Sakamoto T, Ishibashi T, Kimura H, Yoshikawa H, Spee C, Harris M S, Hinton D R, Ryan S J
Doheny Eye Institute, University of Southern California School of Medicine, Los Angeles 90033, USA.
Invest Ophthalmol Vis Sci. 1995 May;36(6):1076-83.
To examine the possible inhibitory effect of tecogalan sodium, derived from bacteria, on three important components of in vitro angiogenesis (endothelial proliferation, migration, and tube formation in a collagen gel) using bovine choroidal endothelial cells (CECs).
The effects of tecogalan sodium (1, 5, 25, 125, and 250 micrograms/ml) on cultured CECs were examined when basic fibroblast growth factor (bFGF, 10 ng/ml), vascular endothelial growth factor (VEGF, 50 ng/ml), a combination of bFGF (10 ng/ml) and VEGF (50 ng/ml) (bFGF/VEGF) and 10% fetal calf serum (FCS) were used as angiogenic stimulants. For the proliferation assay, CECs were cultured and the cell numbers counted on days 1, 3, and 5. For migration assay, CECs were seeded in the upper half of a Boyden chamber while an angiogenic growth factor was loaded in the lower half. After 6 hours of incubation, cell migration was evaluated by counting the numbers of migrated cells per microscopic field on the lower side of the filter. For the tube-forming assay, CECs were seeded in a type I collagen gel, and the length of the tube-like structures (an indicator of angiogenesis) formed by CECs per microscopic field was quantified by image analysis. The effect of neutralizing antibody for bFGF also was tested in these three assays.
All tested angiogenic stimulants induced CEC proliferation. The stimulatory effect of bFGF and bFGF/VEGF was reduced by tecogalan sodium (IC50 for bFGF effect, 26.1 micrograms/ml). However, the effect of VEGF and of 10% FCS was not altered by low doses of tecogalan sodium (< 25 micrograms/ml). Chemotaxis of CECs was stimulated by bFGF alone and by bFGF/VEGF, and this effect was inhibited by tecogalan sodium (IC50 for bFGF, 3.2 micrograms/ml). Stimulation of chemotaxis by VEGF alone and by 10% FCS was not affected by tecogalan sodium in low doses but was inhibited by high doses. Tube formation was stimulated by administration of each of the factors. Stimulation of tube formation by bFGF and by bFGF/VEGF was inhibited by tecogalan sodium (IC50 for bFGF, 18.2 micrograms/ml). High doses of tecogalan sodium (125 and 250 micrograms/ml) also inhibited 10% FCS-induced proliferation, migration, and tube formation.
bFGF, VEGF, and a combination of bFGF and VEGF stimulated proliferation, migration, and tube formation by CECs in vitro. These stimulatory effects, but especially those of bFGF, were inhibited by tecogalan sodium. If tecogalan sodium can be shown to have a similar effect in vivo, it might have the potential for pharmacologic control of subretinal neovascularization.
使用牛脉络膜内皮细胞（CECs）研究源自细菌的替考加兰钠对体外血管生成的三个重要组成部分（内皮细胞增殖、迁移以及在胶原凝胶中形成管腔）的潜在抑制作用。
当使用碱性成纤维细胞生长因子（bFGF，10 ng/ml）、血管内皮生长因子（VEGF，50 ng/ml）、bFGF（10 ng/ml）与VEGF（50 ng/ml）的组合（bFGF/VEGF）以及10%胎牛血清（FCS）作为血管生成刺激剂时，检测替考加兰钠（1、5、25、125和250微克/毫升）对培养的CECs的影响。对于增殖试验，培养CECs并在第1、3和5天计数细胞数量。对于迁移试验，将CECs接种在博伊登小室的上半部分，而在下半部分加载血管生成生长因子。孵育6小时后，通过计数滤膜下侧每个显微镜视野中迁移的细胞数量来评估细胞迁移。对于管腔形成试验，将CECs接种在I型胶原凝胶中，通过图像分析量化每个显微镜视野中CECs形成的管状结构的长度（血管生成的指标）。在这三种试验中还测试了bFGF中和抗体的作用。
所有测试的血管生成刺激剂均诱导CECs增殖。替考加兰钠降低了bFGF和bFGF/VEGF的刺激作用（bFGF作用的IC50为26.1微克/毫升）。然而，低剂量的替考加兰钠（<25微克/毫升）并未改变VEGF和10% FCS 的作用。单独的bFGF以及bFGF/VEGF刺激CECs的趋化性，且这种作用被替考加兰钠抑制（bFGF的IC50为3.2微克/毫升）。单独的VEGF以及10% FCS刺激的趋化性在低剂量时不受替考加兰钠影响，但在高剂量时受到抑制。每种因子的给药均刺激管腔形成。替考加兰钠抑制了bFGF和bFGF/VEGF刺激的管腔形成（bFGF 的IC50为18.2微克/毫升）。高剂量的替考加兰钠（125和250微克/毫升）也抑制了10% FCS诱导的增殖、迁移和管腔形成。
bFGF、VEGF以及bFGF与VEGF的组合在体外刺激CECs的增殖、迁移和管腔形成。这些刺激作用，尤其是bFGF的作用，被替考加兰钠抑制。如果替考加兰钠在体内能显示出类似的作用，它可能具有药理控制视网膜下新生血管形成的潜力。
