VEGFR-3 is expressed on megakaryocyte precursors in the murine bone marrow and plays a regulatory role in megakaryopoiesis

Introduction VEGFR-3 is a member of the VEGFR receptor tyrosine kinase family. It is expressed on lymphatic endothelial cells (LECs) and plays a central role in the regulation of lymphangiogenesis. 1 On binding to its ligands, VEGF-C and VEGF-D, VEGFR-3 is activated and orchestrates the outgrowth of lymphatic vessels. During murine hematopoiesis, Sca-1 ϩ hematopoietic stem cells give rise to the precursors of all hematopoietic lineages. 10 Megakaryocytes develop from CD34 ϩ progenitors. Methods Cell culture HEL cells were obtained from DSMZ and cultivated in RPMI (Gibco-BRL) containing 10% FCS and 1% penicillin-streptomycin. Differentiation was induced with 10nM tetradecanoyl phorbol acetate (TPA; Sigma-Aldrich). Primary human microvascular LECs (Cambrex) from the dermis (HMVECdLyNeo) were cultivated in EGM-2MV (Lonza) and 5% FCS supplemented with growth factors provided by the manufacturer. Bovine lymphatic endothelial cells were cultivated in DMEM (Gibco-BRL) containing 20% FCS and 1% penicillin-streptomycin on gelatin-coated plastic. HEK-293 cells were cultivated in DMEM supplemented with 10% FCS and 1% penicillin-streptomycin. Western blot analysis Cell lysates were analyzed using standard Western blotting techniques. The membranes were probed with Abs specific for VEGFR-3 (R&D Systems), CD31 (Santa Cruz Biotechnology), CD34 (Abcam), CD42a (Santa Cruz Biotechnology), CD61 (R&D Systems), CD144 (Santa Cruz Biotechnology), or GpA (International Blood Group Reference Laboratory). Probing with hypoxanthine phosphoribosyltransferase (HPRT) Abs (Santa Cruz Biotechnology) served as a loading control. PCR analysis RNA was prepared using peqGOLD RNAPure (PeqLab). Synthesis of cDNA using Superscript II (Invitrogen) was performed according to the manufacturer's recommendations. For PCR, cDNAs were amplified as follows: 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 90 seconds (VEGFR-2, Prox1, LYVE-1, Podoplanin, HPRT, Fli-1, Fog-2, Gata-2, and Elf-1) or 94°C for 30 seconds, 54°C for 30 seconds, and 72°C for 90 seconds (VEGFR-3). Details of the primers used are in supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Tubule formation on collagen gels Collagen type 1 was prepared from rat tails. Tendons were isolated, dissolved in acetic acid, then filtered, lyophilized, and redissolved in 0.1% acetic acid at 4 mg/mL. Cells were seeded on collagen gels (2 mg/mL) and cultured in the presence of 30 ng/mL of VEGF 165 (Promokine) for 8 days. Tubule formation was analyzed as described previously. 22 Immunohistochemistry For the immunohistochemical analysis of VEGFR-3 expression in the BM, cryosections of decalcified murine femurs embedded in tissue-freezing medium (Leica) were fixed in acetone and stained with VEGFR-3 Abs (eBiosciences). The stained sections were then analysed at room temperature using an Axioskop (Zeiss) equipped with a PlanNeoflur 20ϫ/0.50 and an Axiocam (Zeiss) and Axiovision software (Ziess). MACS BM cells isolated from femurs and tibias of C57BL/6 mice were treated with Fc-block (BD Biosciences) and then incubated with Abs against VEGFR-3 (R&D Systems), Sca-1, CD41, or CD38 (BD Biosciences), followed by specific secondary MACS Abs (Miltenyi-Biotec) according to the manufacturerЈs recommendations. Cell populations were then either enriched or depleted for the labeled epitope using LS or LD columns (Miltenyi-Biotec), respectively. The purity of the sorted populations was controlled by flow cytometry. CD42 FACS BM was isolated from femurs and tibias of C57BL/6 mice and stained with Abs specific for VEGFR-3 (R&D Systems) and/or CD42a (Emfret) and analyzed by FACS. Lethal irradiation and BM transplantation C57BL/6 mice were irradiated with lethal doses (9 Gy) from a ␥ source. After 24 hours, the mice were all transplanted in parallel by IV injection with either complete BM, BM depleted of VEGFR-3 ϩ cells, or BM mock depleted with an appropriate isotype control using MACS. EDTA blood samples were taken from all animals on days 0, Isolation and culture of primary murine BM cells BM was isolated from femurs and tibias of C57BL6 mice. After lysis of RBCs with ammonium-chloride-potassium buffer, the cells were transferred to IMDM (Gibco-BRL) supplemented with 1% penicillin/streptomycin, 10% HEK-293 cell-conditioned DMEM, Nutridoma SP (Roche), L-glutamine, and 100 pg/mL of recombinant murine TPO (RDI Diagnostics). Depending on the experiment, the cells were cultured with either 100 g/mL of mF4-31C1 VEGFR-3-blocking Abs (kindly provided by ImClone Systems), 100 g/mL of rat IgG isotype control, or 400 ng/mL of VEGF-C-Cys, a mutant form of VEGF-C that activates VEGFR-3 but not VEGFR-2. Long-term injections C57BL/6 mice were injected daily with 25 g of VEGF-C-Cys for 3 weeks. Blood was taken on days 0, 3, 7, 10, 14, 17, and 21. In the blocking Ab experiments, mice were injected with 600 g/animal/injection of mF4-31C1 VEGFR-3-blocking Ab, isotype control Ig, or PBS on a MondayWednesday-Friday schedule for 6 weeks. Blood was taken on days 0, Recovery kinetics after sublethal irradiation Experimental C57BL/6 mice were sublethally irradiated (4.5 Gy) in a ␥ source. They were then either injected daily with VEGF-C-Cys (25 g/animal/injection) or PBS or were intraperitoneally injected with 600 g/animal/injection of mF4-31C1 VEGFR-3-blocking Abs, isotype control Ig, or PBS every other day. Blood was taken on days 0, 7, 11, 14, 18, and 21 after irradiation and analyzed. In each experiment, all animals were treated at the same time and on the same day and all animals were bled at each time point. BM was isolated from femurs and tibias 20 days after irradiation, and the number and ploidy of CD41 ϩ cells in the BM was assessed. Significance was tested using 2-tailed unpaired t tests assuming equal variance. TPO administration C57BL/6 mice were administered with 5 g of recombinant murine TPO (RDI), followed by daily injections of either 25 g of VEGF-C-Cys or PBS. One group received only PBS throughout. Blood was taken and analyzed 0, 3, 5, 7, and 10 days after TPO administration. All animals were treated at the same time and on the same day and all animals were bled at each time point. After 10 days, the animals were killed and the number and ploidy of CD41 ϩ 1900 THIELE et al BLOOD, 30 AUGUST 2012 ⅐ VOLUME 120, NUMBER 9 For personal use only. on October 6, 2016. by guest www.bloodjournal.org From cells in the BM was assessed. Significance was tested using 2-tailed unpaired t tests assuming equal variance. 5-FU treatment C57BL/6 mice were intraperitoneally injected with a single dose of 5-FU (Sigma-Aldrich) at 150 mg/kg. Control mice remained untreated. The 5-FU-treated mice then received daily injections of either 25 g of VEGF-C-Cys or PBS throughout the experiment. Blood was taken and analyzed 0, All animal experiments were approved by the local regulatory authorities and were performed according to German legal requirements. Results Expression of VEGFR-3 and other lymphatic endothelial markers is up-regulated on phorbol diester-induced megakaryocytic differentiation of HEL cells VEGFR-3 is widely used as a marker for lymphatic endothelium. Originally, however, the receptor was cloned from the HEL cell line. 7 This cell line can be induced to differentiate into the erythrocyte lineage by EPO treatment 23 and into the megakaryocyte lineage in response to TPA. Consistent with the notion that HEL cells differentiate into the megakaryocyte lineage on TPA treatment, we detected strong up-regulation of several markers and transcription factors associated with megakaryocytic differentiation A survey of the literature revealed that virtually all markers described to date as being expressed on megakaryocytes can also be expressed on endothelial cells (supplemental These observations raised the question of whether HEL cells really undergo megakaryocytic differentiation after TPA treatment or if they adopt an endothelial phenotype with LEC characteristics. To address this point, we investigated whether TPA-treated HEL cells are capable of forming capillaries, reasoning that if the cells differentiated into endothelial cells, this should be the case. However, in contrast to control bovine LECs, TPA-treated HEL cells could not be induced to form capillaries VEGFR-3 IN MEGAKARYOPOIESIS 1901 BLOOD, 30 AUGUST 2012 ⅐ VOLUME 120, NUMBER 9 For personal use only. on October 6, 2016. by guest www.bloodjournal.org From VEGFR-3 is expressed on megakaryocytic progenitors through to the promegakaryoblast stage in the BM The up-regulation of VEGFR-3 during HEL cell megakaryocytic differentiation suggested to us that VEGFR-3 may play a role in megakaryopoiesis. Because of the limited megakaryocytic differentiation capacity of HEL cells and their cancerous nature, we explored this possibility further using murine BM. First we characterized VEGFR-3 expression in the BM. FACS staining revealed that approximately 2% of murine BM cells were VEGFR-3 ϩ ( To define further the stages of megakaryopoiesis during which VEGFR-3 is expressed, costainings with the stem cell marker Sca-1 and with CD38, CD41, and VEGFR-3 were performed. Expression of Sca-1 is lost during myeloid differentiation. 25 CD38 expression, in turn, is increased early in megakaryopoiesis from the BFU-MK stage on. These observations suggested to us that VEGFR-3 might be expressed on hematopoietic stem cells through to the promegakaryoblast stage. However, Sca1 is not just expressed on hematopoietic stem cells, but also on the immediate progenitors arising from the stem cells. These data are consistent with the notion that VEGFR-3 is not expressed on hematopoietic stem cells, but rather on megakaryocyte precursors through to the premegakaryoblast stage, and that VEGFR-3 expression is lost as megakaryocytes further mature. This notion is further substantiated by the observation that VEGFR-3 ϩ BM cells coexpressed CD42, a marker for megakaryocytes that is not expressed on hematopoietic precursor cells (supplemental Manipulation of VEGFR-3 influences megakaryopoiesis in vitro To examine the role that VEGFR-3 plays during megakaryopoiesis, we cultivated primary murine BM cells with physiologic concentrations of TPO to maintain the megakaryocyte precursors. The cells were grown for 3 days in the presence or absence of VEGF-C-Cys, a mutant form of VEGF-C that specifically activates VEGFR-3 but not VEGFR-2, 20 because VEGFR-2 is also present on megakaryocytic cells. Our data suggest that the specific activation of VEGFR-3 during megakaryopoiesis impairs the transition to polyploid stages, whereas blocking the receptor promotes differentiation and endoreplication. For personal use only. on October 6, 2016. by guest www.bloodjournal.org From Neither activation nor blocking of VEGFR-3 influences steady-state megakaryopoiesis or thrombopoiesis in vivo To study the potential effects of VEGFR-3 manipulation on megakaryopoiesis and thrombopoiesis in vivo, we first injected VEGF-C-Cys to activate VEGFR-3, or PBS as a control, into mice on a daily basis for 3 weeks. Thrombocyte concentrations in the blood were monitored regularly. After 3 weeks of treatment, the mice were killed. BM cells were isolated and stained for CD41 and DNA content to evaluate the number and ploidy of the CD41 ϩ population. We observed a significant decrease in apoptotic CD41 ϩ BM cells in the VEGF-C-Cys-treated group (P Ͻ .01), a trend toward reduced polyploidy, and an increase in 2n CD41 ϩ cells, which were consistent with our in vitro observations. VEGF-C-Cys had no effect on platelet counts or the number of CD41 ϩ cells in the BM (supplemental To determine the effect of inhibiting VEGFR-3 activation on megakaryopoiesis and thrombopoiesis in vivo, mice were injected daily with VEGFR-3-blocking Abs or an appropriate isotype control for 6 weeks. Platelet counts were monitored regularly and the numbers and ploidy distribution of CD41 ϩ BM cells were analyzed at the end of the experiment. Under these conditions, no effects on the measured parameters were observed (supplemental Activation of VEGFR-3 increases platelet counts in TPO-stimulated animals, modulates 5-FU-induced thrombocytopenia and thrombocytosis, and influences ploidy distribution and numbers of CD41 ؉ BM cells after sublethal irradiation Thrombocyte homeostasis is tightly controlled in mammals, and alternative mechanisms exist that can compensate for perturbation . FACS analysis showed that 1.85% Ϯ 0.31% SEM (n ϭ 9) of the murine BM cells expressed VEGFR-3. Dot plots of 1 representative experiment are depicted. Density plots were used to define a region in which 95% (the 2 outer contours) of the negative control events were excluded. The region was then applied to a plot displaying the stained sample. The number of positive events in both the negative control and the actual sample was then assessed. The percentage of true positive cells was calculated by subtraction of the number of events in the negative control within the defined region from the number of events found in the same region for the actual sample. Identical numbers of events were acquired. (B) VEGFR-3 is expressed on isolated mononuclear cells in the murine BM. Sections of murine femurs were stained with VEGFR-3-specific Abs (left panel, VEGFR-3; right panel, control). MK indicates megakaryocyte. Scale bars indicate 100 m. (C) Ploidy of VEGFR-3 ϩ cells in the murine BM. VEGFR-3 ϩ BM cells were enriched by MACS and then analyzed in FACS. As a control, cells were treated with an appropriate isotype control. Clumping cells mimicking polyploidy were excluded from the analysis by appropriate gating strategies. The resulting histogram plot shows the DNA content of VEGFR-3 ϩ cells. Dot plots of the DNA content of the cells were used for the quantification of VEGFR-3 ϩ and isotype-treated cells within different ploidy classes or cell cycle stages, respectively (a detailed scheme of the gating strategy is provided in supplementa

Loss of ASAP1 in mice impairs adipogenic and osteogenic differentiation of mesenchymal progenitor cells through dysregulation of FAK/Src and AKT signaling

ASAP1 is a multi-domain adaptor protein that regulates cytoskeletal dynamics, receptor recycling and intracellular vesicle trafficking. Its expression is associated with poor prognosis for a variety of cancers, and promotes cell migration, invasion and metastasis. Little is known about its physiological role. In this study, we used mice with a gene-trap inactivated ASAP1 locus to study the functional role of ASAP1 in vivo, and found defects in tissues derived from mesenchymal progenitor cells. Loss of ASAP1 led to growth retardation and delayed ossification typified by enlarged hypertrophic zones in growth plates and disorganized chondro-osseous junctions. Furthermore, loss of ASAP1 led to delayed adipocyte development and reduced fat depot formation. Consistently, deletion of ASAP1 resulted in accelerated chondrogenic differentiation of mesenchymal cells in vitro, but suppressed osteo- and adipogenic differentiation. Mechanistically, we found that FAK/Src and PI3K/AKT signaling is compromised in Asap1GT/GT MEFs, leading to impaired adipogenic differentiation. Dysregulated FAK/Src and PI3K/AKT signaling is also associated with attenuated osteogenic differentiation. Together these observations suggest that ASAP1 plays a decisive role during the differentiation of mesenchymal progenitor cells

VEGFR-3 is expressed on megakaryocyte precursors in the murine bone marrow and plays a regulatory role in megakaryopoiesis.

VEGFR-3 is a transmembrane receptor tyrosine kinase that is activated by its ligands VEGF-C and VEGF-D. Although VEGFR-3 has been linked primarily to the regulation of lymphangiogenesis, in the present study, we demonstrate a role for VEGFR-3 in megakaryopoiesis. Using a human erythroleukemia cell line and primary murine BM cells, we show that VEGFR-3 is expressed on megakaryocytic progenitor cells through to the promegakaryoblast stage. Functionally, specific activation of VEGFR-3 impaired the transition to polyploidy of CD41(+) cells in primary BM cultures. Blockade of VEGFR-3 promoted endoreplication consistently. In vivo, long-term activation or blockade of VEGFR-3 did not affect steady-state murine megakaryopoiesis or platelet counts significantly. However, activation of VEGFR-3 in sublethally irradiated mice resulted in significantly elevated numbers of CD41(+) cells in the BM and a significant increase in diploid CD41(+) cells, whereas the number of polyploid CD41(+) cells was reduced significantly. Moreover, activation of VEGFR-3 increased platelet counts in thrombopoietin-treated mice significantly and modulated 5-fluorouracil-induced thrombocytosis strongly, suggesting a regulatory role for VEGFR-3 in megakaryopoiesis

Loss of ASAP1 in mice impairs adipogenic and osteogenic differentiation of mesenchymal progenitor cells through dysregulation of FAK/Src and AKT signaling.

ASAP1 is a multi-domain adaptor protein that regulates cytoskeletal dynamics, receptor recycling and intracellular vesicle trafficking. Its expression is associated with poor prognosis for a variety of cancers, and promotes cell migration, invasion and metastasis. Little is known about its physiological role. In this study, we used mice with a gene-trap inactivated ASAP1 locus to study the functional role of ASAP1 in vivo, and found defects in tissues derived from mesenchymal progenitor cells. Loss of ASAP1 led to growth retardation and delayed ossification typified by enlarged hypertrophic zones in growth plates and disorganized chondro-osseous junctions. Furthermore, loss of ASAP1 led to delayed adipocyte development and reduced fat depot formation. Consistently, deletion of ASAP1 resulted in accelerated chondrogenic differentiation of mesenchymal cells in vitro, but suppressed osteo- and adipogenic differentiation. Mechanistically, we found that FAK/Src and PI3K/AKT signaling is compromised in Asap1GT/GT MEFs, leading to impaired adipogenic differentiation. Dysregulated FAK/Src and PI3K/AKT signaling is also associated with attenuated osteogenic differentiation. Together these observations suggest that ASAP1 plays a decisive role during the differentiation of mesenchymal progenitor cells