour. We evaluate the frequency of circulating endothelial colony forming cells (ECFCs), as the best EPC surrogate, in patients with IH at diagnosis and while receiving propranolol by an ex vivo 12-month longitudinal study. Biological aspects of the ECFCs, such as their in vitro angiogenic potential, membrane CXCR4 expression and Ca2+ signalling, were investigated. Circulating ECFCs were isolated by in vitro culture and expanded for 2 to 3 passages in 23 patients with IH (median age: 5.5 months, range: 5.5 weeks-11 months) before and 3, 6, 9 and 12 months after receiving propranolol. Twenty-four healthy subjects comparable for age were also assessed (CTRLs). Untreated patients with IH had a circulating ECFC frequency lower (p = 0.001) than CTRLs; nevertheless, in in vitro starving conditions, ECFCs showed enhanced capacity to form tube-like structures than those of CTRLs. Patients with IH following the therapy with propranolol had a significantly increased (p = 0.022) circulating ECFC frequency, that showed a diminished tube-like formation capacity in vitro, and an altered constitutive store-operated Ca2+ entry. ECFCs play a role in IH pathogenesis; the response to propranolol therapy is associated with their increased frequency in the peripheral blood and a reduction of their vasculogenic activity.

Primary myelofibrosis: Older age and high JAK2V617F allele burden are associated with elevated plasma high-sensitivity C-reactive protein levels and a phenotype of progressive disease.

We measured plasma levels of high-sensitivity C-reactive protein (hs-CRP) in 526 subjects with primary myelofibrosis (PMF). Thirty-eight percent had an elevated hs-CRP level (≥0.3mg/dL). Elevated hs-CRP levels were associated with a progressive disease phenotype, including anemia, high white blood cell count, low platelet count, increased splenomegaly, increased risk of blast transformation, and worse survival. Age≥52years, but no other demographic characteristics, was associated with an elevated hs-CRP level in multivariable logistic regression (odds ratio [OR], 4.29; 95% CI, 2.73-6.77; P <0.001). Subjects with JAK2V617F mutation and an allele burden≥50% had an age-independent higher incidence of elevated hs-CRP level (OR=1.97; 95% CI,1.21-3.22; P=0.006) compared with a combined cohort of subjects with JAK2V617F <50% allele burden, CALR, MPL mutations, or no detectable driver mutations. Neither ASXL1 or EZH2 sub-clonal mutations, nor JAK2 46/1 haplotype or the A3669G polymorphism of glucocorticoid receptor were significantly associated with increased hs-CRP levels. Subjects with age≥52years and JAK2V617F with≥50% allele burden had a phenotype of progressive disease. Our data indicate that older age and high JAK2V617 allele burden are major determinants of inflammation in PMF, and are associated with disease progression.

Tie2 Expressing Monocytes in the Spleen of Patients with Primary Myelofibrosis.

Primary myelofibrosis (PMF) is a Philadelphia-negative (Ph-) myeloproliferative disorder, showing abnormal CD34+ progenitor cell trafficking, splenomegaly, marrow fibrosis leading to extensive extramedullary haematopoiesis, and abnormal neoangiogenesis in either the bone marrow or the spleen. Monocytes expressing the angiopoietin-2 receptor (Tie2) have been shown to support abnormal angiogenic processes in solid tumors through a paracrine action that takes place in proximity to the vessels. In this study we investigated the frequency of Tie2 expressing monocytes in the spleen tissue samples of patients with PMF, and healthy subjects (CTRLs), and evaluated their possible role in favouring spleen angiogenesis. We show by confocal microscopy that in the spleen tissue of patients with PMF, but not of CTRLs, the most of the CD14+ cells are Tie2+ and are close to vessels; by flow cytometry, we found that Tie2 expressing monocytes were Tie2+CD14lowCD16brightCDL62-CCR2- (TEMs) and their frequency was higher (p = 0.008) in spleen tissue-derived mononuclear cells (MNCs) of patients with PMF than in spleen tissue-derived MNCs from CTRLs undergoing splenectomy for abdominal trauma. By in vitro angiogenesis assay we evidenced that conditioned medium of immunomagnetically selected spleen tissue derived CD14+ cells of patients with PMF induced a denser tube like net than that of CTRLs; in addition, CD14+Tie2+ cells sorted from spleen tissue derived single cell suspension of patients with PMF show a higher expression of genes involved in angiogenesis than that found in CTRLs. Our results document the enrichment of Tie2+ monocytes expressing angiogenic genes in the spleen of patients with PMF, suggesting a role for these cells in starting/maintaining the pathological angiogenesis in this organ.

Increase of circulating endothelial cells in patients with Hereditary Hemorrhagic Telangiectasia.

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disorder characterized by vascular malformations. The genes known to be associated with HHT include ENG (HHT1), ACVRL1 (HHT2) and SMAD4 (JPHT). It has been reported that circulating CD34(+) cell subsets repair damaged vessels. To investigate whether mobilization of these cells is present in the peripheral blood (PB) of HTT patients, we analyzed CD34(+) cells, CD34(+)VEGFR-2(+) progenitor or mature endothelial cells, and CD34(+)CD133(+)VEGFR-2(-) hematopoietic progenitor cells (HPCs). Cytofluorimetric analysis was performed in 150 HTT patients and 43 healthy subjects (CTRLs). In HTT patients, PB CD34(+) cells were significantly increased; the frequency of endothelial cells was higher (P = 0.002), while the frequency of CD34(+)CD133(+)VEGFR-2(-) HPCs was lower (P = 0.00007) than in CTRLs. Results were comparable in patients with ENG or ACVRL1 gene mutation; in patients with ENG mutation, the frequency of the cell subsets inversely correlated with the age of the patients at time of sampling (CD34(+)), disease duration (CD34(+)VEGFR-2(+)), and age at disease onset (CD34(+)CD133(+) VEGFR-2(-)). In conclusion, HHT patients show an increase of circulating endothelial cells and a decrease of HPCs. In patients with ENG mutation, the frequency of CD34(+) endothelial cells correlates with specific clinical characteristics suggesting that their active turnover characterizes the initial phase of the disease.