Therapeutic potential of endothelial colony‐forming cells in ischemic disease: Strategies to improve their regenerative efficacy

Cardiovascular disease (CVD) comprises a range of major clinical cardiac and circulatory diseases, which produce immense health and economic burdens worldwide. Currently, vascular regenerative surgery represents the most employed therapeutic option to treat ischemic disorders, even though not all the patients are amenable to surgical revascularization. Therefore, more efficient therapeutic approaches are urgently required to promote neovascularization. Therapeutic angiogenesis represents an emerging strategy that aims at reconstructing the damaged vascular network by stimulating local angiogenesis and/or promoting de novo blood vessel formation according to a process known as vasculogenesis. In turn, circulating endothelial colony‐forming cells (ECFCs) represent truly endothelial precursors, which display high clonogenic potential and have the documented ability to originate de novo blood vessels in vivo. Therefore, ECFCs are regarded as the most promising cellular candidate to promote therapeutic angiogenesis in patients suffering from CVD. The current briefly summarizes the available information about the origin and characterization of ECFCs and then widely illustrates the preclinical studies that assessed their regenerative efficacy in a variety of ischemic disorders, including acute myocardial infarction, peripheral artery disease, ischemic brain disease, and retinopathy. Then, we describe the most common pharmacological, genetic, and epigenetic strategies employed to enhance the vasoreparative potential of autologous ECFCs by manipulating crucial pro‐angiogenic signaling pathways, e.g., extracellular‐signal regulated kinase/Akt, phosphoinositide 3‐kinase, and Ca2+ signaling. We conclude by discussing the possibility of targeting circulating ECFCs to rescue their dysfunctional phenotype and promote neovascularization in the presence of CVD

Circulating endothelial progenitor cells from patients with renal cell carcinoma display aberrant VEGF regulation, reduced apoptosis and altered ultrastructure

Endothelial colony forming cells (ECFCs) are the only endothelial progenitor cells (EPCs) subtype belonging to the endothelial phenotype and capable of forming neovessels in vivo. We have recently shown that the intracellular Ca2+ machinery plays a key role in ECFC activation and is remodeled in ECFCs isolated from patients suffering from renal cellular carcinoma (RCC-ECFCs). More specifically, ECFCs upregulate the store-operated Ca2+ entry (SOCE) machinery, while they seemingly show a reduction in the Ca2+ concentration within the endoplasmic reticulum ([Ca2+]ER). Metastatic RCC patients are commonly treated with an anti-vascular endothelial growth factor (VEGF) therapy, but they show either intrinsic or adaptive refractoriness, which ultimately leads to their death. Herein, we assessed whether and how the rearrangement of the Ca2+ machinery impacts on the pro-angiogenic Ca2+ response to VEGF, which stimulates normal ECFCs (N-ECFCs) through an oscillatory Ca2+ response. We found that VEGF stimulates the nuclear translocation of p65/RelA, a major component of the Ca2+-dependent transcription fac- tor NF-kB, in N-ECFCs. This process is blocked by the pharmacological abrogation of VEGF-induced Ca2+ oscillations. We further showed that NF-kB controls VEGF-induced protein expression of E-selectin, VCAM-1 and MMP9. Likewise, VEGF-induced expression was also inhibited by the pharmacological suppression of the accompanying Ca2+ spikes. Thus, VEGF induces a Ca2+-dependent, NF-kB-mediated protein expression in N-ECFCs. VEGF did not trigger protein expression in RCC-ECFCs despite the fact that VEGFR-2 was normally expressed and auto-phosphorylated. Our subsequent studies employed the tar- geted recombinant Ca2+-sensitive photoprotein aequorin to confirm that [Ca2+]ER is lower in RCC-ECFCs; surprisingly, electron microscopy analysis revealed that the endoplasmic reticulum cisternae are enlarged rather than shrinked in these cells. These results show for the first time that VEGF fails to stimulate tumor-derived ECFCs: these findings could therefore help to understand the relative failure of anti-VEGF treatment in RCC patients. Reference

Shared and Distinctive Ultrastructural Abnormalities Expressed by Megakaryocytes in Bone Marrow and Spleen From Patients With Myelofibrosis

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

Arachidonic acid-evoked Ca^{2+} signals promote nitric oxide release and proliferation in human endothelial colony forming cells

Arachidonic acid (AA) stimulates endothelial cell (EC) proliferation through an increase in intracellular Ca^{2+} concentration ([Ca^{2+}]_{i}), that, in turn, promotes nitric oxide (NO) release. AA-evoked Ca^{2+} 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 [Ca^{2+}]_{i} and the following activation of the endothelial NO synthase (eNOS). AA induced a dose-dependent [Ca^{2+}]_{i} raise that was mimicked by its non-metabolizable analogue eicosatetraynoic acid. AA-evoked Ca^{2+} signals required both intracellular Ca^{2+} release and external Ca^{2+} inflow. AA-induced Ca^{2+} 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 Ca^{2+} entry was, in turn, mediated by TRPV4, while it did not involve store-operated Ca^{2+} entry. Moreover, AA caused an increase in NO levels which was blocked by preventing the concomitant increase in [Ca^{2+}]_{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 Ca^{2+} - and NO-dependent manner. Therefore, AA-evoked Ca^{2+} signals emerge as an additional target to prevent cancer vascularisation, which may be sustained by ECFC recruitment

Defective iron supply for erythropoiesis and adequate endogenous erythropoietin production in the anemia associated with systemic-onset juvenile chronic arthritis.

peer reviewedSystemic-onset juvenile chronic arthritis (SoJCA) is associated with high levels of circulating interleukin-6 (IL-6) and is frequently complicated by severe microcytic anemia whose pathogenesis is unclear. Therefore, we studied 20 consecutive SoJCA patients with hemoglobin (Hb) levels <12 g/dL, evaluating erythroid progenitor proliferation, endogenous erythropoietin production, body iron status, and iron supply for erythropoiesis. Hb concentrations ranged from 6.5 to 11.9 g/dL. Hb level was directly related to mean corpuscular volume (r = .82, P < .001) and inversely related to circulating transferrin receptor (r = -.81, P < .001) suggesting that the severity of anemia was directly proportional to the degree of iron-deficient erythropoiesis. Serum ferritin ranged from 18 to 1,660 microgram/L and was unrelated to Hb level. Bone marrow iron stores wore markedly reduced in the three children investigated, and they also showed increased serum transferrin receptor and normal-to-high serum ferritin. All 20 patients had elevated IL-6 levels and normal in vitro growth of erythroid progenitors. Endogenous erythropoietin (epo) production was appropriate for the degree of anemia as judged by both the observed to predicted log (serum epo) ratio 10.95 +/- 0.12) and a comparison of the serum epo-Hb regression found in these subjects with that of thalassemia patients. Multiple regression analysis showed that serum transferrin receptor was the parameter most closely related to hemoglobin concentration: variation in circulating transferrin receptor explained 61% of the variation in Hb level (P < .001). In 10 severely anemic patients, amelioration of anemia following intravenous iron administration resulted in normalization of serum transferrin receptor. Defective iron supply to the erythron rather than blunted epo production is the major cause of the microcytic anemia associated with SoJCA. A true body-iron deficiency caused by decreased iron absorption likely complicates long-lasting inflammation in the most anemic children, and this can be recognized by high serum transferrin receptor levels. Although oral iron is of no benefit, intravenous iron saccharate is a safe and effective means for improving iron availability for erythropoiesis and correcting this anemia. Thus, while chronically high endogenous IL-6 levels do not appear to blunt epo production, they are probably responsible for the observed abnormalities in iron metabolism. Anemia of chronic disease encompasses a variety of anemic conditions whose peculiar features may specifically correlate with the type of cytokine(s) predominantly released

Conjugated polymers mediate intracellular Ca2+ signals in circulating endothelial colony forming cells through the reactive oxygen species-dependent activation of Transient Receptor Potential Vanilloid 1 (TRPV1)

Endothelial colony forming cells (ECFCs) represent the most suitable cellular substrate to induce revascularization of ischemic tissues. Recently, optical excitation of the light-sensitive conjugated polymer, regioregular Poly (3-hexyl-thiophene), rr-P3HT, was found to stimulate ECFC proliferation and tube formation by activating the non-selective cation channel, Transient Receptor Potential Vanilloid 1 (TRPV1). Herein, we adopted a multidisciplinary approach, ranging from intracellular Ca2+ imaging to pharmacological manipulation and genetic suppression of TRPV1 expression, to investigate the effects of photoexcitation on intracellular Ca2+ concentration ([Ca2+](i)) in circulating ECFCs plated on rr-P3HT thin films. Polymer-mediated optical excitation induced a long-lasting increase in [Ca2+](i) that could display an oscillatory pattern at shorter light stimuli. Pharmacological and genetic manipulation revealed that the Ca2+ response to light was triggered by extracellular Ca2+ entry through TRPV1, whose activation required the production of reactive oxygen species at the interface between rr-P3HT and the cell membrane. Light-induced TRPV1-mediated Ca2+ entry was able to evoke intracellular Ca2+ release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate receptors, followed by store-operated Ca2+ entry on the plasma membrane. These data show that TRPV1 may serve as a decoder at the interface between rr-P3HT thin films and ECFCs to translate optical excitation in pro-angiogenic Ca2+ signals

Consensus conference on the management of tumor lysis syndrome.

Tumor lysis syndrome is a potentially life threatening complication of massive cellular lysis in cancers. Identification of high-risk patients and early recognition of the syndrome is crucial in the institution of appropriate treatments. Drugs that act on the metabolic pathway of uric acid to allantoin, like allopurinol or rasburicase, are effective for prophylaxis and treatment of tumor lysis syndrome. Sound recommendations should regulate diagnosis and drug application in the clinical setting. The current article reports the recommendations on the management of tumor lysis syndrome that were issued during a Consensus Conference project, and which were endorsed by the Italian Society of Hematology (SIE), the Italian Association of Pediatric Oncologists (AIEOP) and the Italian Society of Medical Oncology (AIOM). Current concepts on the pathophysiology, clinical features, and therapy of tumor lysis syndrome were evaluated by a Panel of 8 experts. A consensus was then developed for statements regarding key questions on tumor lysis syndrome management selected according to the criterion of relevance by group discussion. Hydration and rasburicase should be administered to adult cancer patients who are candidates for tumor-specific therapy and who carry a high risk of tumor lysis syndrome. Cancer patients with a low-risk of tumor lysis syndrome should instead receive hydration along with oral allopurinol. Hydration and rasburicase should also be administered to patients with clinical tumor lysis syndrome and to adults and high-risk children who develop laboratory tumor lysis syndrome. In conclusion, the Panel recommended rasburicase for tumor lysis syndrome prophylaxis in selected patients based on the drug efficacy profile. Methodologically rigorous studies are needed to clarify its cost-effectiveness profile

Impact of the rs1024611 polymorphism of ccl2 on the pathophysiology and outcome of primary myelofibrosis

Single nucleotide polymorphisms (SNPs) can modify the individual pro-inflammatory background and may therefore have relevant implications in the MPN setting, typified by aberrant cytokine production. In a cohort of 773 primary myelofibrosis (PMF), we determined the contribution of the rs1024611 SNP of CCL2—one of the most potent immunomodulatory chemokines—to the clinical and biological characteristics of the disease, demonstrating that male subjects carrying the homozygous genotype G/G had an increased risk of PMF and that, among PMF patients, the G/G genotype is an independent prognostic factor for reduced overall survival. Functional characterization of the SNP and the CCL2-CCR2 axis in PMF showed that i) homozygous PMF cells are the highest chemokine producers as compared to the other genotypes; ii) PMF CD34+ cells are a selective target of CCL2, since they uniquely express CCR2 (CCL2 receptor); iii) activation of the CCL2-CCR2 axis boosts pro-survival signals induced by driver mutations via Akt phosphorylation; iv) ruxolitinib effectively counteracts CCL2 production and down-regulates CCR2 expression in PMF cells. In conclusion, the identification of the role of the CCL2/CCR2 chemokine system in PMF adds a novel element to the pathophysiological picture of the disease, with clinical and therapeutic implications