13
1.3.7 Critical Functional Role for MT1-MMP in EC Lumen
and Tube Formation in 3D Collagen Matrices
Matrix metalloproteinases (MMPs) are a family of zinc-dependent metalloendopeptidases that degrade a variety of substrates, to affect the vasculature and other tissues
[33, 47]. Their targets include the ECM, cytokines, and cell surface receptors, to
affect vascularization as well as many other cellular responses [33, 47]. Our work
and that of others have demonstrated a role for MT1-MMP (i.e., MMP-14) in EC
morphogenic events in 3D matrix environments [21, 87, 95]. MT1-MMP is a transmembrane protein, and its cell surface expression is required for it to perform the
localized ECM degradation necessary to control cell movement in 3D matrices.
MT2-MMP is also able to participate in these types of events, while the function of
MT3-MMP is less clear, although several studies show that it does not play a major
role. Mouse knockout of MT1-MMP is compatible with embryogenesis, but the
mice are small and ill and die within a month or two of birth [108]. Attempts to
induce angiogenesis in these mice reveal that these responses do not occur [108].
Furthermore, aortic ring assays in 3D collagen matrices show no sprouting, in either
3D collagen or fibrin matrices using MT1-MMP knockout tissues compared to control [21]. Thus, this work demonstrates an important role for MT1-MMP in vascular
morphogenic events in 3D matrices and during in vivo angiogenic responses
(Fig. 1.4).
To elucidate the molecular mechanisms by which MT1-MMP controls vascular
morphogenesis as well as cellular invasive events, our laboratory has examined this
question using models of vasculogenesis and angiogenesis [9, 87, 95]. We have
utilized protein and chemical MMP inhibitors as well as siRNA suppression
approaches. In our first studies, we demonstrated that EC sprouting in response to
sphingosine-1-phosphate (incorporated into the collagen matrices) was blocked by
the broad spectrum inhibitor, GM6001, as well as tissue inhibitor of metalloproteinase (TIMP)-2, TIMP-3, and TIMP-4, but not TIMP-1 (Fig. 1.4a) [9]. Interestingly,
MT-MMPs are insensitive to TIMP-1, but the other inhibitors utilized block their
activity. When EC lumen and tube formation assays were performed, GM6001,
TIMP-2, TIMP-3, and TIMP-4 blocked completely, while TIMP-1 had no influence.
One important distinction among the TIMPs is that TIMP-3 is able to block the
activity of MT-MMPs, but also many other members of the ADAM family of cell
surface expressed metalloproteinases [7]. To functionally dissect which EC surface
expressed metalloproteinases are relevant during EC sprouting and lumen formation, we performed siRNA suppression analysis. Our results suggest that the dominant metalloproteinase controlling these events is MT1-MMP (Fig. 1.4b), with a
lesser influence of MT2-MMP during both sprouting and lumen formation [87, 95].
We observed a partial blocking effect of ADAM-15 siRNA knockdown in EC
sprouting assays, using stromal-derived factor-1α as the invasion stimulus [87]. We
did not observe an effect of either MT3-MMP or ADAM-17 siRNAs in our assays
[87], although a recent study using a similar system revealed a potential role for
ADAM-17 in modulating the invasion response [69]. The fact that TIMP-2 and
1 Molecular Control of Capillary Tube Morphogenesis and Maturation Through…
1.3.7 Critical Functional Role for MT1-MMP in EC Lumen
and Tube Formation in 3D Collagen Matrices
Matrix metalloproteinases (MMPs) are a family of zinc-dependent metalloendopeptidases that degrade a variety of substrates, to affect the vasculature and other tissues
[33, 47]. Their targets include the ECM, cytokines, and cell surface receptors, to
affect vascularization as well as many other cellular responses [33, 47]. Our work
and that of others have demonstrated a role for MT1-MMP (i.e., MMP-14) in EC
morphogenic events in 3D matrix environments [21, 87, 95]. MT1-MMP is a transmembrane protein, and its cell surface expression is required for it to perform the
localized ECM degradation necessary to control cell movement in 3D matrices.
MT2-MMP is also able to participate in these types of events, while the function of
MT3-MMP is less clear, although several studies show that it does not play a major
role. Mouse knockout of MT1-MMP is compatible with embryogenesis, but the
mice are small and ill and die within a month or two of birth [108]. Attempts to
induce angiogenesis in these mice reveal that these responses do not occur [108].
Furthermore, aortic ring assays in 3D collagen matrices show no sprouting, in either
3D collagen or fibrin matrices using MT1-MMP knockout tissues compared to control [21]. Thus, this work demonstrates an important role for MT1-MMP in vascular
morphogenic events in 3D matrices and during in vivo angiogenic responses
(Fig. 1.4).
To elucidate the molecular mechanisms by which MT1-MMP controls vascular
morphogenesis as well as cellular invasive events, our laboratory has examined this
question using models of vasculogenesis and angiogenesis [9, 87, 95]. We have
utilized protein and chemical MMP inhibitors as well as siRNA suppression
approaches. In our first studies, we demonstrated that EC sprouting in response to
sphingosine-1-phosphate (incorporated into the collagen matrices) was blocked by
the broad spectrum inhibitor, GM6001, as well as tissue inhibitor of metalloproteinase (TIMP)-2, TIMP-3, and TIMP-4, but not TIMP-1 (Fig. 1.4a) [9]. Interestingly,
MT-MMPs are insensitive to TIMP-1, but the other inhibitors utilized block their
activity. When EC lumen and tube formation assays were performed, GM6001,
TIMP-2, TIMP-3, and TIMP-4 blocked completely, while TIMP-1 had no influence.
One important distinction among the TIMPs is that TIMP-3 is able to block the
activity of MT-MMPs, but also many other members of the ADAM family of cell
surface expressed metalloproteinases [7]. To functionally dissect which EC surface
expressed metalloproteinases are relevant during EC sprouting and lumen formation, we performed siRNA suppression analysis. Our results suggest that the dominant metalloproteinase controlling these events is MT1-MMP (Fig. 1.4b), with a
lesser influence of MT2-MMP during both sprouting and lumen formation [87, 95].
We observed a partial blocking effect of ADAM-15 siRNA knockdown in EC
sprouting assays, using stromal-derived factor-1α as the invasion stimulus [87]. We
did not observe an effect of either MT3-MMP or ADAM-17 siRNAs in our assays
[87], although a recent study using a similar system revealed a potential role for
ADAM-17 in modulating the invasion response [69]. The fact that TIMP-2 and
1 Molecular Control of Capillary Tube Morphogenesis and Maturation Through…