Le Ricousse-Roussanne Sophie, Barateau Véronique, Contreres Jean-Olivier, Boval Bernadette, Kraus-Berthier Laurence, Tobelem Gérard
Institut des Vaisseaux et du Sang, Centre de Recherche de l'association Claude Bernard, Hôpital Lariboisière, 8 rue Guy Patin, 75475 Paris Cedex 10, France.
Cardiovasc Res. 2004 Apr 1;62(1):176-84. doi: 10.1016/j.cardiores.2004.01.017.
Recent studies have provided increasing evidence that postnatal neovascularization does not rely exclusively on sprouting of preexisting vessels, but also involves bone marrow-derived circulating endothelial precursors (BM-EPCs). Animal studies revealed that neovascularization of ischemic tissue can be enhanced by BM-EPCs transplantation. But a possible limitation to the use of vascular precursors for therapeutic angiogenesis is the relatively low number of these cells. In this study, we demonstrate that ex vivo expanded differentiated endothelial cells (ECs) and smooth muscle cells (SMCs), may home to the tumor vasculature allowing targeting of transgene expression to the neoangiogenic site.
Mononuclear cells (MNCs) or CD34+ -enriched cells were purified from cord blood. We have defined culture conditions in which we observed two types of clones easily differentiated according to their morphology: cobblestone or spindle-shaped. Phenotypic characterization was assessed by immunocytochemistry, flow cytometry analysis and polymerase reaction with reverse transcription. Formation of capillary-like network in vitro was studied in three-dimensional collagen culture. And recruitment of these cells to a tumoral neoangiogenic site was assessed into tumor-bearing Severe Combined Immunodeficient (SCID) mouse model.
The cobblestone cells uniformly positive for CD31, VE-cadherin, vWF, VEGF R1 and R2, ecNOS and incorporating acetylated LDL were ECs. Spindle-shaped cells expressed alpha-smooth muscle actin (alpha-SMA), Smooth Muscle Heavy Chain (SMHC), SM22 and calponin. They also displayed a carbachol-induced contractility in a medium containing IGF1. So we concluded that spindle-shaped cells were SMCs. ECs and SMCs interacted with each other to form a capillary like network in three-dimensional type I collagen culture. Moreover, these ex vivo differentiated cells are able to home to the tumor vasculature.
We provide evidence that progenitors for ECs and SMCs circulate in human cord blood and differentiate into functional ECs and SMCs. These differentiated cells could provide a biomaterial for vascular cell therapy, because of their homing capacity to the neovascularization site.
最近的研究提供了越来越多的证据表明，出生后新血管形成并非仅依赖于已有血管的芽生，还涉及骨髓来源的循环内皮前体细胞（BM-EPCs）。动物研究显示，BM-EPCs移植可增强缺血组织的新血管形成。但是，使用血管前体细胞进行治疗性血管生成的一个可能限制是这些细胞数量相对较少。在本研究中，我们证明了体外扩增的分化内皮细胞（ECs）和平滑肌细胞（SMCs）可能归巢至肿瘤血管系统，从而使转基因表达靶向至新生血管生成部位。
从脐带血中纯化单核细胞（MNCs）或富集CD34+的细胞。我们确定了培养条件，在此条件下我们观察到两种易于根据形态区分的克隆类型：鹅卵石样或纺锤形。通过免疫细胞化学、流式细胞术分析和逆转录聚合酶反应评估表型特征。在三维胶原培养中研究体外毛细血管样网络的形成。并将这些细胞募集至荷瘤重症联合免疫缺陷（SCID）小鼠模型的肿瘤新生血管生成部位进行评估。
鹅卵石样细胞CD31、VE-钙黏蛋白、vWF、VEGF R1和R2、内皮型一氧化氮合酶（ecNOS）均呈阳性，并摄取乙酰化低密度脂蛋白（LDL），这些细胞为ECs。纺锤形细胞表达α-平滑肌肌动蛋白（α-SMA）、平滑肌重链（SMHC）、SM22和钙调蛋白。它们在含有胰岛素样生长因子1（IGF1）的培养基中也表现出卡巴胆碱诱导的收缩性。因此我们得出结论，纺锤形细胞为SMCs。ECs和SMCs相互作用，在三维I型胶原培养中形成毛细血管样网络。此外，这些体外分化的细胞能够归巢至肿瘤血管系统。
我们提供证据表明，ECs和SMCs的祖细胞在人脐带血中循环，并分化为功能性ECs和SMCs。由于这些分化细胞具有归巢至新生血管生成部位的能力，它们可为血管细胞治疗提供一种生物材料。
