技术资料

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

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

Emerging studies suggested that murine podoplanin-positive monocytes (PPMs) are involved in lymphangiogenesis. The goal of this study was to demonstrate the therapeutic lymphangiogenesis of human PPMs by the interaction with platelets. Aggregation culture of human peripheral blood mononuclear cells (PBMCs) resulted in cellular aggregates termed hematospheres. During 5-day culture,PPMs expanded exponentially and expressed several lymphatic endothelial cell-specific markers including vascular endothelial growth factor receptor (VEGFR)-3 and well-established lymphangiogenic transcription factors. Next,we investigated the potential interaction of PPMs with platelets that had C-type lectin-like receptor-2 (CLEC-2),a receptor of podoplanin. In vitro coculture of PPMs and platelets stimulated PPMs to strongly express lymphatic endothelial markers and upregulate lymphangiogenic cytokines. Recombinant human CLEC-2 also stimulated PPMs through Akt and Erk signaling. Likewise,platelets in coculture with PPMs augmented secretion of a lymphangiogenic cytokine,interleukin (IL)-1-β,via podoplanin/CLEC-2 axis. The supernatant obtained from coculture was able to enhance the migration,viability,and proliferation of lymphatic endothelial cell. Local injection of hematospheres with platelets significantly increased lymphatic neovascularization and facilitated wound healing in the full-thickness skin wounds of nude mice. Cotreatment with PPMs and platelets augments lymphangiogenesis through podoplanin/CLEC-2 axis,which thus would be a promising novel strategy of cell therapy to treat human lymphatic vessel disease. 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

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

Neonatal hyperoxia impairs vascular and alveolar growth in mice and decreases endothelial progenitor cells. To determine the role of bone marrow-derived cells in restoration of neonatal lung structure after injury,we studied a novel bone marrow myeloid progenitor cell population from Tie2-green fluorescent protein (GFP) transgenic mice (bone marrow-derived angiogenic cells; BMDAC). We hypothesized that treatment with BMDAC would restore normal lung structure in infant mice during recovery from neonatal hyperoxia. Neonatal mice (1-day-old) were exposed to 80% oxygen for 10 days. BMDACs (1 x 10(5)),embryonic endothelial progenitor cells,mouse embryonic fibroblasts (control),or saline were then injected into the pulmonary circulation. At 21 days of age,saline-treated mice had enlarged alveoli,reduced septation,and a reduction in vascular density. In contrast,mice treated with BMDAC had complete restoration of lung structure that was indistinguishable from room air controls. BMDAC comprised 12% of distal lung cells localized to pulmonary vessels or alveolar type II (AT2) cells and persist (8.8%) for 8 wk postinjection. Coculture of AT2 cells or lung endothelial cells (luEC) with BMDAC augmented AT2 and luEC cell growth in vitro. We conclude that treatment with BMDAC after neonatal hyperoxia restores lung structure in this model of bronchopulmonary dysplasia. View Publication

AIMS: Endothelial progenitor cells (EPC) have been shown to repair pulmonary endothelium,although they can also migrate into the arterial intima and differentiate into smooth muscle-like (mesenchymal) cells contributing to intimal hyperplasia. The molecular mechanisms by which this process proceeds have not been fully elucidated. Here,we study whether genes involved in the endothelial-to-mesenchymal transition (EnMT) may contribute to the mesenchymal phenotype acquisition of EPC and we evaluate whether transforming growth factor β1 (TGFβ1) is involved in this process. METHODS AND RESULTS: Our results show that co-culture of EPC with smooth muscle cells (SMC) increases the expression of the mesenchymal cell markers α-smooth muscle actin,sm22-α,and myocardin,and decreases the expression of the endothelial cell marker CD31. In the same conditions,we also observed a concomitant increase in the gene expression of the EnMT-related transcription factors: slug,snail,zeb1,and endothelin-1. This indicates that mesenchymal phenotype acquisition occurred through an EnMT-like process. Inhibition of TGFβ receptor I (TGFβRI) downregulated snail gene expression,blocked the EnMT,and facilitated the differentiation of EPC to the endothelial cell lineage. Furthermore,TGFβRI inhibition decreased migration of EPC stimulated by SMC without affecting their functionality and adhesion capacity. CONCLUSION: These results indicate that EPC may differentiate into SMC-like cells through an EnMT-like process and that TGFβI plays an important role in the fate of EPC. View Publication

It is known that umbilical cord blood (UCB) is a rich source of stem cells with practical and ethical advantages. Three important types of stem cells which can be harvested from umbilical cord blood and used in disease treatment are hematopoietic stem cells (HSCs),mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). Since these stem cells have shown enormous potential in regenerative medicine,numerous umbilical cord blood banks have been established. In this study,we examined the ability of banked UCB collected to produce three types of stem cells from the same samples with characteristics of HSCs,MSCs and EPCs. We were able to obtain homogeneous plastic rapidly-adherent cells (with characteristics of MSCs),slowly-adherent (with characteristics of EPCs) and non-adherent cells (with characteristics of HSCs) from the mononuclear cell fractions of cryopreserved UCB. Using a protocol of 48 h supernatant transferring,we successfully isolated MSCs which expressed CD13,CD44 and CD90 while CD34,CD45 and CD133 negative,had typical fibroblast-like shape,and was able to differentiate into adipocytes; EPCs which were CD34,and CD90 positive,CD13,CD44,CD45 and CD133 negative,adherent with cobble-like shape; HSCs which formed colonies when cultured in MethoCult medium. View Publication