Villaschi S, Nicosia R F
Department of Pathology, Medical College of Pennsylvania, Philadelphia.
Lab Invest. 1994 Aug;71(2):291-9.
Previous data suggest that paracrine interactions between fibroblasts and endothelial cells modulate the formation of granulation tissue during wound healing. The study of these interactions in vivo is hindered by the interference of serum and other cell types. To overcome this limitation, we developed a serum-free in vitro model in which microvessels were cocultured with fibroblasts in a 3-dimensional collagen gel.
Microvascular networks were obtained by culturing rat aortic endothelial cells between two layers of collagen. Microvessels were cultured in serum-free medium with or without fibroblasts embedded in the collagen. The cultures were studied by phase contrast microscopy, conventional electron microscopy, and by light and electron immunohistochemistry. The role of endothelial cells and the endothelial-derived peptide endothelin-1 in the transformation of fibroblasts into myofibroblasts was studied by evaluating fibroblast alpha-smooth muscle actin expression and collagen contraction. The angiogenic properties of fibroblasts were investigated with the rat aorta assay.
Microvessels cultured without fibroblasts degenerated within 3 to 4 days. Fibroblasts stabilized the microvessels greatly prolonging their life span. This effect was associated with an increased deposition of subendothelial extracellular matrix. Both fibroblasts and fibroblast-conditioned medium stimulated angiogenesis in the rat aorta assay. Endothelial cells and endothelin-1 enhanced the expression of alpha-smooth muscle actin in fibroblasts and stimulated fibroblast-mediated collagen contraction.
This study demonstrates that endothelial-fibroblast interactions can be studied in vitro under serum-free conditions. Our results suggest that paracrine mechanisms operating between endothelial cells and fibroblasts play a key role in the formation and contraction of granulation tissue during wound healing. We propose that fibroblasts stimulate angiogenesis and stabilize the neovascular endothelium which in turn promotes the morphological and functional transformation of fibroblasts into myofibroblasts.
先前的数据表明，成纤维细胞与内皮细胞之间的旁分泌相互作用在伤口愈合过程中调节肉芽组织的形成。血清和其他细胞类型的干扰阻碍了对这些相互作用的体内研究。为克服这一限制，我们开发了一种无血清体外模型，其中微血管在三维胶原凝胶中与成纤维细胞共培养。
通过在两层胶原之间培养大鼠主动脉内皮细胞获得微血管网络。微血管在无血清培养基中培养，培养基中含有或不含有包埋在胶原中的成纤维细胞。通过相差显微镜、传统电子显微镜以及光镜和电镜免疫组织化学对培养物进行研究。通过评估成纤维细胞α-平滑肌肌动蛋白表达和胶原收缩，研究内皮细胞和内皮衍生肽内皮素-1在成纤维细胞向肌成纤维细胞转化中的作用。用大鼠主动脉试验研究成纤维细胞的血管生成特性。
在没有成纤维细胞的情况下培养的微血管在3至4天内退化。成纤维细胞使微血管稳定，大大延长了它们的寿命。这种作用与内皮下细胞外基质沉积增加有关。成纤维细胞和成纤维细胞条件培养基在大鼠主动脉试验中均刺激血管生成。内皮细胞和内皮素-1增强了成纤维细胞中α-平滑肌肌动蛋白的表达，并刺激了成纤维细胞介导的胶原收缩。
本研究表明，内皮细胞与成纤维细胞之间的相互作用可在无血清条件下进行体外研究。我们的结果表明，内皮细胞与成纤维细胞之间的旁分泌机制在伤口愈合过程中肉芽组织的形成和收缩中起关键作用。我们提出，成纤维细胞刺激血管生成并稳定新生血管内皮，而新生血管内皮反过来促进成纤维细胞向肌成纤维细胞的形态和功能转化。
