CAPILLARY MORPHOGENESIS DURING HUMAN ENDOTHELIAL CELL INVASION OF THREE-DIMENSIONAL COLLAGEN MATRICES

Endothelial cells are subjected to biochemical and mechanical stimuli, which regulate their angiogenic potential. We determined the synergistic effects of sphingosine-1-phosphate (S1P) and fluid wall shear stress (WSS) on a previously established model of human umbilical vein endothelial cell invasion into three-dimensional collagen matrices. Collagen matrices were incorporated into a parallel-plate flow chamber to apply controlled WSS to the surface of endothelial monolayers over a period of 24 h. Cell invasion required the presence of S1P, with the effects of S1P being enhanced by shear stress to an extent comparable with S1P combined with angiogenic growth factor stimulation. The number of invading cells depended on the magnitude of shear stress, with a maximal induction at a shear stress of ∼5 dyn/cm2, whereas the invasion distance was proportional to the magnitude of shear stress. The enhancement of invasion by 5.3 dyn/cm2 shear stress coincided with elevated phosphorylation of Akt and matrix metalloproteinase (MMP)-2 activation. Furthermore, invasion induced by the combined application of WSS and S1P was attenuated by inhibitors of MMPs (GM6001) and the phosphatidylinositol 3-kinase/Akt signaling pathway (wortmannin). These results provide evidence that shear stress is a positive modulator of S1P-induced endothelial cell invasion into collagen matrices through enhanced Akt and MMP-2 activation.

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Matrix metalloproteinase-1 and -9 activation by plasmin regulates a novel endothelial cell-mediated mechanism of collagen gel contraction and capillary tube regression in three-dimensional collagen matrices

Journal of Cell Science ◽

10.1242/jcs.114.5.917 ◽

2001 ◽

Vol 114 (5) ◽

pp. 917-930 ◽

Cited By ~ 2

Author(s):

G.E. Davis ◽

K.A. Pintar ◽

Allen, R. Salazar ◽

S.A. Maxwell

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Endothelial Cell ◽

Three Dimensional ◽

Collagen Gel ◽

Plasminogen Activators ◽

Collagen Matrices ◽

Collagen Gel Contraction ◽

Blocking Antibodies ◽

Pai 1

Here, we describe a new function for plasmin and matrix metalloproteinases (MMPs), which is to regulate the regression of capillary tubes in three-dimensional extracellular matrix environments. Using a well-described capillary morphogenesis system in three-dimensional collagen matrices, a new model of capillary regression has been established by adding plasminogen to the culture medium. Plasminogen is converted to plasmin by endothelial cell plasminogen activators which then induces matrix metalloproteinase-dependent collagen gel contraction and capillary regression. Plasminogen addition results in activation of MMP-1 and MMP-9, which then results in collagen proteolysis followed by capillary regression. The endothelial cells undergo apoptosis following gel contraction as detected by flow cytometric analysis as well as by detectable caspase-3 cleavage and caspase-dependent cleavage of the actin cytoskeletal regulatory protein, gelsolin. In addition, directly correlating with the contraction response, tyrosine phosphorylation of p130cas, an adapter protein in the focal adhesion complex, is observed followed by disappearance of the protein. Proteinase inhibitors that block MMPs (TIMP-1 or TIMP-2), plasminogen activators (PAI-1) or plasmin (aprotinin) completely block the gel contraction and regression process. In addition, chemical inhibitors of MMPs that block capillary regression also block MMP-1 and MMP-9 activation suggesting that a key element in this regression response is the molecular control of MMP activation by endothelial cells. Blocking antibodies directed to MMP-1 or MMP-9 interfere with capillary regression while blocking antibodies directed to PAI-1 accelerate capillary regression suggesting that endogenous synthesis of PAI-1 negatively regulates this process. These data present a novel system to study a new mechanism that may regulate regression of capillary tubes, namely, plasmin and MMP-mediated degradation of extracellular matrix.

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