Therapeutic angiogenesis represents an emerging strategy to treat ischemic diseases by stimulating blood vessel growth to rescue local blood perfusion. Therefore, injured microvasculature may be ...repaired by stimulating resident endothelial cells or circulating endothelial colony forming cells (ECFCs) or by autologous cell-based therapy. Endothelial Ca
signals represent a crucial player in angiogenesis and vasculogenesis; indeed, several angiogenic stimuli induce neovessel formation through an increase in intracellular Ca
concentration. Several members of the Transient Receptor Potential (TRP) channel superfamily are expressed and mediate Ca
-dependent functions in vascular endothelial cells and in ECFCs, the only known truly endothelial precursor. TRP Vanilloid 1 (TRPV1), a polymodal cation channel, is emerging as an important player in endothelial cell migration, proliferation, and tubulogenesis, through the integration of several chemical stimuli. Herein, we first summarize TRPV1 structure and gating mechanisms. Next, we illustrate the physiological roles of TRPV1 in vascular endothelium, focusing our attention on how endothelial TRPV1 promotes angiogenesis. In particular, we describe a recent strategy to stimulate TRPV1-mediated pro-angiogenic activity in ECFCs, in the presence of a photosensitive conjugated polymer. Taken together, these observations suggest that TRPV1 represents a useful target in the treatment of ischemic diseases.
Polycythaemia vera is a myeloproliferative neoplasm characterised by excessive proliferation of erythroid, myeloid, and megakaryocytic components in the bone marrow due to mutations in the Janus ...kinase 2 (JAK2) gene. Ruxolitinib, a JAK 1 and JAK 2 inhibitor, showed superiority over best available therapy in a phase 2 study in patients with polycythaemia vera who were resistant to or intolerant of hydroxyurea. We aimed to compare the long-term safety and efficacy of ruxolitinib with best available therapy in patients with polycythaemia vera who were resistant to or intolerant of hydroxyurea.
We report the 5-year results for a randomised, open-label, phase 3 study (RESPONSE) that enrolled patients at 109 sites across North America, South America, Europe, and the Asia-Pacific region. Patients (18 years or older) with polycythaemia vera who were resistant to or intolerant of hydroxyurea were randomly assigned 1:1 to receive either ruxolitinib or best available therapy. Patients randomly assigned to the ruxolitinib group received the drug orally at a starting dose of 10 mg twice a day. Single-agent best available therapy comprised hydroxyurea, interferon or pegylated interferon, pipobroman, anagrelide, approved immunomodulators, or observation without pharmacological treatment. The primary endpoint, composite response (patients who achieved both haematocrit control without phlebotomy and 35% or more reduction from baseline in spleen volume) at 32 weeeks was previously reported. Patients receiving best available therapy could cross over to ruxolitinib after week 32. We assessed the durability of primary composite response, complete haematological remission, overall clinicohaematological response, overall survival, patient-reported outcomes, and safety after 5-years of follow-up. This study is registered with ClinicalTrials.gov, NCT01243944.
We enrolled patients between Oct 27, 2010, and Feb 13, 2013, and the study concluded on Feb 9, 2018. Of 342 individuals screened for eligibility, 222 patients were randomly assigned to receive ruxolitinib (n=110, 50%) or best available therapy (n=112, 50%). The median time since polycythaemia vera diagnosis was 8·2 years (IQR 3·9-12·3) in the ruxolitinib group and 9·3 years (4·9-13·8) in the best available therapy group. 98 (88%) of 112 patients initially randomly assigned to best available therapy crossed over to receive ruxolitinib and no patient remained on best available therapy after 80 weeks of study. Among 25 primary responders in the ruxolitinib group, six had progressed at the time of final analysis. At 5 years, the probability of maintaining primary composite response was 74% (95% CI 51-88). The probability of maintaining complete haematological remission was 55% (95% CI 32-73) and the probability of maintaining overall clinicohaematological responses was 67% (54-77). In the intention-to-treat analysis not accounting for crossover, the probability of survival at 5 years was 91·9% (84·4-95·9) with ruxolitinib therapy and 91·0% (82·8-95·4) with best available therapy. Anaemia was the most common adverse event in patients receiving ruxolitinib (rates per 100 patient-years of exposure were 8·9 for ruxolitinib and 8·8 for the crossover population), though most anaemia events were mild to moderate in severity (grade 1 or 2 anaemia rates per 100 patient-years of exposure were 8·0 for ruxolitinib and 8·2 for the crossover population). Non-haematological adverse events were generally lower with long-term ruxolitinib treatment than with best available therapy. Thromboembolic events were lower in the ruxolitinib group than the best available therapy group. There were two on-treatment deaths in the ruxolitinib group. One of these deaths was due to gastric adenocarcinoma, which was assessed by the investigator as related to ruxolitinib treatment.
We showed that ruxolitinib is a safe and effective long-term treatment option for patients with polycythaemia vera who are resistant to or intolerant of hydroxyurea. Taken together, ruxolitinib treatment offers the first widely approved therapeutic alternative for this post-hydroxyurea patient population.
Novartis Pharmaceuticals Corporation.
Endothelial progenitor cells (EPCs) may be recruited from bone marrow to sustain tumor vascularisation and promote the metastatic switch. Understanding the molecular mechanisms driving EPC ...proliferation and tubulogenesis could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca(2+) entry (SOCE), which is activated by a depletion of the intracellular Ca(2+) pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca(2+)-sensor, Stim1, and the plasmalemmal Ca(2+) channel, Orai1. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca(2+) influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients.
The present study employed Ca(2+) imaging, recombinant sub-membranal and mitochondrial aequorin, real-time polymerase chain reaction, gene silencing techniques and western blot analysis to investigate the expression and the role of SOCE in EPCs isolated from peripheral blood of patients affected by renal cellular carcinoma (RCC; RCC-EPCs) as compared to control EPCs (N-EPCs). SOCE, activated by either pharmacological (i.e. cyclopiazonic acid) or physiological (i.e. ATP) stimulation, was significantly higher in RCC-EPCs and was selectively sensitive to BTP-2, and to the trivalent cations, La(3+) and Gd(3+). Furthermore, 2-APB enhanced thapsigargin-evoked SOCE at low concentrations, whereas higher doses caused SOCE inhibition. Conversely, the anti-angiogenic drug, carboxyamidotriazole (CAI), blocked both SOCE and the intracellular Ca(2+) release. SOCE was associated to the over-expression of Orai1, Stim1, and transient receptor potential channel 1 (TRPC1) at both mRNA and protein level The intracellular Ca(2+) buffer, BAPTA, BTP-2, and CAI inhibited RCC-EPC proliferation and tubulogenesis. The genetic suppression of Stim1, Orai1, and TRPC1 blocked CPA-evoked SOCE in RCC-EPCs.
SOCE is remodelled in EPCs from RCC patients and stands out as a novel molecular target to interfere with RCC vascularisation due to its ability to control proliferation and tubulogenesis.
Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by megakaryocyte (MK) hyperplasia, bone marrow fibrosis, and abnormal stem cell trafficking. PMF may be associated with ...somatic mutations in JAK2, MPL, or CALR. Previous studies have shown that abnormal MKs play a central role in the pathophysiology of PMF. In this work, we studied both gene and microRNA (miRNA) expression profiles in CD34+ cells from PMF patients. We identified several biomarkers and putative molecular targets such as FGR, LCN2, and OLFM4. By means of miRNA-gene expression integrative analysis, we found different regulatory networks involved in the dysregulation of transcriptional control and chromatin remodeling. In particular, we identified a network gathering several miRNAs with oncogenic potential (eg, miR-155-5p) and targeted genes whose abnormal function has been previously associated with myeloid neoplasms, including JARID2, NR4A3, CDC42, and HMGB3. Because the validation of miRNA-target interactions unveiled JARID2/miR-155-5p as the strongest relationship in the network, we studied the function of this axis in normal and PMF CD34+ cells. We showed that JARID2 downregulation mediated by miR-155-5p overexpression leads to increased in vitro formation of CD41+ MK precursors. These findings suggest that overexpression of miR-155-5p and the resulting downregulation of JARID2 may contribute to MK hyperplasia in PMF.
•Differential gene and miRNA expression analysis in PMF granulocytes identifies new biomarkers and putative therapeutic targets.•Activation of the miR-155/JARID2 axis in PMF CD34+ cells results in overproduction of MK precursors.
Arachidonic acid (AA) stimulates endothelial cell (EC) proliferation through an increase in intracellular Ca2+ concentration (Ca2+i), that, in turn, promotes nitric oxide (NO) release. AA-evoked Ca2+ ...signals are mainly mediated by Transient Receptor Potential Vanilloid 4 (TRPV4) channels. Circulating endothelial colony forming cells (ECFCs) represent the only established precursors of ECs. In the present study, we, therefore, sought to elucidate whether AA promotes human ECFC (hECFC) proliferation through an increase in Ca2+i and the following activation of the endothelial NO synthase (eNOS). AA induced a dose-dependent Ca2+i raise that was mimicked by its non-metabolizable analogue eicosatetraynoic acid. AA-evoked Ca2+ signals required both intracellular Ca2+ release and external Ca2+ inflow. AA-induced Ca2+ release was mediated by inositol-1,4,5-trisphosphate receptors from the endoplasmic reticulum and by two pore channel 1 from the acidic stores of the endolysosomal system. AA-evoked Ca2+ entry was, in turn, mediated by TRPV4, while it did not involve store-operated Ca2+ entry. Moreover, AA caused an increase in NO levels which was blocked by preventing the concomitant increase in Ca2+i and by inhibiting eNOS activity with NG-nitro-l-arginine methyl ester (l-NAME). Finally, AA per se did not stimulate hECFC growth, but potentiated growth factors-induced hECFC proliferation in a Ca2+- and NO-dependent manner. Therefore, AA-evoked Ca2+ signals emerge as an additional target to prevent cancer vascularisation, which may be sustained by ECFC recruitment.
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The term "angiogenic switch" describes one of the earlier events of tumorigenesis, that occurs when the balance between pro-and anti-angiogenic factors shifts towards a pro-angiogenic outcome. This ...leads to the transition from a microscopic indolent lesion to a macroscopic and vascularised primary tumor, that may eventually metastasize and spread to distant sites. The molecular mechanisms underlying such a critical step in the carcinogenetic process have been extensively investigated. Both local endothelial cells (ECs) and endothelial progenitor cells (EPCs), recruited from bone marrow, have been implicated in the angiogenic switch, which is ultimately triggered by a plethora of growth factors released by cancer cells, pivotal among which is vascular endothelial growth factor (VEGF); indeed, VEGF both activates ECs nearby the growing tumor, and leads to EPC mobilization into the circulation. In kidney, in particular, the frequent mutation of the Von Hippel Lindau tumor suppressor gene leads to an overproduction of pro-angiogenic factors which makes this neoplasm quite sensitive to antiangiogenic drugs. However, it is now evident that the use of VEGF(Rs) inhibitors in everyday clinical practice is not as effective as observed in murine models. The investigation of alternative signaling pathways involved in the angiogenic switch is, therefore, imperative in order to induce tumor regression whereby preventing harmful drawback consequences. Ca(2+) entry across the plasma membrane has long been known to stimulate mature ECs to undergo angiogenesis. Recent work from several groups worldwide has then outlined that members of the Transient Receptor Potential (TRP) super-family of cationic channels and Orai1 provide the pathway for such proangiogenic Ca(2+) signal. In addition, Canonical TRP 1 (TRPC1) and Orai1 channels control proliferation and tubulogenesis in both normal EPCs and EPCs isolated from peripheral blood of tumor patients. As a consequence, TRP channels and Orai1 might serve as novel molecular targets to develop alternative and more effective strategies of angiogenesis inhibition.
An increase in the frequency of circulating endothelial colony forming cells (ECFCs), the only subset of endothelial progenitor cells (EPCs) truly belonging to the endothelial phenotype, occurs in ...patients affected by primary myelofibrosis (PMF). Herein, they might contribute to the enhanced neovascularisation of fibrotic bone marrow and spleen. Store-operated Ca2+ entry (SOCE) activated by the depletion of the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca2+ store drives proliferation in ECFCs isolated from both healthy donors (N-ECFCs) and subjects suffering from renal cellular carcinoma (RCC-ECFCs). SOCE is up-regulated in RCC-ECFCs due to the over-expression of its underlying molecular components, namely Stim1, Orai1, and TRPC1.
We utilized Ca2+ imaging, real-time polymerase chain reaction, western blot analysis and functional assays to evaluate molecular structure and the functional role of SOCE in ECFCs derived from PMF patients (PMF-ECFCs). SOCE, induced by either pharmacological (i.e. cyclopiazonic acid or CPA) or physiological (i.e. ATP) stimulation, was significantly higher in PMF-ECFCs. ATP-induced SOCE was inhibited upon blockade of the phospholipase C/InsP3 signalling pathway with U73111 and 2-APB. The higher amplitude of SOCE was associated to the over-expression of the transcripts encoding for Stim2, Orai2-3, and TRPC1. Conversely, immunoblotting revealed that Stim2 levels remained constant as compared to N-ECFCs, while Stim1, Orai1, Orai3, TRPC1 and TRPC4 proteins were over-expressed in PMF-ECFCs. ATP-induced SOCE was inhibited by BTP-2 and low micromolar La3+ and Gd3+, while CPA-elicited SOCE was insensitive to Gd3+. Finally, BTP-2 and La3+ weakly blocked PMF-ECFC proliferation, while Gd3+ was ineffective.
Two distinct signalling pathways mediate SOCE in PMF-ECFCs; one is activated by passive store depletion and is Gd3+-resistant, while the other one is regulated by the InsP3-sensitive Ca2+ pool and is inhibited by Gd3+. Unlike N- and RCC-ECFCs, the InsP3-dependent SOCE does not drive PMF-ECFC proliferation.
In primary myelofibrosis, extra-domain A fibronectin (EDA-FN), the result of alternative splicing of FN gene, sustains megakaryocyte proliferation and confers a pro-inflammatory phenotype to bone ...marrow cell niches. In this work we assessed the levels of circulating EDA-FN in plasma samples of 122 patients with primary myelofibrosis. Patients with a homozygous JAK2V617F genotype displayed the higher level of plasma EDA-FN. Increased EDA-FN levels were associated with anemia, elevated high-sensitivity C-reactive protein, bone marrow fibrosis and splanchnic vein thrombosis at diagnosis. While no correlation was observed with CD34+ hematopoietic stem cell mobilization, elevated blood level of EDA-FN at diagnosis was a predictor of large splenomegaly (over 10 cm from the left costal margin) outcome. Thus, EDA-FN expression in primary myelofibrosis may represent the first marker of disease progression, and a novel target to treat splenomegaly.
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.
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