Circulating endothelial progenitor cells (EPCs) provide revascularisation for cardiovascular disease and the expansion of these cells opens up the possibility of their use as a cell therapy. Herein ...we show that interleukin-3 (IL3) strongly expands a population of human non-adherent endothelial forming cells (EXnaEFCs) with low immunogenicity as well as pro-angiogenic capabilities in vivo, making their therapeutic utilisation a realistic option. Non-adherent CD133+ EFCs isolated from human umbilical cord blood and cultured under different conditions were maximally expanded by day 12 in the presence of IL3 at which time a 350-fold increase in cell number was obtained. Cell surface marker phenotyping confirmed expression of the hematopoietic progenitor cell markers CD133, CD117 and CD34, vascular cell markers VEGFR2 and CD31, dim expression of CD45 and absence of myeloid markers CD14 and CD11b. Functional experiments revealed that EXnaEFCs exhibited classical properties of endothelial cells (ECs), namely binding of Ulex europaeus lectin, up-take of acetylated-low density lipoprotein and contribution to EC tube formation in vitro. These EXnaEFCs demonstrated a pro-angiogenic phenotype within two independent in vivo rodent models. Firstly, a Matrigel plug assay showed increased vascularisation in mice. Secondly, a rat model of acute myocardial infarction demonstrated reduced heart damage as determined by lower levels of serum creatinine and a modest increase in heart functionality. Taken together, these studies show IL3 as a potent growth factor for human CD133+ cell expansion with clear pro-angiogenic properties (in vitro and in vivo) and thus may provide clinical utility for humans in the future.
•Endothelial progenitor cells (EPCs) are of clinical interest as a cell therapy.•Increasing EPC numbers will provide the cells needed to reach clinical potential.•Interleukin-3 (IL-3) greatly expands human non-adherent endothelial forming cells (EXnaEFCs).•EXnaEFCs exhibit low immunogenicity.•EXnaEFCs are pro-angiogenic and partly restore infarct injury in vivo.
Although integrin engagement initiates signaling events such as focal-adhesion kinase (FAK) and Src kinase activation, the role of phosphoinositide turnover in cell adhesion is less clear. To assess ...PLC-γ1 function in this process, Plcg1-/- fibroblasts (Null) were compared with the same fibroblasts in which PLC-γ1 was re-expressed (Null+). Following plating on fibronectin, Null cells displayed a significantly impaired rate of adhesion compared with Null+ cells. This defect was detected at low concentrations of fibronectin; at high fibronectin concentrations, the Null and Null+ cells displayed equivalent adhesion characteristics. The differences were not due to PLC-γ1-dependent changes in integrin subunit expression, nor was integrin receptor clustering impaired with the absence of PLC-γ1. Experiments with site-specific antibodies and PLC-γ1 mutants showed that fibronectin selectively increased phosphorylation of Tyr783 and that mutagenesis of this residue, but not Tyr771 or Tyr1253, abrogated fibronectin-dependent adhesion. The SH2 domains of PLC-γ1 were also required for maximal adhesion on fibronectin. Adhesion to fibronectin induced PLC-γ1 tyrosine phosphorylation that was inhibited by a Src-kinase inhibitor, but not an epidermal-growth-factor-receptor kinase inhibitor. Moreover, in cells null for Src family members, but not in cells null for FAK family members, integrin-dependent PLC-γ1 tyrosine phosphorylation was greatly reduced. Finally, the data demonstrated that PLC-γ1 co-immunoprecipitated with Src following fibronectin-induced integrin activation, and this association did not depend on FAK expression.
BACKGROUND—Hennekam lymphangiectasia–lymphedema syndrome (Online Mendelian Inheritance in Man 235510) is a rare autosomal recessive disease, which is associated with mutations in the CCBE1 gene. ...Because of the striking phenotypic similarity of embryos lacking either the Ccbe1 gene or the lymphangiogenic growth factor Vegfc gene, we searched for collagen- and calcium-binding epidermal growth factor domains 1 (CCBE1) interactions with the vascular endothelial growth factor-C (VEGF-C) growth factor signaling pathway, which is critical in embryonic and adult lymphangiogenesis.
METHODS AND RESULTS—By analyzing VEGF-C produced by CCBE1-transfected cells, we found that, whereas CCBE1 itself does not process VEGF-C, it promotes proteolytic cleavage of the otherwise poorly active 29/31-kDa form of VEGF-C by the A disintegrin and metalloprotease with thrombospondin motifs-3 protease, resulting in the mature 21/23-kDa form of VEGF-C, which induces increased VEGF-C receptor signaling. Adeno-associated viral vector–mediated transduction of CCBE1 into mouse skeletal muscle enhanced lymphangiogenesis and angiogenesis induced by adeno-associated viral vector–VEGF-C.
CONCLUSIONS—These results identify A disintegrin and metalloprotease with thrombospondin motifs-3 as a VEGF-C–activating protease and reveal a novel type of regulation of a vascular growth factor by a protein that enhances its proteolytic cleavage and activation. The results suggest that CCBE1 is a potential therapeutic tool for the modulation of lymphangiogenesis and angiogenesis in a variety of diseases that involve the lymphatic system, such as lymphedema or lymphatic metastasis.
Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth ...factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2(+/-);Vegfr3(+/-) compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts.
Somatic Mutations of ErbB4 Tvorogov, Denis; Sundvall, Maria; Kurppa, Kari ...
The Journal of biological chemistry,
02/2009, Letnik:
284, Številka:
9
Journal Article
Recenzirano
Odprti dostop
Cancer drugs targeting ErbB receptors, such as epidermal growth factor receptor and ErbB2, are currently in clinical use. However, the role of ErbB4 as a potential cancer drug target has remained ...controversial. Recently, somatic mutations altering the coding region of ErbB4 were described in patients with breast, gastric, colorectal, or non-small cell lung cancer, but the functional significance of these mutations is unknown. Here we demonstrate that 2 of 10 of the cancer-associated mutations of ErbB4 lead to loss of ErbB4 kinase activity due to disruption of functionally important structural features. Interestingly, the kinase-dead ErbB4 mutants were as efficient as wild-type ErbB4 in forming a heterodimeric neuregulin receptor with ErbB2 and promoting phosphorylation of Erk1/2 and Akt in an ErbB2 kinase-dependent manner. However, the mutant ErbB4 receptors failed to phosphorylate STAT5 and suppressed differentiation of MDA-MB-468 mammary carcinoma cells. These findings suggest that the somatic ErbB4 mutations have functional consequences and lead to selective changes in ErbB4 signaling.
Introduction: Mutations within the gene encoding calreticulin (CALR) are the second most common genetic aberration associated with primary myelofibrosis (PMF), observed in 70% of non-JAK2 V617F ...cases. Importantly, patients with CALR mutations do not effectively respond to JAK inhibitor therapy and no mutation specific therapy is currently in use. Virtually all CALR mutations identified in PMF are small insertions or deletions clustered within exon 9 leading to a neo-epitope peptide sequence which is thought to directly or indirectly activate the thrombopoietin receptor (TpoR) by a poorly defined mechanism. Here we engineered a neo-epitope specific monoclonal antobody that has striking biological activity against ruxolitinib persistent cells.
Methods TF-1 TpoR cells expressing TpoR were supplemented with 20 ng/mL of TPO. Rats were immunised with a CALR mutant peptide coupled to KLH. Serum from the immunised rats was screened by enzyme linked immunoassay, to verify a strong titre to the peptide immunogen. Primary PMF CD34+ cells were cultured in StemCell Pro with human SCF, IL-6 and IL-9. NSG mice were used to for engraftment studies after 150 cGy irradiation.
Results: We engineered a panel of rat monoclonal antibodies after immunization with a 30 amino acid peptide corresponding to the C-terminal mutant CALR neoepitope sequence with an extra cysteine residue. Clone 4D7 showed superior activity of detecting mutant but not wild type CALR protein with a binding affinity of 13.5 pM and dissociation constant of 1.53 nM as measured by I 125-Scatchard. Treatment with 4D7 resulted in a significant (5-7-fold) increase in the amount of full-length mutant CALR protein in conditioned media. 4D7 inhibited Tpo-independent cell growth over 6 days in TF-1 cells expressing MPL and mutant CALR at 2, 10 and 20 µg. 4D7 blocked constitutive factor-independent phospho-STAT5 and phospho-ERK after incubation exclusively in mutant CALR cells but not in TF-1 cells expressing TpoR alone and increased the sub-G 0 fraction was observed compared to IgG control (P = 0.001, n = 3 independent experiments) consistent with induction of an apoptotic response. We tested activity in purified primary CD34+ cells obtained from patients with CALR mutant myelofibrosis using two orthogonal assays: - (i) Tpo-independent megakaryocyte differentiation in liquid culture and (ii) Tpo-independent megakaryocyte colony formation on a collagen-based medium. 4 out of 4 patient samples that displayed robust Tpo-independent growth of CD41+CD61+ megakaryocyte progenitors showed inhibition by 4D7 of at least 50%. Similarly, we saw dramatic reduction in the absolute numbers of primary Tpo-independent megakaryocyte colonies cultured on collagen (colony-forming unit-mega) treated with 4D7 in multiple patient samples (decrease of 46%, P = 0.0001, Student's t-test, n = 4 independent patient samples) Importantly, secretion of mutant CALR protein was neither upregulated nor downregulated by ruxolitinib, indicating ruxolitinib is unlikely to alter mutant CALR trafficking in patients. 4D7 had strong inhibitory activity on cells that were resistant to ruxolitinib, in both liquid culture at 96 hours or colony formation. To test whether 4D7 could block mutant CALR-dependent proliferation in vivo, we developed two distinct xenograft models, a bone marrow engraftment model, which measures mutant CALR dependent proliferation in the bone marrow microenvironment, and a chloroma model, which mimics extravascular infiltration of mutant CALR leukaemia, by injection of TPO-independent TF-1 cells in NSG mice. In the bone marrow engraftment model 4D7 treatment (12 mg/kg twice weekly via intraperitoneal injection) lowered peripheral blood engraftment of human CD33 myeloid cells at 3 weeks, bone marrow engraftment and significantly prolonged survival compared to IgG control (P=0.004, HR=0.2). In the chloroma model, 4D7 treatment resulted in significant decrease in tumour growth measured at 3 weeks (P<0.01) and improved overall survival (P=0.02, HR=0.07) compared to IgG control
Conclusion: Together, these results suggest an immunotherapeutic approach may have clinical utility CALR-driven myeloproliferative neoplasms and CALR mutant acute myeloid leukaemia, as well as activity in CALR mutant patients that develop resistance/persistence to ruxolitinib.
Ross: Bristol Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Keros Therapeutics: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.
Calreticulin (CALR) is recurrently mutated in myelofibrosis via a frameshift that removes an endoplasmic reticulum retention signal, creating a neoepitope potentially targetable by immunotherapeutic ...approaches. We developed a specific rat monoclonal IgG2α antibody, 4D7, directed against the common sequence encoded by both insertion and deletion mutations. 4D7 selectively bound to cells co‐expressing mutant CALR and thrombopoietin receptor (TpoR) and blocked JAK‐STAT signalling, TPO‐independent proliferation and megakaryocyte differentiation of mutant CALR myelofibrosis progenitors by disrupting the binding of CALR dimers to TpoR. Importantly, 4D7 inhibited proliferation of patient samples with both insertion and deletion CALR mutations but not JAK2 V617F and prolonged survival in xenografted bone marrow models of mutant CALR‐dependent myeloproliferation. Together, our data demonstrate a novel therapeutic approach to target a problematic disease driven by a recurrent somatic mutation that would normally be considered undruggable.
Synopsis
CALR is recurrently mutated in myelofibrosis, creating a surface‐exposed neoepitope that activates the thrombopoietin receptor. A monoclonal antibody (4D7) with mutation‐specific efficacy in CALR mutant progenitors disrupts the receptor‐CALR complex and inhibits proliferation.
4D7 specifically blocks mutant CALR‐dependent proliferation and TPO‐independent megakaryocyte differentiation.
Neoepitope targeting is a feasible and attractive strategy to overcome ruxolitinib‐persistence.
Neoepitope targeting disrupts CALR oligomerisation with MPL and subsequent signal transduction.
4D7 inhibits proliferation of patient samples with CALR mutations and prolongs survival in xenograft models.
CALR is recurrently mutated in myelofibrosis, creating a surface‐exposed neoepitope that activates the thrombopoietin receptor. A monoclonal antibody (4D7) with mutation‐specific efficacy in CALR mutant progenitors disrupts the receptor‐CALR complex and inhibits proliferation.
•GM-CSF and IL-3 are pleiotropic cytokines with roles in health and disease.•New crystal structure of IL3Rα reveals novel open and closed conformations.•Membrane proximal and transmembrane domain ...movement likely to initiate signaling.
Granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and IL-5 are members of a small family of cytokines that share a beta receptor subunit (βc). These cytokines regulate the growth, differentiation, migration and effector function activities of many hematopoietic cells in bone marrow, blood and sites of inflammation. Excessive or aberrant signaling can result in chronic inflammatory conditions and myeloid leukemias. The crystal structures of the GM-CSF ternary complex, the IL-5 binary complex and the very recent IL-3 receptor alpha subunit build upon decades of structure–function studies, giving new insights into cytokine–receptor specificity and signal transduction. Selective modulation of receptor function is now a real possibility and the structures of the βc receptor family are being used to discover novel and disease-specific therapeutics.
ERBB4 is a member of the epidermal growth factor receptor (EGFR)/ERBB subfamily of receptor tyrosine kinases that regulates cellular processes including proliferation, migration, and survival. ERBB4 ...signaling is involved in embryogenesis and homeostasis of healthy adult tissues, but also in human pathologies such as cancer, neurological disorders, and cardiovascular diseases. Here, an MS-based analysis revealed the Vav guanine nucleotide exchange factor 3 (VAV3), an activator of Rho family GTPases, as a critical ERBB4-interacting protein in breast cancer cells. We confirmed the ERBB4–VAV3 interaction by targeted MS and coimmunoprecipitation experiments and further defined it by demonstrating that kinase activity and Tyr-1022 and Tyr-1162 of ERBB4, as well as the intact phosphotyrosine-interacting SH2 domain of VAV3, are necessary for this interaction. We found that ERBB4 stimulates tyrosine phosphorylation of the VAV3 activation domain, known to be required for guanine nucleotide exchange factor (GEF) activity of VAV proteins. In addition to VAV3, the other members of the VAV family, VAV1 and VAV2, also coprecipitated with ERBB4. Analyses of the effects of overexpression of dominant-negative VAV3 constructs or shRNA-mediated down-regulation of VAV3 expression in breast cancer cells indicated that active VAV3 is involved in ERBB4-stimulated cell migration. These results define the VAV GEFs as effectors of ERBB4 activity in a signaling pathway relevant for cancer cell migration.
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