FLT3 Receptor Research Articles

The Soluble VEGF Receptor sFlt-1 Contributes to Impaired Neovascularization in Aged Mice.

The mechanism by which angiogenesis declines with aging is not fully understood. Soluble vascular endothelial growth factor receptor 1 (VEGFR1) form (sFlt1) contributes to endothelial dysfunction in pathological conditions. However, the roles of sFlt1 in ischemia-induced neovascularizationof aged animals have not been investigated. To study aging-related sFlt1 change and its impact on ischemia-induced neovascularization, a hindlimb ischemia model was applied to young and aged mice. Blood flow imaging assay revealed that the blood flow recovery remained impaired throughout the follow-up period. At day 14, immunostaining showed lesser capillary formation in the aged mice. An ELISA showed that the aged mice had increased plasma sFlt-1 levels at indicated time points after surgery. On operative day 4, the aged ischemic muscles had decreased levels of p-VEGFR2 and p-Akt and increased levels of sFlt-1, Wnt5a, and SC35 genes or/and protein as well as increased numbers of inflammatory cells (macrophages and leucocytes) and matrix metalloproteinase-9 activity. Immnunofluorescence showed that Flt-1 was co-localized with CD11b+ macrophages of aged ischemic muscles. Hypoxia stimulated sFlt1 expression in CD11b+ cells of aged bone-marrow (BM), and this effect was diminished by siWnt5a. The cultured medium of aged mice BM-derived CD11b+ cells suppressed human endothelial cell (EC) and endothelial progenitor cell (EPC) angiogenic actions induced by VEGF, and these decreases were improved by treatment with siWnt5a-conditioned medium. Thus, aging appears to decline neovascularization in response to ischemic stress via the VEGFR2/Akt signaling inactivation in ECs and ECPs that is mediated by Wnt5a/SC35 axis activated macrophages-derived sFlt1 production in advanced age.

VEGF, Flt-1, and microvessel density in primary tumors as predictive factors of colorectal cancer prognosis.

Angiogenesis in the primary tumor is known to be necessary for tumor progression in adenocarcinomas of the colon. However, whether angiogenesis in the primary tumors of patients with colorectal cancer affects their prognosis has yet to be fully elucidated. The aim of the present study was to assess the association between selected pathoclinical parameters and overall survival of resectable colorectal cancer patients with the expression of angiogenesis-promoting factors, including vascular endothelial growth factor (VEGF) and Fms-like tyrosine kinase receptor (Flt-1), and microvessel density (MVD) in the primary tumor. VEGF and Flt-1 expression were assessed, as well as MVD (with anti-CD34) by immunohistochemistry in 139 archived primary colorectal cancer tissue samples. These results were compared with the overall survival of the patients and potential prognostic pathoclinical parameters. A higher MVD in the tumors expressing Flt-1 (P=0.04) was identified. However, there was no correlation between the pathoclinical parameters of colon cancer and Flt-1 expression, VEGF expression, or MVD in the tumor. Furthermore, the intensity of VEGF expression, Flt-1 expression and tumor MVD did not correlate with the overall survival of the patients. Therefore, although increased expression of VEGF and Flt-1 was correlated with an increased expression of MVD in the primary tumors of resectable colorectal cancer patients, these factors were not correlated with prognostic pathoclinical factors and overall survival.

Cystic endometrial hyperplasia-pyometra syndrome in bitches: identification of hemodynamic, inflammatory, and cell proliferation changes.

Cystic endometrial hyperplasia (CEH)-pyometra syndrome is one of the most common diseases of noncastrated female dogs. However, determination of etiological mechanisms and differential diagnosis of CEH-pyometra syndrome are undefined. The aim of this study is to compare immunohistochemically the expression of cyclooxygenase-2 (COX-2) inflammatory mediator, Ki-67 antigen proliferation marker, vascular endothelial growth factor (VEGF-A) angiogenesis mediator and its FLT-1 and KDR receptors, and correlate with Doppler velocimetry of uterine artery and endometrial vascularization in bitches with CEH-pyometra syndrome. Bitches were allocated into CEH-mucometra Group (n = 13), Pyometra Group (n = 11), and Control Group (n = 8). Pyometra Group presented cytoplasmatic staining intensity for COX-2, VEGF-A, and FLT-1 and KDR receptors in luminal epithelium cells significantly higher compared to CEH-mucometra and Control groups. For the glandular epithelium, Pyometra Group had higher immunostaining score for VEGF-A and its receptors (FLT-1 and KDR). Hemodynamic indexes showed negative correlation with VEGF-A and its receptors as well as with COX-2. On the other hand, uterine vascularization score showed positive correlation in relation to immunostaining of COX-2, VEGF-A, and receptors in the endometrium luminal epithelium. In conclusion, uterus of bitches with CEH-pyometra syndrome show inflammatory process characterized by COX-2 expression, resulting in greater expression of proliferative Ki-67 marker as tissue response against the infectious agent. Furthermore, the increased VEGF-A expression and its receptors in CEH-pyometra reflect the increased blood flow and lower vascular resistance. Therefore, canine pyometra is characterized by an inflammatory, proliferative, and vascular disorder.

Src-like Adaptor Protein Promotes the CD103+ dendritic Cell Homeostasis in the Lung

Dendritic cells (DCs) along with mast cells function as sentinels for the innate immune system and perform as antigen presenting cells (APCs) to mount an adaptive immune response against invading pathogen. FLT3 receptor tyrosine kinase signaling has been shown to regulate the homeostatic mechanisms of subsets of DCs particularly, CD103+DCs compared to CD11b+DCs. CD103+DCs are regarded as APCs with superior capabilities to mount an effective immune response, thus understanding their homeostasis mechanism(s)/function is of paramount importance to devise effective therapeutics including DC vaccines. The Src-like adapter protein (SLAP) has been shown to dampen the signaling downstream of receptor tyrosine kinases including FLT3, cKit, and immune cell receptors including T cell receptor, B cell receptor, and Granulocyte-monocyte colony stimulating factor receptor via by recruiting c-Cbl, an ubiquitin ligase.Here, we report that SLAP deficient mice (KO) have reduced numbers of CD103+DC in lung while equal numbers in liver and kidney compared to control mice. To further confirm reduced CD103+DC in the lung, efferocytosis assays that are dependent upon CD+103 DC in lung epithelium to cleanse the apoptotic cells were performed. Flow cytometric quantification of CD103+DCs that uptake fluorescently labeled apoptotic cells administered via intranasal route and migrate to mediastinal lymph nodes confirmed reduced number of CD103+DCs in SLAP KO mice. Further analysis of DC progenitor populations showed reduced pre-DC progenitor in the lung in SLAP KO mice while bone marrow compartment showed equal progenitor populations including pre-DC and common dendritic progenitors suggesting the role of SLAP in localized FLT3 signaling in the lung. Consistently, DCs in lymphoid compartment including spleen, thymus, inguinal and popliteal lymph node did not show any defects. Upon further dissecting the cellular mechanism, SLAP KO DCs showed increased apoptosis while having similar proliferation potential in vivo at steady state.Bone marrow progenitors from SLAP KO mice failed to generate mature DCs in the presence of FLT3 ligand in vitrodue to enhanced apoptosis at early time points. Also, submaximal inhibition of FLT3 with an inhibitor, quizartinib partially rescues the apoptotic phenotype of SLAP KO bone marrow progenitors suggesting a cell-intrinsic role of SLAP in the survival of DCs. Biochemical analysis revealed that SLAP is directly recruited to the juxta-membrane residues of the FLT3 receptor in an inducible manner suggesting a direct role of SLAP in the regulation of FLT3 signaling. Phosphoflow analysis of DCs generated in the combined presence of GMCSF and FLT3 ligands showed that SLAP promotes the signaling to SHP2 while perturbs signaling to the mTOR pathway. Together these results suggest that SLAP is a critical regulator of CD103+DCs homeostasis in selective peripheral organs including the lung. DisclosuresNo relevant conflicts of interest to declare.

Circulating Adiponectin and Markers of Endothelial and Cardiovascular Dysfunction Correlate with Disease Burden in Newly Diagnosed Patients with Multiple Myeloma; Increase of Adiponectin after Bortezomib- and IMiD-Based Regimens

Adiponectin is a multimeric protein of the white adipose tissue which has anti-inflammatory, anti-atherogenic, and possibly anti-neoplastic properties. Low levels of adiponectin are associated with endothelial dysfunction in several disorders, while low levels of circulating adiponectin have been recently associated with increased risk for multiple myeloma (MM) in overweight individuals. Furthermore, decreased host-derived adiponectin promotes myeloma tumor growth and osteolysis in myeloma animal models. There is no information in the literature for adiponectin levels in MM patients. The aim of this study was to evaluate circulating adiponectin in MM patients who responded to first line anti-myeloma therapy and explore possible correlations with disease features, including bone disease, endothelial dysfunction which is present in several plasma cell dyscrasias (Waldenstrom's macroglobulinemia and AL amyloidosis), angiogenesis and inflammation.We studied 76 newly-diagnosed MM patients (46M/30F, median age 70.5 years) before the administration of any kind of therapy and after best response to front-line therapy. Forty-five patients received bortezomib-based regimens, while 31 patients were treated with IMiD based regimens (16 with lenalidomide-based therapies). Evidence of bone involvement was documented using plain radiographs. Adiponectin was measured in the serum of all patients and of 24 healthy individuals of similar age, gender and body index along with a series of biochemical markers of: i) endothelial (vWF:antigen and GDF-15) and cardiovascular dysfunction (hs-troponin and NTproBNP); ii) angiogenesis (placental-growth factor, PlGF and its receptor sFlt-1); iii) renal impairment (cystatin-C and NGAL); iv) bone disease (CTX and P1NP); and v) inflammation (hs-CRP and IL-6).Adiponectin levels were elevated in MM patients at diagnosis compared to healthy individuals (mean±SD: 16.2±2.6 mg/L vs. 11.8±0.8 mg/L; p=0.03). The circulating vWF concentrations were markedly elevated in patients compared to controls (mean±SD: 215±16.3 IU/dl vs. 85.3±5.1; p<0.001). Similarly, all markers of cardiovascular dysfunction were elevated in MM patients compared to controls (GDF-15: 3893±970 pg/ml vs 665±50.9 pg/ml, p<0.001; hs-troponin 22.3±3.3 ng/l vs. 6.9±0.7 ng/l, p<0.001 and NTproBNP 623±153 pg/ml vs 44.2±7.2 pg/ml, p<0.001). Both cystatin-C and NGAL, sFlt-1 as well as hs-CRP, IL-6 and CTX were also elevated in MM patients (p<0.01 for all comparisons).Adiponectin negatively correlated with M-protein (r=-0.408, p=0.01), serum calcium (r=-0.391, p=0.015) and GDF-15 (r=-0.40, p=0.01) and positively with NGAL (p=0.395, p=0.014) and CTX (p=0.385, p=0.018). VWF:antigen strongly correlated with beta2-microglobulin (r=0.524, p=0.001) and all factors of renal impairment: urea (r=0.416, p=0.009), creatinine (r=0.431, p=0.007), cystatin-C (r=0.400, p=0.012), NGAL (r=0.400, p=0.013) and eGFR (based on CKD-EPI; r=-0.416, p=0.009). Both adiponectin and vWF correlated with bone marrow plasma cell infiltration (r=-0.376, p=0.031 and r=0.354, p=0.043, respectively). Notably, all markers of endothelial and cardiovascular dysfunction increased with the increase of ISS stage: p-ANOVA for hs-troponin, NTproBNP and GDF-15 <0.001 and for vWF 0.03.The studied population included 16 (21%) patients who achieved a CR, 19 (25%) with vgPR and 41 with PR. Response to anti-myeloma therapy resulted in a dramatic increase of adiponectin (from 16.2±2.6 mg/l to 24.2±4.2 mg/l, p<0.01). Interestingly response to IMiD-based therapies was followed by a higher increase in circulating adiponectin (from 18±1.7 mg/L to 27±2.7 mg/L; p<0.001). vWF was reduced in responders and this reduction was higher in patients who received bortezomib-based regimens (from 240±86 IU/dl to 165±78 IU/dl; p=0.018). There were no alterations in responding patients regarding GDF-15, hs-troponin and NTproBNP after treatment.We conclude that adiponectin and markers of endothelial and cardiovascular dysfunction are elevated in newly-diagnosed patients with MM. Adiponectin and vWF correlates with bone marrow plasma cell infiltration, while all markers of endothelial and cardiovascular dysfunction correlated with ISS stage. Response to therapy increased circulating adiponectin. These results support a role of adiponectin in disease biology as suggested by experiments in murine myeloma models. DisclosuresTerpos:Genesis: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Takeda: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Celgene: Honoraria; Novartis: Honoraria. Kastritis:Genesis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Dimopoulos:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Genesis: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Preclinical Evidence That Trametinib Enhances the Response to Tyrosine Kinase Inhibitors in Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is the most common type of acute leukemia in adults and the second in children. The overall survival is less than 35% and 60% for adults and children respectively. Activating mutations of FLT3 are now recognized as the most common molecular abnormality in this disease, and the poor prognosis of patients harboring these mutations renders FLT3 an obvious target of therapy. Although different tyrosine kinase inhibitors (TKI) have been used for this purpose, the ability of these drugs to extend progression-free and overall survival in this patient population is limited by drug resistance. This strategy could be improved by rationally combining TKIs with other agents. In this work, we have explored by phosphoproteomics the alternative pathways activated after TKI treatment in vitro and ex vivo.The phosphoproteome profile of the bone marrow from a FLT3-AML patient before and after TKI treatment, studied by LC-MSMS after IMAC enrichment, suggested the activation of Ras-Raf-MEK-ERK1/2 pathway as a possible mechanism for TKI resistance, which could be avoided by dual inhibition using the MEK inhibitor trametinib. Therefore, we characterized the effect of trametinib in combination with the TIK pazopanib and sorafenib by the in vitro cell viability assay using WST8in the FLT3-ITD AML cell line MOLM13. As it is presented in figure 1a, trametinib showed an IC50 value in the low-nanomolar range (5.4 nM) and this MEKI produced a strong synergy (0.5<Ci) with the two TKI tested (figure1b). Moreover, when we analized the activation of the three main pathways downstream the FLT3 receptor by western blot (figure 1c), we observed that the combination of trametinib with both pazopanib and sorafenib showed a complete inhibition of phospho-ERK1/2 compared to the DMSO control (P≤0.0001). Interestingly, we also observed some differences between the two combinations: while trametinib in combination with sorafenib inhibited STAT5 phosphorilation (P≤0.05), the MEKI combination with pazopanib produced a significant decrease on phospho-AKT levels (P≤0.01). In conclusion, we provide preclinical evidence that combining a TKI, such as sorafenib or pazopanib, with a MEKI, such as trametinib, is a rational and efficacious treatment regimen for AML. As trametinib has previously shown good results when combined with pazopanib in clinical trials for other kind of tumors, we expect similar results in AML. On the other hand, trametinib+sorafenib could offer an optimal combination in those patients with elevated levels of phospho-STAT5, as it has been described for patients which present FLT3-ITD mutations.M.L. holds a postdoctoral Fellowship of the Spanish Ministry of Economy and Competitiveness (FPDI-2013-16409) y ML.M. holds a Fellowship of the Spanish Ministry of Education, Culture and Sport (REF-91442). [Display omitted] DisclosuresMartínez-López:Novartis: Honoraria, Speakers Bureau.

The Ex-Vivo Expansion of Megakaryocytic Progenitors from Hematopoietic Stem Cells for Thrombocytopenia in NOD/SCID Mice

Objective: Thrombocytopenia is a common clinical problem in patients with cancer or bone marrow transplantation. Currently it is mainly managed by platelet transfusion. Repeated platelet transfusions are associated with the risks of transfusion reactions/alloimmunisation and may lead to platelet refractoriness. Infusion of ex vivo expanded megakaryocytic (MK) progenitor cells is other strategy for the treatment of thrombocytopenia. This study aimed to establish efficient conditions for the expansion of the MK progenitors from enriched CD34(+) cells of umbilical cord blood.Methods: This study investigated the effect of flt-3 ligang (FL), stem cell factor (SCF) and platelet-derived growth factor (PDGF) in combination with other megakaryocyte-promoting cytokines such as thrombopoietin (TPO) on the differentiation and proliferation of megakaryocytic progenitors. As an early acting growth factor, FL may promote the ex vivo expansion of hematopoietic stem and progenitor cells. We compared the effects of FL and SCF in combination with other megakaryocyte-promoting cytokines in megakaryocytic progenitors.Results: In liquid cultures of enriched CD34+ cells from human umbilical cord blood for 14 days, FL plus TPO, interleukin-3 (IL-3), and IL-6 promoted the expansion of nucleated cells, CD34+ cells, CD34+ CD38- cells, and megakaryocyte colony-forming units (CFU-MK) by 300 +/- 115-, 23.8 +/- 11.3-, 33.9 +/- 28.6-, and 584 +/- 220-fold, respectively. Replacing FL with SCF significantly decreased the yield of all cell types. While one human acute lymphoblastic leukemia sample expressed high levels of flt-3 receptor, the four megakaryocytic cell lines (Meg-01, CHRF-288-11, M-07e, and Dami) did not show any positive expression. Our data suggest that the effect of FL in augmenting the expansion of MK progenitors might be due to the early action of FL at the pluripotent stem cell stage. Our results also demonstrated that TPO alone produced a high proportion of CD61(+)CD41(+) cells but a low total cell count and high cell death, resulting in an inferior expansion. The addition of in IL-1 beta, FL and to a lesser extent IL-3 improved the expansion outcome. The treatment groups with three to five cytokines produced efficient expansions of CFU-MK up to 400-fold with the highest yield observed in the presence of TPO, IL-1 beta, IL-3, IL-6 and FL. CD34(+) cells were expanded by five to 22-fold. PDGF improved the expansion of all cell types with CD61(+)CD41(+) cells, CFU-MK and CD34(+) cells increased by 101%, 134% and 70%, respectively. More significantly, PDGF enhanced the engraftment of human CD45+ cells and their myeloid subsets (CD33+, CD14+ cells) in NOD/SCID mice.Conclusions: This study showed that the present cytokine combination and expansion conditions provide an effective and potentially useful system for the clinical expansion of cord blood for bone marrow transplantation (BMT). PDGF might be a suitable growth factor to improve the ex vivo expansion of MK progenitors for clinical applications. DisclosuresYang:National Natural Science Foundation of China: Other: National Natural Science Foundation of China(81270580).

Interim Results of a Phase 1b/2 Study of Entospletinib (GS-9973) Monotherapy and in Combination with Chemotherapy in Patients with Acute Myeloid Leukemia

Background:Spleen tyrosine kinase (SYK) is a nonreceptor cytoplasmic tyrosine kinase primarily expressed in cells of hematopoietic lineage. Constitutive activation of SYK in acute myeloid leukemia (AML) has been reported and targeted inhibition of SYK induced differentiation in vitro and demonstrated anti-leukemia activity in AML mouse models. SYK has also been shown to directly phosphorylate the FLT3 receptor, modulating its activation and possibly promoting its role in leukemogenesis. Entospletinib is an orally bioavailable, selective inhibitor of SYK shown to be clinically active in B-cell malignancies. Here we evaluate the combination of entospletinib in patients with untreated AML using a 14-day window phase to assess single-agent activity, then adding standard intensive chemotherapy.Methods: In this phase 1b/2 study (NCT02343939), patients age 18 to 70 years with previously untreated AML, preserved organ function, and ECOG ≤ 2 were eligible to receive dose escalated entospletinib for 14 days as monotherapy (days -14 to 0) followed by combination with daunorubicin 60 mg/m2/d, cycle 1 day 1 to 3, and cytarabine 100 mg/m2/d, cycle 1 day 1 to 7. All patients received entospletinib monotherapy for up to 14 days prior to starting induction. Chemotherapy could be initiated after 5 days of monotherapy (and entospletinib continued for 4+ weeks) in patients with leukemia-related complications necessitating chemotherapy. Patients enrolled to dose level (DL) 0 and DL 1 received entospletinib 200 mg po BID and 400 mg po BID, respectively. Patients with residual disease two weeks after chemotherapy received a second induction cycle identical to the first. Entospletinib was continued without interruption until remission was assessed at count recovery.Results:Twelve patients enrolled with a median age of 54 (range, 18-69) years. Patients were in the following European LeukemiaNet genetic risk groups: favorable (n=1), intermediate I (n=3), intermediate II (n=2), and adverse (n=4), respectively. Three patients were not evaluable for dose limiting toxicity (DLT) assessment and were replaced (due to detection of CNS disease requiring non-study therapy (n=1), and withdrawal of consent unrelated to drug toxicity (n=2)). Single-agent entospletinib during the window period was well tolerated; toxicities after combination with intensive chemotherapy were common and typical. Among 3 patients treated at 200mg BID, no DLT was observed. Of 3 patients treated at 400mg BID, a patient with documented fungal pneumonia developed grade 3 pneumonitis that was possibly related to entospletinib. Although this did not meet DLT criteria, DL 1 was expanded with 3 additional patients, none of whom experienced DLT. Overall, the most common non hematologic adverse events (inclusive of intensive chemotherapy periods) were febrile neutropenia, nausea, and diarrhea. Based on this clinical experience and compiled pharmacokinetic data demonstrating lack of benefit to further dose escalation, 400 mg BID was selected as the recommended phase 2 dose. Responses were seen at both levels. Among the 3 patients treated at 200 mg BID, two required a second induction but all achieved a complete remission (CR) (3/3; 100%). Of the 6 patients treated at 400mg BID, none required a second induction and the CR rate was also 100%. Remarkably, an 18 year old male with 11q23-rearranged AML achieved morphologic and cytogenetic CR after only the 14 day entospletinib monotherapy window (prior to chemotherapy). Another patient with 11q23-rearranged AML had significant platelet response during the window period (this patient refused disease evaluation by marrow aspiration prior to chemotherapy).Conclusions: Entospletinib appears to have significant clinical activity in AML, and its combination at doses up to 400mg BID with intensive chemotherapy is well tolerated. An extended phase 2 program is now underway. Patients with 11q23-rearranged AML may be uniquely sensitive to SYK inhibition by entospletinib. Detailed molecular analysis of these patients is ongoing and will be presented. DisclosuresWalker:Gilead Sciences: Research Funding. Bhatnagar:Karyopharm: Research Funding. Marcondes:Gilead Sciences: Employment, Equity Ownership. DiPaolo:Gilead Sciences: Employment, Equity Ownership. Abella-Dominicis:Gilead Sciences: Employment, Equity Ownership.

Radial glia regulate vascular patterning around the developing spinal cord.

Vascular networks surrounding individual organs are important for their development, maintenance, and function; however, how these networks are assembled remains poorly understood. Here we show that CNS progenitors, referred to as radial glia, modulate vascular patterning around the spinal cord by acting as negative regulators. We found that radial glia ablation in zebrafish embryos leads to excessive sprouting of the trunk vessels around the spinal cord, and exclusively those of venous identity. Mechanistically, we determined that radial glia control this process via the Vegf decoy receptor sFlt1: sflt1 mutants exhibit the venous over-sprouting observed in radial glia-ablated larvae, and sFlt1 overexpression rescues it. Genetic mosaic analyses show that sFlt1 function in trunk endothelial cells can limit their over-sprouting. Together, our findings identify CNS-resident progenitors as critical angiogenic regulators that determine the precise patterning of the vasculature around the spinal cord, providing novel insights into vascular network formation around developing organs.

MEK/ERK signaling is involved in the role of VEGF and IGF1 in cardiomyocyte differentiation of mouse adipose tissue-derived stromal cells

BackgroundWe found that mouse brown adipose tissue-derived stromal cells (BATDCs), but not white adipose tissue-derived stromal cells (WATDCs), could spontaneously differentiate into cardiomyocyte-like cells in a simple culture medium. This study would find out some critical trophic factors that were responsible for such difference in differentiation, and further determine the involved signaling pathway. Methods and resultsThe cardiomyocyte differentiation capacity of cells was identified by morphological observations, immunofluorescence staining, and evaluation of expression of cardiomyocyte specific markers. The amount of vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF1) secreted by cells was determined by ELISA analysis. Results indicated that BATDCs secreted higher levels of VEGF and IGF1 than WATDCs. Supplementation of BATDCs with antibodies against VEGF receptor Flt-1 or IGF1 receptor Igf-1rα significantly suppressed the cardiac differentiation capacity of the cells. Additionally, anti-Flt-1 and anti-Igf-1rα antibodies decreased phosphorylation of ERK1/2 in BATDCs. Inhibition of MEK/ERK activity by the inhibitor PD0325901 or by RNA interference blunted the cardiac differentiation of BATDCs. Loading recombinant VEGF and IGF1, or transfecting their expression vectors into WATDCs, promoted cardiac differentiation of the cells. Preincubation with PD0325901, before VEGF and IGF1 supplementation or vector transfections, blocked the stimulation of cardiac differentiation in WATDCs. ConclusionsThese findings indicated that VEGF and IGF1 were critical factors for the spontaneous cardiac differentiation of BATDCs, and MEK/ERK signaling was involved in the role of VEGF and IGF1. VEGF and IGF1 could be used to promote the development of cardiomyocyte phenotype in WATDCs.

Angiogenic Imbalance and Residual Myocardial Injury in Recovered Peripartum Cardiomyopathy Patients.

Recent studies suggest that angiogenic imbalance during pregnancy may lead to acute peripartum cardiomyopathy (PPCM). We propose that angiogenic imbalance and residual cardiac dysfunction may exist even after recovery from PPCM. Twenty-nine women at least 12 months after presentation with PPCM, who exhibited recovery of left ventricular (LV) ejection fraction (≥50%), were included in the study (mean age 35±6 years, LV ejection fraction 61.0±3.9%). The number of circulating endothelial progenitor cells (EPCs) and plasma levels of proangiogenic vascular endothelial growth factor and of soluble vascular endothelial growth factor receptor Flt1 (sFlt1) were measured. All patients underwent comprehensive cardiac function assessment, including tissue Doppler imaging and 2-dimensional (2D) strain echocardiography. All measurements were compared with healthy controls. Patients with a history of PPCM have significantly higher sFlt1 concentrations (median [25th-75th percentile]; 149.57, [63.14-177.89] versus 20.29, [15.00-53.89] pg/mL, P<0.001) and significantly decreased vascular endothelial growth factor/sFlt1 ratio (P=0.012) compared with controls, with a trend toward lower concentration of circulating CD34+/KDR+ levels. In addition, patients with PPCM had lower early velocities E' septal (9.9±2.1 versus 11.0±1.5 cm/s, P=0.02), with a significantly lower systolic velocity S' septal (7.6±1.2 versus 8.5±1.2 cm/s, P=0.003) by tissue Doppler imaging. Significantly lower LV global longitudinal (-19.1±3.3 versus -22.7±2.2%, P<0.001) and apical circumferential 2D strain (-16.6±4.9 versus -21.2±7.9, P=0.02) were present in patients with PPCM compared with controls. Higher concentration of sFlt1 with concomitant decreased circulating endothelial progenitor cell levels along with inappropriate attenuated vascular endothelial growth factor levels may imply an angiogenic imbalance that exists even after recovery and may thus predispose to PPCM. In addition, tissue Doppler imaging and 2D strain were able to identify residual myocardial injury in post-PPCM women with apparent recovery of LV systolic function. Both angiogenic imbalance and residual myocardial injury may play an important role in the recurrence of LV dysfunction during subsequent pregnancies.

Differentiation of nerve-derived adult pluripotent stem cells into osteoblastic and endothelial cells

Background ContextStem cell-involved tissue engineering has gained dramatic attention as a therapeutic strategy for tissue regeneration including bone repair. However, the currently available possibilities to use embryonic stem cells and induced pluripotent stem cells (iPCs) face potential ethical issues, as well as risks of malignant transformation and immune rejection. Recently identified peripheral nerve-derived adult pluripotent stem cells (NEDAPS) that quickly proliferate after exposure to bone morphogenetic protein-2 (BMP-2) or nerve trauma and exhibit many embryonic stem cell characteristics may provide an attractive source cells for a variety of regenerative therapies. PurposeThe study aimed to examine the differentiation potential of the NEDAPS cells into osteoblastic cells and endothelial cells. Study Design/SettingAn in vitro investigation was undertaken to induce mouse NEDAPS cells into the phenotypes of osteoblastic and endothelial cells. MethodsNEDAPS cells were isolated from low-dose BMP-2-exposed mouse sciatic nerves by collagenase and trypsin extraction. The cells were cultured in a stem cell maintenance medium, and the expression of KLF4, Sox2, c-Myc, and Oct4 before differentiation was confirmed. The cells were then subcultured in a complete osteogenic cell induction medium or endothelial cell growth medium, respectively, at 37°C and 5%CO2 atmosphere. Histologic, morphologic, and molecular assessments were performed 7 days later. ResultsThe cells propagated in complete osteogenic medium for 7 days showed strong staining for type I collagen and alkaline phosphatase, suggesting the structural and functional properties of the osteoblastic cells. Further, real-time polymerase chain reaction (RT-PCR) revealed a significant expression of the osteoblast markers osteocalcin, osteopontin, and type I collagen. Similarly, the cells in endothelial growth medium were successfully differentiated into cobblestone-shaped endothelial cells expressing vascular endothelial growth factor (VEGF) receptors Flk-1 and Flt-1 demonstrated by RT-PCR. ConclusionsNEDAPS cells are readily induced to osteoblastic and endothelial cells, suggesting therapeutic potential for bone repair and other regenerative therapies.

Leukocyte trafficking is not affected by multikinase inhibitors sunitinib or sorafenib in mice.

Sunitinib and sorafenib are broad-spectrum tyrosine kinase inhibitors (TKI) targeting, for example, VEGF1-3, PDGFRb, RET, FLT3, CD117 (c-KIT) and CSF-1R cell membrane receptors thus suppressing tumor angiogenesis and cancer cell growth. Recently it has been suggested that the kinases targeted by Sunitinib and/or Sorafenib regulate leukocyte transmigration, which might in part be responsible for the often-observed reduction in tumor-associated myeloid derived suppressor cells and regulatory T cells. The aim of the current study is to determine whether sunitinib or sorafenib inhibit leukocyte extravasation. Sunitinib, sorafenib, or vehicle treated animals did not show any difference in leukocyte trafficking either in peritonitis or in vivo homing experiments, although sunitinib treatment effectively inhibited growth of B16 melanoma tumors in WT, SCID and SCID beige mice. Inhibition of tumor growth was associated with an increased number of infiltrating CD11b+ cells in the tumor, while the numbers of CD8, Gr-1 and F4/80 expressing cells were unchanged. In conclusion, the findings suggest that despite multiple targets with a potential role in leukocyte extravasation, neither sunitinib nor sorafenib effectively inhibits this process in vivo. Thus, the observed specific effect on CD11b cells among tumor infiltrating leukocytes is most likely an indirect effect.

Expression of PIGF and Its receptor Flt-1 in Patients with Multiple Myeloma and their Correlation with Chemotherapeutic Efficacy

To investigate the expression of PIGF and its receptor Flt-1 in patients with multiple myeloma, and to analyze their correlation with the efficacy of thalidomide-based chemotherapy so as to provide further theoretical basis for individualized treatment. A total of 35 patients diagnosed as multiple myeloma from June 2012 to March 2013 in our hospital and 15 non-tumor patients as controls were enrolled in this study. MM patients were treated with thalidomide-based chemotherapy for 3 months, and then were grouped according to the curative effecacy. The expression levels of PIGF and Flt-1 were detected in bone marrow of control and MM patient group by RT-PCR and Western blot before and after chemotherapy, their correlation with chemotherapeutic efficacy was analyzed. Serum concentrations of PITG and Flt-1 were detected in control and MM patients before and after chemotherapy by ELISA and their correlation with the chemotherapeutic efficacy was analyzed. The effective rate of three-months-thalidomide-based chemotherapy was 54.3% in MM patients. The expression levels of PIGF and Flt-1 in MM patients' bone marrow were obviously higher than those in controls. After chemotherapy, PIGF and Flt-1 expression levels significantly reduced and the decline level was positively correlated with curative effecacy(r=0.71). The serum concentrations of PIGF and Flt-1 in MM patients' bone marrow were obviously higher than those in control. After chemotherapy, serum concentrations of PIGF and Flt-1 were significantly decreased and the decline level positively correlated with curative effecacy(r=0.87). PIGF and FLT-1 are highly expressed in patients with multiple myeloma, and their expression levels positively correlates with curative effecacy of thalidomide-based chemotherapy.

45 On the origin and disappearance of sFlt-1 and PlGF in preeclampsia

Background The pro-angiogenic placental growth factor (PlGF) and the anti-angiogenic, PlGF-sequestering soluble receptor (sFlt-1) are both released from the placenta into the maternal circulation during pregnancy. In preeclampsia (PE), the balance shifts towards sFlt-1, resulting in an elevated sFlt-1/PlGF ratio. It is currently unknown how fast these proteins disappear after delivery, and to what degree extraplacental sources contribute to their levels. Objective To investigate the changes in sFlt-1 and PlGF levels in PE women after delivery. Methods Serum sFlt-1 and PlGF levels were measured in 23 women aged 20–41 years with a median duration of labor of 27 weeks (range 24–32 weeks) using the automated Elecsys system. The women were randomly chosen from a big PE cohort, and samples were taken both before and after delivery. Concentrations after delivery were expressed as a percentage of the concentration in the last sample that was taken during pregnancy. Results Median sFlt-1 and PlGF concentrations at the final stage of pregnancy were 11007 (3593–85000) and 26 (6–237) pg/mL, respectively, and the ratio was 544 (83–1034). SFlt-1 decreased with a half-life of 1.2 ± 0.4 days, and then stabilized at levels corresponding with 0.3 ± 0.2% of the level during pregnancy. PlGF decreased with a half-life of 4.8 ± 1.6 and then stabilized at levels corresponding with 14.7 ± 4.1% of the level during pregnancy. Changes in the ratio paralleled those in sFlt-1. Conclusions sFlt-1, but not PlGF, almost exclusively ( > 99%) originates in the placenta, and disappears more rapidly after delivery, thus allowing a quick normalization of the sFlt-1/PlGF ratio. Future studies should address the kinetics of these parameters in women with an uncomplicated pregnancy, and investigate whether sFlt-1 levels, despite a massive reduction, remain elevated in PE women vs. healthy pregnant women after delivery.

Adipose-derived stem cells sustain prolonged angiogenesis through leptin secretion

Recent studies suggest that adipose-derived stem cells (ASCs) play a role in tissue remodeling through the release of cytokines and growth factors. We compared the secreted cytokine profile of hypoxia-conditioned ASCs (hASCs) with normoxic ASCs (nASCs) and we analyzed the effect of ASCs conditioned medium (CM) on endothelial cells. We found that hypoxia induced a transient upregulation of VEGF in ASCs and a notable and enduring upregulation of leptin mRNA expression 30-fold greater than control after 24 h and up to 60-fold greater than control at day 7. CM from hASC stimulated EC tube formation to a significantly greater extent than CM from nASC. This might be due to leptin-secreted factor. Indeed, exogenous leptin stimulated the expression of HIF2-α, but not HIF1-α, and upregulated the expression of Flt-1 and Tie-1 proangiogenic receptors. In conclusion, hASCs may be particularly efficient in sustaining angiogenesis through the release of leptin.

Palbociclib treatment of FLT3-ITD+ AML cells uncovers a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6

AbstractUp to 30% of patients with acute myeloid leukemia have constitutively activating internal tandem duplications (ITDs) of the FLT3 receptor tyrosine kinase. Such mutations are associated with a poor prognosis and a high propensity to relapse after remission. FLT3 inhibitors are being developed as targeted therapy for FLT3-ITD+ acute myeloid leukemia; however, their use is complicated by rapid development of resistance, which illustrates the need for additional therapeutic targets. We show that the US Food and Drug Administration–approved CDK4/6 kinase inhibitor palbociclib induces apoptosis of FLT3-ITD leukemic cells. The effect is specific for FLT3-mutant cells and is ascribed to the transcriptional activity of CDK6: CDK6 but not its functional homolog CDK4 is found at the promoters of the FLT3 and PIM1 genes, another important leukemogenic driver. There CDK6 regulates transcription in a kinase-dependent manner. Of potential clinical relevance, combined treatment with palbociclib and FLT3 inhibitors results in synergistic cytotoxicity. Simultaneously targeting two critical signaling nodes in leukemogenesis could represent a therapeutic breakthrough, leading to complete remission and overcoming resistance to FLT3 inhibitors.

Flt1/VEGFR1 heterozygosity causes transient embryonic edema.

Vascular endothelial growth factor-A is a major player in vascular development and a potent vascular permeability factor under physiological and pathological conditions by binding to a decoy receptor Flt1 and its primary receptor Flk1. In this study, we show that Flt1 heterozygous (Flt1+/−) mouse embryos grow up to adult without life-threatening abnormalities but exhibit a transient embryonic edema around the nuchal and back regions, which is reminiscent of increased nuchal translucency in human fetuses. Vascular permeability is enhanced and an intricate infolding of the plasma membrane and huge vesicle-like structures are seen in Flt1+/− capillary endothelial cells. Flk1 tyrosine phosphorylation is elevated in Flt1+/− embryos, but Flk1 heterozygosity does not suppress embryonic edema caused by Flt1 heterozygosity. When Flt1 mutants are crossed with Aspp1−/− mice which exhibit a transient embryonic edema with delayed formation and dysfunction of lymphatic vessels, only 5.7% of Flt1+/−; Aspp1−/− mice survive, compared to expected ratio (25%). Our results demonstrate that Flt1 heterozygosity causes a transient embryonic edema and can be a risk factor for embryonic lethality in combination with other mutations causing non-lethal vascular phenotype.

Osmotic induction of placental growth factor in retinal pigment epithelial cells in vitro: contribution of NFAT5 activity.

One risk factor of neovascular age-related macular degeneration is systemic hypertension; hypertension is mainly caused by extracellular hyperosmolarity after consumption of dietary salt. In retinal pigment epithelial (RPE) cells, high extracellular osmolarity induces vascular endothelial growth factor (VEGF)-A (Hollborn et al. in Mol Vis 21:360-377, 2015). The aim of the present study was to determine whether extracellular hyperosmolarity and chemical hypoxia trigger the expression of further VEGF family members including placental growth factor (PlGF) in human RPE cells. Hyperosmotic media were made up by addition of 100mM NaCl or sucrose. Chemical hypoxia was induced by CoCl2. Gene expression was quantified by real-time RT-PCR, and secretion of PlGF-2 was investigated with ELISA. Nuclear factor of activated T cell 5 (NFAT5) was depleted using siRNA. Extracellular hyperosmolarity triggered expression of VEGF-A, VEGF-D, and PlGF genes, and secretion of PlGF-2. Hypoosmolarity decreased PlGF gene expression. Hypoxia induced expression of VEGF-A, VEGF-B, VEGF-D, and PlGF genes. Extracellular hyperosmolarity and hypoxia produced additive PlGF gene expression. Both hyperosmolarity and hypoxia induced expression of KDR and FLT-4 receptor genes, while hyperosmolarity caused neuropilin-2 and hypoxia neuropilin-1 gene expression. The hyperosmotic, but not the hypoxic, PlGF gene expression was in part mediated by NFAT5. The expression of PlGF in RPE cells depends on the extracellular osmolarity. The data suggest that high consumption of dietary salt may exacerbate the angiogenic response of RPE cells in the hypoxic retina via transcriptional activation of various VEGF family member genes.

Flt-1 (VEGFR-1) coordinates discrete stages of blood vessel formation.

In developing blood vessel networks, the overall level of vessel branching often correlates with angiogenic sprout initiations, but in some pathological situations, increased sprout initiations paradoxically lead to reduced vessel branching and impaired vascular function. We examine the hypothesis that defects in the discrete stages of angiogenesis can uniquely contribute to vessel branching outcomes. Time-lapse movies of mammalian blood vessel development were used to define and quantify the dynamics of angiogenic sprouting. We characterized the formation of new functional conduits by classifying discrete sequential stages-sprout initiation, extension, connection, and stability-that are differentially affected by manipulation of vascular endothelial growth factor-A (VEGF-A) signalling via genetic loss of the receptor flt-1 (vegfr1). In mouse embryonic stem cell-derived vessels genetically lacking flt-1, overall branching is significantly decreased while sprout initiations are significantly increased. Flt-1(-/-) mutant sprouts are less likely to retract, and they form increased numbers of connections with other vessels. However, loss of flt-1 also leads to vessel collapse, which reduces the number of new stable conduits. Computational simulations predict that loss of flt-1 results in ectopic Flk-1 signalling in connecting sprouts post-fusion, causing protrusion of cell processes into avascular gaps and collapse of branches. Thus, defects in stabilization of new vessel connections offset increased sprout initiations and connectivity in flt-1(-/-) vascular networks, with an overall outcome of reduced numbers of new conduits. These results show that VEGF-A signalling has stage-specific effects on vascular morphogenesis, and that understanding these effects on dynamic stages of angiogenesis and how they integrate to expand a vessel network may suggest new therapeutic strategies.