Analysis of the biological function of vascular smooth muscle cells regulated by fibrinolytic factors

纤溶因子调控血管平滑肌细胞生物学功能分析

• 批准号: 11670054
• 负责人: MATSUO Osamu
• 金额: $ 2.24万
• 依托单位: Department of Physiology, Kinki University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670054/
• 关键词: Fibrinolytic system; vascular smooth muscle cells; gene targeting mouse; t-PA; u-PA; PAI-1; u-PAR; carcinoma cells; uーPA; ノックアウトマウス

Vascular smooth muscle cells (SMC) were successfully isolated from thoracic aorta of normal and fibrinolytic factor gene deficient mice. The wild-type SMC (WT/SMC) and the four SMC cultures, lacking urokinase-type plasminogen activator (u-PA^<-/->/SMC), tissue-type plasminogen activator (t-PA^<-/->/SMC), type 1 plasminogen activator inhibitor (PAI-1^<-/->/SMC), and u-PA receptor (u-PAR^<-/->/SMC) were employed to analyze their proliferative activities in the presence of mouse melanoma cells (B16). The growth rates of u-PA^<-/->/SMC, t-PA^<-/->/SMC, and PAI-1^<-/->/SMC as well as WT/SMC were not changed when they were co-cultured with B16 (mixed culture and two-chamber culture) in the presence of 10% fetal calf serum (FCS). On the other hand, the FCS-free conditioned medium of confluent B16 promoted the growth of these SMC to the same extent (〜200%) each of which was associated with both increased tyrosine phosphorylation of 77-kDa protein (7.5〜11-fold) and mitogen-activated protein kinase (MAPK) activity (2-fold). In contrast, only the growth of u-PAR^<-/->/SMC was arrested in these co-cultures. The B16 conditioned medium also suppressed the growth of the SMC of which the phosphorylation of 77- kDa protein and MAPK activity were not altered. These results indicate that these SMC were stimulated by B16-derived growth factor-like substance (s) and that the expressions of u-PA, t-PA and PAI-1 were not involved in these events. However, it is suggested that u-PAR plays an important role in the growth mechanism possibly by forming a functional unit with integrins. Thus, one of the biological responses of SMC to carcinoma cells may be the defense by increased growth of cell-mass against the metastatic process of carcinoma cells at the vascular wall.

从正常小鼠和纤溶因子基因缺陷小鼠胸主动脉中成功分离出血管平滑肌细胞。采用野生型SMC （WT/SMC）和4种缺乏尿激酶型纤溶酶原激活剂（u-PA^<-/->/SMC）、组织型纤溶酶原激活剂（t-PA^<-/->/SMC）、1型纤溶酶原激活剂抑制剂（PAI-1^<-/->/SMC）和u-PA受体（u-PAR^<-/->/SMC）的SMC培养物，分析了它们在小鼠黑色素瘤细胞（B16）存在下的增殖活性。在10%胎牛血清（FCS）的条件下，u-PA^<-/->/SMC、t-PA^<-/->/SMC、PAI-1^<-/->/SMC和WT/SMC与B16共培养（混合培养和双室培养）时，其生长率均无变化。另一方面，无fcs的confluent B16条件培养基对这些SMC的生长促进程度相同（~ 200%），每一个都与77-kDa蛋白酪氨酸磷酸化增加（7.5 ~ 11倍）和丝裂原活化蛋白激酶（MAPK）活性增加（2倍）有关。相比之下，在这些共培养中，只有u-PAR^<-/->/SMC的生长被阻止。B16条件培养基也抑制了77- kDa蛋白磷酸化和MAPK活性未发生变化的SMC的生长。这些结果表明，这些SMC受到b16衍生的生长因子样物质(s)的刺激，而u-PA、t-PA和PAI-1的表达不参与这些事件。然而，这表明u-PAR可能通过与整合素形成功能单元在生长机制中发挥重要作用。因此，SMC对癌细胞的生物学反应之一可能是通过增加细胞团的生长来防御癌细胞在血管壁的转移过程。

项目成果

Fukao, H., Ueshima, S., Okada, K.and Matsuo, O.: "Biological properties of cultured vascular smooth muscle cells from fibrinolytic factor gene deficient mice."Thrombosis and Haemostasis. (submitted).

Fukao, H.、Ueshima, S.、Okada, K. 和 Matsuo, O.：“纤溶因子基因缺陷小鼠培养的血管平滑肌细胞的生物学特性。”血栓形成和止血。

Fukao, H., Ueshima, S., Okada, K.and Matsuo, O.: "Cross-talk of murine vascular smooth muscle cells with carcinoma cells in culture."Cell Structure and Function. (submitted).

Fukao, H.、Ueshima, S.、Okada, K. 和 Matsuo, O.：“小鼠血管平滑肌细胞与培养物中癌细胞的串扰。”细胞结构和功能。

Fukao, H., Ueshima, S., Okada, K.and Matsuo, O.: "Effects of mouse melanoma cell line (B16) on the intracellular signal transductions of wild-type and fibrinolytic factor gene deficient mice."Experimental Cell Research. (submitted).

Fukao, H.、Ueshima, S.、Okada, K. 和 Matsuo, O.：“小鼠黑色素瘤细胞系 (B16) 对野生型和纤溶因子基因缺陷型小鼠细胞内信号转导的影响。”实验细胞研究