Laboratório de Ultra-Estrutura Celular, Rio de Janeiro, Brazil.
Exp Cell Res. 2009 Nov 15;315(19):3406-18. doi: 10.1016/j.yexcr.2009.09.016. Epub 2009 Sep 19.
In embryogenesis, coronary blood vessels are formed by vasculogenesis from epicardium-derived progenitors. Subsequently, growing or regenerating myocardium increases its vasculature by angiogenesis, forming new vessels from the pre-existing ones. Recently, cell therapies for myocardium ischemia that used different protocols have given promising results, using either extra-cardiac blood vessel cell progenitors or stimulating the cardiac angiogenesis. We have questioned whether cardiomyocytes could sustain both vasculogenesis and angiogenesis. We used a 3D culture model of tissue-like spheroids in co-cultures of cardiomyocytes supplemented either with endothelial cells or with bone marrow-derived mesenchymal stroma cells. Murine foetal cardiomyocytes introduced into non-adherent U-wells formed 3D contractile structures. They were coupled by gap junctions. Cardiomyocytes segregated inside the 3D structure into clumps separated by connective tissue septa, rich in fibronectin. Three vascular endothelial growth factor isoforms were produced (VEGF 120, 164 and 188). When co-cultured with human umbilical cord endothelial cells, vascular structures were produced in fibronectin-rich external layer and in radial septa, followed by angiogenic sprouting into the cardiomyocyte microtissue. Presence of vascular structures led to the maintenance of long-term survival and contractile capacity of cardiac microtissues. Conversely, bone marrow mesenchymal cells formed isolated cell aggregates, which progressively expressed the endothelial markers von Willebrand's antigen and CD31. They proceeded to typical vasculogenesis forming new blood vessels organised in radial pattern. Our results indicate that the in vitro 3D model of cardiomyocyte spheroids provides the two basic elements for formation of new blood vessels: fibronectin and VEGF. Within the myocardial environment, endothelial and mesenchymal cells can proceed to formation of new blood vessels either through angiogenesis or vasculogenesis, respectively.
在胚胎发生过程中，冠状动脉血管通过心脏外胚层来源的祖细胞的血管发生形成。随后，生长或再生的心肌通过血管生成增加其脉管系统，从预先存在的血管形成新的血管。最近，使用不同方案的用于心肌缺血的细胞治疗已经取得了有希望的结果，使用心脏外血管细胞祖细胞或刺激心脏血管生成。我们质疑心肌细胞是否能够维持血管发生和血管生成。我们使用组织样球体的 3D 培养模型，在补充内皮细胞或骨髓间充质基质细胞的心肌细胞共培养物中进行。引入非附着 U 孔的鼠胎心肌细胞形成 3D 收缩结构。它们通过缝隙连接连接。心肌细胞在 3D 结构内分离成由富含纤维连接蛋白的结缔组织隔分开的团块。产生了三种血管内皮生长因子同工型（VEGF120、164 和 188）。当与人脐带内皮细胞共培养时，血管结构在富含纤维连接蛋白的外层和放射状隔中产生，随后血管生成芽进入心肌细胞微组织中。血管结构的存在导致心脏微组织的长期存活和收缩能力得以维持。相反，骨髓间充质细胞形成孤立的细胞聚集物，这些细胞逐渐表达血管内皮标志物 von Willebrand 抗原和 CD31。它们开始形成新的血管，呈放射状排列，形成典型的血管发生。我们的结果表明，心肌细胞球体的体外 3D 模型提供了形成新血管的两个基本要素：纤维连接蛋白和 VEGF。在心肌环境中，内皮细胞和间充质细胞可以分别通过血管生成或血管发生形成新的血管。
