Live-cell imaging demonstrates extracellular matrix degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation.
Live-cell imaging demonstrates extracellular matrix degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation.
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DOI：
10.1016/j.yexcr.2009.01.021
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发表时间：
2009-04-15
影响因子：
3.7
通讯作者：
Sloane BF
中科院分区：
文献类型：
--
作者：
Cavallo-Medved D;Rudy D;Blum G;Bogyo M;Caglic D;Sloane BF
蛋白水解酶在内皮细胞表面的定位和细胞外基质(ECM)的重塑是内皮细胞管形成和血管生成所必需的。在这里，我们将活性组织蛋白酶B及其细胞表面结合伙伴膜联蛋白II异四聚体(AIIt)的S100A/p11(P11)部分定位于人脐静脉内皮细胞(HUVEC)的小窝。通过活细胞蛋白分解实验，我们观察到在单层培养或体外成管实验中，猝灭荧光(DQ)蛋白(即明胶和IV型胶原)的降解产物与HUVEC的小凹蛋白-1(Cav-1)共定位于细胞内。活性探针与活性半胱氨酸组织蛋白和DQ-IV型胶原降解产物共价结合，部分定位于含有Cav-1和活性半胱氨酸组织蛋白的细胞内小泡。生化分析表明，在体外试管形成过程中，活性组织蛋白酶B在腔泡组分中的分布增加。与组织蛋白酶B到ECM降解途径相关联的Pro-uPA、uPAR、基质金属蛋白酶-2和基质金属蛋白酶-14在体外成管过程中也被发现在空泡部分增加。我们的发现首次通过活细胞成像证明了在管子形成过程中内皮细胞小窝中ECM的降解与活跃的组织蛋白酶B有关。
Localization of proteases to the surface of endothelial cells and remodeling of the extracellular matrix (ECM) are essential to endothelial cell tube formation and angiogenesis. Here, we partially localized active cathepsin B and its cell surface binding partners, S100A/p11 (p11) of the annexin II heterotetramer (AIIt), to caveolae of human umbilical vein endothelial cells (HUVEC). Via a live-cell proteolysis assay, we observed that degradation products of quenched-fluorescent (DQ)-proteins (i.e. gelatin and collagen IV) colocalized intracellularly with caveolin-1 (cav-1) of HUVEC grown in either monolayer cultures or in vitro tube formation assays. Activity-based probes that bind covalently to active cysteine cathepsins and degradation products of DQ-collagen IV partially localized to intracellular vesicles that contained cav-1 and active cysteine cathepsins. Biochemical analyses revealed that the distribution of active cathepsin B in caveolar fractions increased during in vitro tube formation. Pro-uPA, uPAR, MMP-2 and MMP-14, which have been linked with cathepsin B to ECM degradation pathways, were also found to increase in caveolar fractions during in vitro tube formation. Our findings are the first to demonstrate through live-cell imaging ECM degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation.
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影响因子：
14.8
作者：
Blum, Galia;von Degenfeld, Georges;Bogyo, Matthew
通讯作者：
Bogyo, Matthew
影响因子：
4.1
作者：
Bernardo, MM;Fridman, R
通讯作者：
Fridman, R
影响因子：
7.8
作者：
Esser, S;Wolburg, K;Wolburg, H;Breier, G;Kurzchalia, T;Risau, W
通讯作者：
Risau, W
影响因子：
3.3
作者：
Gálvez, BG;Matías-Román, S;Arroyo, AG
通讯作者：
Arroyo, AG
影响因子：
4
作者：
Cavallo-Medved, D;Mai, JX;Sloane, BF
通讯作者：
Sloane, BF