技术资料

Endoglin is an accessory receptor for TGF-beta signaling and is required for normal hemangioblast,early hematopoietic,and vascular development. We have previously shown that an upstream enhancer,Eng -8,together with the promoter region,mediates robust endothelial expression yet is inactive in blood. To identify hematopoietic regulatory elements,we used array-based methods to determine chromatin accessibility across the entire locus. Subsequent transgenic analysis of candidate elements showed that an endothelial enhancer at Eng +9 when combined with an element at Eng +7 functions as a strong hemato-endothelial enhancer. Chromatin immunoprecipitation (ChIP)-chip analysis demonstrated specific binding of Ets factors to the promoter as well as to the -8,+7+9 enhancers in both blood and endothelial cells. By contrast Pu.1,an Ets factor specific to the blood lineage,and Gata2 binding was only detected in blood. Gata2 was bound only at +7 and GATA motifs were required for hematopoietic activity. This modular assembly of regulators gives blood and endothelial cells the regulatory freedom to independently fine-tune gene expression and emphasizes the role of regulatory divergence in driving functional divergence. View Publication

Antiangiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane. Bortezomib's inhibitory effects on chorioallantoic membrane vascularization were abrogated in the presence of distinct tumor xenografts,thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy,we identified GRP-78,a chaperone protein of the unfolded protein response,as being responsible for bortezomib resistance. Indeed,a variety of bortezomib-resistant solid tumor cell lines (PC-3,HRT-18),but not myeloma cell lines (U266,OPM-2),were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78 gene expression in tumor cells and immunodepletion of GRP-78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib but induced prosurvival signals by phosphorylation of extracellular signal-related kinase and inhibited p53-mediated expression of proapoptotic Bok and Noxa proteins in endothelial cells. From our data,we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment,thus demonstrating a hitherto unknown mechanism of resistance to bortezomib. View Publication

Tumor angiogenesis is essential for malignant growth and metastasis. Bone marrow (BM)-derived endothelial progenitor cells (EPC) contribute to angiogenesis-mediated tumor growth. EPC ablation can reduce tumor growth; however,the lack of a marker that can track EPCs from the BM to tumor neovasculature has impeded progress in understanding the molecular mechanisms underlying EPC biology. Here,we report the use of transgenic mouse and lentiviral models to monitor the BM-derived compartment of the tumor stroma; this approach exploits the selectivity of the transcription factor inhibitor of DNA binding 1 (Id1) for EPCs to track EPCs in the BM,blood,and tumor stroma,as well as mature EPCs. Acute ablation of BM-derived EPCs using Id1-directed delivery of a suicide gene reduced circulating EPCs and yielded significant defects in angiogenesis-mediated tumor growth. Additionally,use of the Id1 proximal promoter to express microRNA-30-based short hairpin RNA inhibited the expression of critical EPC-intrinsic factors,confirming that signaling through vascular endothelial growth factor receptor 2 is required for EPC-mediated tumor biology. By exploiting the selectivity of Id1 gene expression in EPCs,our results establish a strategy to track and target EPCs in vivo,clarifying the significant role that EPCs play in BM-mediated tumor angiogenesis. View Publication

The physiologic roles of angiopoietin-like proteins (Angptls) in the hematopoietic system remain unknown. Here we show that hematopoietic stem cells (HSCs) in Angptl3-null mice are decreased in number and quiescence. HSCs transplanted into Angptl3-null recipient mice exhibited impaired repopulation. Bone marrow sinusoidal endothelial cells express high levels of Angptl3 and are adjacent to HSCs. Importantly,bone marrow stromal cells or endothelium deficient in Angptl3 have a significantly decreased ability to support the expansion of repopulating HSCs. Angptl3 represses the expression of the transcription factor Ikaros,whose unregulated overexpression diminishes the repopulation activity of HSCs. Angptl3,as an extrinsic factor,thus supports the stemness of HSCs in the bone marrow niche. View Publication

Hematopoietic cells (HCs) promote blood vessel formation by producing various proangiogenic cytokines and chemokines and matrix metalloproteinases. We injected mouse colon26 colon cancer cells or human PC3 prostate adenocarcinoma cells into mice and studied the localization of HCs during tumor development. HCs were distributed in the inner tumor mass in all of the tumor tissues examined; however,the localization of HCs in the tumor tissue differed depending on the tumor cell type. In the case of colon26 tumors,as the tumor grew,many mature HCs migrated into the tumor mass before fine capillary formation was observed. On the other hand,although very few HCs migrated into PC3 tumor tissue,c-Kit+ hematopoietic stem/progenitor cells accumulated around the edge of the tumor. Bone marrow suppression induced by injection of anti-c-Kit neutralizing antibody suppressed tumor angiogenesis by different mechanisms according to the tumor cell type: bone marrow suppression inhibited the initiation of sprouting angiogenesis in colon26 tumors,while it suppressed an increase in the caliber of newly developed blood vessels at the tumor edge in PC3 tumors. Our findings suggest that HCs are involved in tumor angiogenesis and regulate the angiogenic switch during tumorigenesis. View Publication

TEL-TRKC is a fusion gene generated by chromosomal translocation and encodes an activated tyrosine kinase. Uniquely,it is found in both solid tumors and leukemia. However,a single exon difference (in TEL) in TEL-TRKC fusions is associated with the two sets of cancer phenotypes. We expressed the two TEL-TRKC variants in vivo by using the 3' regulatory element of SCL that is selectively active in a subset of mesodermal cell lineages,including endothelial and hematopoietic stem cells and progenitors. The leukemia form of TEL-TRKC (-exon 5 of TEL) enhanced hematopoietic stem cell renewal and initiated leukemia. In contrast,the TEL-TRKC solid tumor variant (+ TEL exon 5) elicited an embryonic lethal phenotype with impairment of both angiogenesis and hematopoiesis indicative of an effect at the level of the hemangioblasts. The ability of TEL-TRKC to repress expression of Flk1,a critical regulator of early endothelial and hematopoietic cells,depended on TEL exon 5. These data indicate that related oncogenic fusion proteins similarly expressed in a hierarchy of early stem cells can have selective,cell type-specific developmental impacts. View Publication

OBJECTIVE: To investigate the kinetics of myocardial engraftment of bone marrow-derived mononuclear cells (BMNCs) after intracoronary injection using 99mTc-d,l-hexamethylpropylene amine oxime (99mTc-HMPAO) nuclear imaging in patients with acute and chronic anterior myocardial infarction. DESIGN: Nuclear imaging-derived tracking of BMNCs at 2 and 20 h after injection in the left anterior descending (LAD) coronary artery. SETTING: Academical cardiocentre. PATIENTS: Five patients with acute (mean (SD) age 58 (11) years; ejection fraction range 33-45%) and five patients with chronic (mean (SD) age 50 (6) years; ejection fraction range 28-34%) anterior myocardial infarction. INTERVENTIONS: A total of 24.2 x 10(8)-57.0 x 10(8) BMNCs (20% labelled with 700-1000 MBq 99mTc-HMPAO) were injected in the LAD coronary artery. RESULTS: At 2 h after BMNC injection,myocardial activity was observed in all patients with acute (range 1.31-5.10%) and in all but one patient with chronic infarction (range 1.10-3.0%). At 20 h,myocardial engraftment was noted only in three patients with acute myocardial infarction,whereas no myocardial activity was noted in any patient with chronic infarction. CONCLUSIONS: Engraftment of BMNCs shows dynamic changes within the first 20 h after intracoronary injection. Persistent myocardial engraftment was noted only in a subset of patients with acute myocardial infarction. View Publication

Retinal vascular damages are the cardinal hallmarks of retinopathy of prematurity (ROP),a leading cause of vision impairment and blindness in childhood. Both angiogenesis and vasculogenesis are disrupted in the hyperoxia-induced vaso-obliteration phase,and recapitulated,although aberrantly,in the subsequent ischemia-induced neovessel formation phase of ROP. Yet,whereas the histopathological features of ROP are well characterized,many key modulators with a therapeutic potential remain unknown. The CCN1 protein also known as cysteine-rich protein 61 (Cyr61) is a dynamically expressed,matricellular protein required for proper angiogenesis and vasculogenesis during development. The expression of CCN1 becomes abnormally reduced during the hyperoxic and ischemic phases of ROP modeled in the mouse eye with oxygen-induced retinopathy (OIR). Lentivirus-mediated re-expression of CCN1 enhanced physiological adaptation of the retinal vasculature to hyperoxia and reduced pathological angiogenesis following ischemia. Remarkably,injection into the vitreous of OIR mice of hematopoietic stem cells (HSCs) engineered to express CCN1 harnessed ischemia-induced neovessel outgrowth without adversely affecting the physiological adaptation of retinal vessels to hyperoxia. In vitro exposure of HSCs to recombinant CCN1 induced integrin-dependent cell adhesion,migration,and expression of specific endothelial cell markers as well as many components of the Wnt signaling pathway including Wnt ligands,their receptors,inhibitors,and downstream targets. CCN1-induced Wnt signaling mediated,at least in part,adhesion and endothelial differentiation of cultured HSCs,and inhibition of Wnt signaling interfered with normalization of the retinal vasculature induced by CCN1-primed HSCs in OIR mice. These newly identified functions of CCN1 suggest its possible therapeutic utility in ischemic retinopathy. View Publication

Excessive accumulation of smooth-muscle cells (SMCs) has a key role in the pathogenesis of vascular diseases. It has been assumed that SMCs derived from the outer medial layer migrate,proliferate and synthesize extracellular matrix components on the luminal side of the vessel. Although much effort has been devoted to targeting migration and proliferation of medial SMCs,there is no effective therapy that prevents occlusive vascular remodeling. We show here that in models of post-angioplasty restenosis,graft vasculopathy and hyperlipidemia-induced atherosclerosis,bone-marrow cells give rise to most of the SMCs that contribute to arterial remodeling. Notably,purified hematopoietic stem cells differentiate into SMCs in vitro and in vivo. Our findings indicate that somatic stem cells contribute to pathological remodeling of remote organs,and may provide the basis for the development of new therapeutic strategies for vascular diseases through targeting mobilization,homing,differentiation and proliferation of bone marrow-derived vascular progenitor cells. View Publication

Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression,and were sorted as a CD34+ and CD45- fraction. Cells in this fraction were approximately 94% positive for Sca-1,and mostly negative (textless3% positive) for CD14,31,49,144,c-kit,and FLK-1. The CD34+/45- cells formed colonies in clonal cell cultures and colony-forming units displayed the potential to differentiate into adipocytes,endothelial,and myogenic cells. The CD34+/45- cells fully differentiated into vascular endothelial cells and skeletal muscle fibers in vivo after transplantation. Immediately after sorting,CD34+/45- cells expressed only c-met mRNA,and did not express any other myogenic cell-related markers such as MyoD,myf-5,myf-6,myogenin,M-cadherin,Pax-3,and Pax-7. However,after 3 d of culture,these cells expressed mRNA for all myogenic markers. CD34+/45- cells were distinct from satellite cells,as they expressed Bcrp1/ABCG2 gene mRNA (Zhou et al.,2001). These findings suggest that myo-endothelial progenitors reside in the interstitial spaces of mammalian skeletal muscles,and that they can potentially contribute to postnatal skeletal muscle growth. View Publication