Investigating Metalloproteinases MMP-2 and MMP-9 Mechanosensitivity to Feedback Loops Involved in the Regulation of In Vitro Angiogenesis by Endogenous Mechanical Stresses

Abstract

Angiogenesis is a complex morphogenetic process regulated by growth factors, but also by the force balance between endothelial cells (EC) traction stresses and extracellular matrix (ECM) viscoelastic resistance. Studies conducted with in vitro angiogenesis assays demonstrated that decreasing ECM stiffness triggers an angiogenic switch that promotes organization of EC into tubular cords or pseudo-capillaries. Thus, mechano-sensitivity of EC with regard to proteases secretion, and notably matrix metalloproteinases (MMPs), should likely play a pivotal role in this switching mechanism. While most studies analysing strain regulation of MMPs used cell cultured on stretched membranes, this work focuses on MMP expression during self-assembly of EC into capillary-like structures within fibrin gels, i.e. on conditions that mimics more closely the in vivo cellular mechanical microenvironment. The activity of MMP-2 and MMP-9, two MMPs that have a pivotal role in capillaries formation, has been monitored in pace with the progressive elongation of EAhy926 cells that takes place during the emergence of cellular cords. We found an increase of the zymogen proMMP-2 that correlates with the initial stages of EC cords formation. However, MMP-2 was not detected. ProMMP-9 secretion decreased, with levels of MMP-9 kept at a rather low value. In order to analyse more precisely the observed differences of EAhy926 response on fibrin and plastic substrates, we proposed a theoretical model of the mechano-regulation of proMMP-2 activation in the presence of type 2 tissue inhibitor of MMPs (TIMP-2). Using association/dissociation rates experimentally reported for this enzymatic network, the model adequately describes the synergism of proMMP-2 and TIMP-2 strain activation during pseudo-capillary morphogenesis. All together, these results provide a first step toward a systems biology approach of angiogenesis mechano-regulation by cell-generated extracellular stresses and strains.

Blockade of Apoptosis Signal-Regulating Kinase 1 Attenuates Matrix Metalloproteinase 9 Activity in Brain Endothelial Cells and the Subsequent Apoptosis in Neurons after Ischemic Injury

“…The MMP-9 that is secreted from endothelial cells plays pivotal roles in BBB disruption, endothelial cell morphogenesis, and capillary formation (Dong et al, 2009; Dao Thi et al, 2012). Thus, we performed MMP-9 activity assay in cell media and cultured endothelial cells after 6 h (hypoxia)/24 h (reperfusion; Figures 3A,B ).…”

Angiogenic responses are enhanced in mechanically and microscopically characterized, microbial transglutaminase crosslinked collagen matrices with increased stiffness

“…For example, cell traction magnitude and focal adhesion size have been shown to depend on ECM stiffness [81], and cell traction has been shown to be necessary for focal adhesion growth and maintenance [82]. More frequent sprout invasion in stiffer matrices may result from higher VEGFR expression, which is governed by two antagonistic transcription factors, TFII-1 and GATA2, of a mechanosensitive pathway [83] and from mechanosensitive protease secretion [84]. Cell–ECM traction modulates the tension in cell–cell contacts [85] and the size of cell–cell junctions [86].…”

Extracellular matrix compression temporally regulates microvascular angiogenesis

“…Many of these individual processes are known to be responsive to mechanical stimuli. For example, key molecular regulators of tip cell selection have recently been identified as mechanosensitive ( 5 ), and expression and secretion of proteases that are involved in angiogenesis are also mechanosensitive ( 6 ). Intracellular mechanotransducers, such as YAP and TAZ, also regulate a number of processes crucial to angiogenesis, including vascular endothelial growth factor (VEGF) signaling ( 7 ), cell migration ( 8 ), proliferation, and cell-cell junction formation ( 9 ).…”