参考文献

Chemokine stromal derived factor 1 (SDF-1) is involved in trafficking of hematopoietic stem cells (HSCs) from the bone marrow (BM) to peripheral blood (PB) and has been found to enhance postischemia angiogenesis. This study was aimed at investigating whether SDF-1 plays a role in differentiation of BM-derived c-kit(+) stem cells into endothelial progenitor cells (EPCs) and in ischemia-induced trafficking of stem cells from PB to ischemic tissues. We found that SDF-1 enhanced EPC number by promoting alpha(2),alpha(4),and alpha(5) integrin-mediated adhesion to fibronectin and collagen I. EPC differentiation was reduced in mitogen-stimulated c-kit(+) cells,while cytokine withdrawal or the overexpression of the cyclin-dependent kinase (CDK) inhibitor p16(INK4) restored such differentiation,suggesting a link between control of cell cycle and EPC differentiation. We also analyzed the time course of SDF-1 expression in a mouse model of hind-limb ischemia. Shortly after femoral artery dissection,plasma SDF-1 levels were up-regulated,while SDF-1 expression in the bone marrow was down-regulated in a timely fashion with the increase in the percentage of PB progenitor cells. An increase in ischemic tissue expression of SDF-1 at RNA and protein level was also observed. Finally,using an in vivo assay such as injection of matrigel plugs,we found that SDF-1 improves formation of tubulelike structures by coinjected c-kit(+) cells. Our findings unravel a function for SDF-1 in increase of EPC number and formation of vascular structures by bone marrow progenitor cells. View Publication

BACKGROUND AND OBJECTIVES: Interactions between acute myelogenous leukemia (AML) blasts and non-leukemic cells in the bone marrow seem to be important for both disease development and susceptibility to chemotherapy. Recent studies have focused on the endothelial cells,but other non-leukemic cells may also be involved. In the present study we investigated how osteoblasts affect native human AML blasts. DESIGN AND METHODS: AML cells were derived from a large group of consecutive patients. The AML blasts and osteoblastic sarcoma cell lines (Cal72,SJSA-1) were incubated together in different chambers separated by a semipermeable membrane. We investigated effects of co-culture on proliferation,apoptosis and cytokine release. RESULTS: The cross-talk between these two cell populations,achieved via release of soluble mediators,resulted in increased AML blast proliferation,including increased proliferation of clonogenic progenitors,but did not affect spontaneous in vitro apoptosis. Both interleukin (IL) 1-b and granulocyte-macrophage colony-stimulating factor were involved in this growth-enhancing cross-talk,and normal osteoblasts could also increase the AML blast proliferation. Furthermore,co-culture of AML blasts with osteoblastic sarcoma cells as well as normal osteoblasts increased the levels of the pro-angiogenic mediator IL8. INTERPRETATION AND CONCLUSIONS: Our in vitro results suggest that the release of soluble mediators by osteoblasts supports leukemic hematopoiesis through two major mechanisms: (i) direct enhancement of AML blast proliferation; and (ii) enhanced angiogenesis caused by increased IL8 levels. View Publication

Human umbilical cord blood (UCB) contains high numbers of endothelial progenitors cells (EPCs) characterized by coexpression of CD34 and CD133 markers. Prior studies have shown that CD34+/CD133+ EPCs from the cord or peripheral blood (PB) can give rise to endothelial cells and induce angiogenesis in ischemic tissues. In the present study,it is shown that freshly isolated human cord blood CD34+ cells injected into ischemic adductor muscles gave rise to endothelial and,unexpectedly,to skeletal muscle cells in mice. In fact,the treated limbs exhibited enhanced arteriole length density and regenerating muscle fiber density. Under similar experimental conditions,CD34- cells did not enhance the formation of new arterioles and regenerating muscle fibers. In nonischemic limbs CD34+ cells increased arteriole length density but did not promote formation of new muscle fibers. Endothelial and myogenic differentiation ability was maintained in CD34+ cells after ex vivo expansion. Myogenic conversion of human cord blood CD34+ cells was also observed in vitro by coculture onto mouse myoblasts. These results show that human cord blood CD34+ cells differentiate into endothelial and skeletal muscle cells,thus providing an indication of human EPCs plasticity. The full text of this article is available online at http://www.circresaha.org. View Publication

Vascular endothelial growth factor (VEGF) and stem cell factor (SCF) act as growth factors for the hemangioblast,an embryonic progenitor of the hematopoietic and endothelial lineages. Because thrombopoietin (TPO) and its receptor,c-Mpl,regulate primitive hematopoietic populations,including bone marrow hematopoietic stem cells,we investigated whether TPO acts on the hemangioblasts that derive from differentiation of embryonic stem cells in vitro. Reverse transcriptase polymerase chain reaction analysis detected expression of c-Mpl beginning on day 3 of embryoid body differentiation when the hemangioblast first arises. In assays of the hemangioblast colony-forming cell (BL-CFC),TPO alone supported BL-CFC formation and nearly doubled the number of BL-CFC when added together with VEGF and SCF. When replated under the appropriate conditions,TPO-stimulated BL-CFC gave rise to secondary hematopoietic colonies,as well as endothelial cells,confirming their nature as hemangioblasts. Addition of a neutralizing anti-VEGF antibody did not block TPO enhancement of BL-CFC formation,suggesting that TPO acts independently of VEGF. These results establish that Mpl signaling plays a role in the earliest stages of hematopoietic development and that TPO represents a third growth factor influencing hemangioblast formation. 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

Large scale purification of endothelial cells is of great interest as it could improve tissue transplantation,reperfusion of ischemic tissues and treatment of pathologies in which an endothelial cell dysfunction exists. In this study,we describe a novel genetic approach that selects for endothelial cells from differentiating embryonic stem (ES) cells. Our strategy is based on the establishment of ES-cell clones that carry an integrated puromycin resistance gene under the control of a vascular endothelium-specific promoter,tie-1. Using EGFP as a reporter gene,we first confirmed the endothelial specificity of the tie-1 promoter in the embryoid body model and in cells differentiated in 2D cultures. Subsequently,tie-1-EGFP ES cells were used as recipients for the tie-1-driven puror transgene. The resulting stable clones were expanded and differentiated for seven days in the presence of VEGF before puromycin selection. As expected,puromycin-resistant cells were positive for EGFP and also expressed several endothelial markers,including CD31,CD34,VEGFR-1,VEGFR-2,Tie-1,VE-cadherin and ICAM-2. Release from the puromycin selection resulted in the appearance of alpha-smooth muscle actin-positive cells. Such cells became more numerous when the population was cultured on laminin-1 or in the presence of TGF-beta1,two known inducers of smooth muscle cell differentiation. The hypothesis that endothelial cells or their progenitors may differentiate towards a smooth muscle cell phenotype was further supported by the presence of cells expressing both CD31 and alpha-smooth muscle actin markers. Finally,we show that purified endothelial cells can incorporate into the neovasculature of transplanted tumors in nude mice. Taken together,these results suggest that application of endothelial lineage selection to differentiating ES cells may become a useful approach for future pro-angiogenic and endothelial cell replacement therapies. 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

Interaction between endothelial cells and mural cells (pericytes and vascular smooth muscle) is essential for vascular development and maintenance. Endothelial cells arise from Flk1-expressing (Flk1+) mesoderm cells,whereas mural cells are believed to derive from mesoderm,neural crest or epicardial cells and migrate to form the vessel wall. Difficulty in preparing pure populations of these lineages has hampered dissection of the mechanisms underlying vascular formation. Here we show that Flk1+ cells derived from embryonic stem cells can differentiate into both endothelial and mural cells and can reproduce the vascular organization process. Vascular endothelial growth factor promotes endothelial cell differentiation,whereas mural cells are induced by platelet-derived growth factor-BB. Vascular cells derived from Flk1+ cells can organize into vessel-like structures consisting of endothelial tubes supported by mural cells in three-dimensional culture. Injection of Flk1+ cells into chick embryos showed that they can incorporate as endothelial and mural cells and contribute to the developing vasculature in vivo. Our findings indicate that Flk1+ cells can act as 'vascular progenitor cells' to form mature vessels and thus offer potential for tissue engineering of the vascular system. View Publication