FIBRONECTIN, HYALURONAN, AND A HYALURONAN BINDING-PROTEIN CONTRIBUTE TO INCREASED DUCTUS-ARTERIOSUS SMOOTH-MUSCLE CELL-MIGRATION
FIBRONECTIN, HYALURONAN, AND A HYALURONAN BINDING-PROTEIN CONTRIBUTE TO INCREASED DUCTUS-ARTERIOSUS SMOOTH-MUSCLE CELL-MIGRATION
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DOI：
10.1016/0012-1606(91)90074-d
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发表时间：
1991-02-01
影响因子：
2.7
通讯作者：
RABINOVITCH, M
中科院分区：
文献类型：
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作者：
BOUDREAU, N;TURLEY, E;RABINOVITCH, M
妊娠晚期羔羊动脉导管（DA）中发育的“内膜垫”的特征是平滑肌细胞迁移到一个大的内皮下空间。我们先前的体外研究，比较DA细胞与来自主动脉（Ao）的DA细胞，已经显示，即使在妊娠早期，DA内皮细胞将透明质酸掺入内皮下基质中增加了10倍，平滑肌纤维连接蛋白合成增加了2倍，并且响应于内皮条件培养基，硫酸软骨素增加了2倍。为了确定这些细胞外基质成分是否可能在诱导DA平滑肌迁移中发挥作用，我们将DA或Ao平滑肌细胞接种到三维胶原（2.0 mg/ml）凝胶上，并在2、5和8天后评估迁移。8d后，DA细胞侵入凝胶的比例明显高于Ao细胞（23.1 ± 3.1%vs16.2 ± 2.3%P <0.01）。添加GRGDS肽（0.5 mM）或针对纤连蛋白的抗体显着降低DA细胞中的迁移，但对Ao中的迁移没有影响。添加内皮条件培养基诱导平滑肌硫酸软骨素的产生对DA细胞迁移没有影响。然而，凝胶中包含透明质酸（0.5 - 1.5 mg）进一步增强了DA细胞迁移，在1 mg/ml浓度下最大（31.9 ± 3.1%）。海洛宁对主动脉平滑肌细胞迁移无影响。通过向细胞表面透明质酸结合蛋白（HABP）添加抗体（1：100稀释，1 μ g/ml），完全阻断透明质酸促进DA平滑肌细胞培养物迁移的能力。同样，在含有胶原蛋白的凝胶上向细胞中加入抗HABP仅显著降低DA中的迁移，而不是Ao。免疫荧光染色显示，在DA细胞中，HABP更集中在板状伪足和前缘比在Ao细胞。同时，DA平滑肌细胞合成了大量的HABP，如通过使用HABP的多克隆抗血清的Western免疫印迹和免疫沉淀所确定的。因此，我们的研究表明，增加纤连蛋白和HABP有助于增强DA平滑肌细胞的迁移。这些结果，加上我们以前的研究表明，DA内皮基质中的透明质酸积累增加了10倍，这表明了体内DA平滑肌迁移到内皮下基质中的机制。
“Intimal cushions” which develop in the late gestation lamb ductus arteriosus (DA) are characterized by smooth muscle cells migrating into a large subendothelial space. Our previousin vitrostudies, comparing DA cells with those from the aorta (Ao), have shown, even in early gestation, a 10-fold increase in DA endothelial incorporation of hyaluronan into the subendothelial matrix, a 2-fold increase in smooth muscle fibronectin synthesis and, in response to endothelial conditioned medium, a 2-fold increase in chondroitin sulfate. To determine whether these extracellular matrix components may be playing a role in inducing DA smooth muscle migration, we seeded DA or Ao smooth muscle cells onto three-dimensional collagen (2.0 mg/ml) gels and assessed migration 2, 5, and 8 days later. After 8 days, significantly greater numbers of DA compared to Ao cells were found invading the gels (23.1 ± 3.1% vs 16.2 ± 2.3%P< 0.01). Addition of GRGDS peptides (0.5 mM) or antibodies against fibronectin significantly decreased migration in the DA cells, but had no effect on migration in the Ao. Addition of endothelial conditioned medium to induce smooth muscle chondroitin sulfate production had no effect on DA cell migration. Inclusion of hyaluronan in the gel (0.5–1.5 mg), however, further enhanced DA cell migration, being greatest (31.9 ± 3.1%) at a concentration of 1 mg/ml. Hyaluronan was without effect on Ao smooth muscle cell migration. The ability of hyaluronan to promote migration in cultures of DA smooth muscle cells was blocked completely by the addition of antibodies (1:100 dilution, 1 μg/ml) to a cell surface hyaluronan binding protein (HABP). As well, addition of anti-HABP to cells on gels containing collagen only significantly reduced migration in the DA but not the Ao. Immunofluorescent staining revealed that in DA cells, HABP was more concentrated in lamellipodia and leading edges than in Ao cells. As well, DA smooth muscle cells synthesized greater amounts of HABP as determined by Western immunoblotting and immunoprecipitation using polyclonal antisera to HABP. Thus, our studies indicate that both increased fibronectin and HABP contribute to the enhanced migration of DA smooth muscle cells. These results, together with our previous studies showing a 10-fold increase in hyaluronan accumulation in the DA endothelial matrix, would suggest a mechanism for increased DA smooth muscle migration into the subendothelial matrix observedin vivo.