Modular control of endothelial sheet migration

Philip Vitorino and Tobias Meyer

Genes & Dev. 2008. 22: 3268-3281 doi: 10.1101/gad.1725808

Growth factor-induced migration of endothelial cell monolayers enables embryonic development, wound healing, and angiogenesis. Although collective migration is widespread and therapeutically relevant, the underlying mechanism by which cell monolayers respond to growth factor, sense directional signals, induce motility, and coordinate individual cell movements is only partially understood. Here we used RNAi to identify 100 regulatory proteins that enhance or suppress endothelial sheet migration into cell-free space. We measured multiple live-cell migration parameters for all siRNA perturbations and found that each targeted protein primarily regulates one of four functional outputs: cell motility, directed migration, cell–cell coordination, or cell density. We demonstrate that cell motility regulators drive random, growth factor-independent motility in the presence or absence of open space. In contrast, directed migration regulators selectively transduce growth factor signals to direct cells along the monolayer boundary toward open space. Lastly, we found that regulators of cell–cell coordination are growth factor-independent and reorient randomly migrating cells inside the sheet when boundary cells begin to migrate. Thus, cells transition from random to collective migration through a modular control system, whereby growth factor signals convert boundary cells into pioneers, while cells inside the monolayer reorient and follow pioneers through growth factor-independent migration and cell–cell coordination.

Molecular components that direct sheet migration into cell-free space

The existence of a directed migration control module (Fig. 3E) argues strongly against a simple diffusive model for sheet migration whereby cells move randomly into unobstructed space. Because FGFR1 was identified as a member of this module, we compared the effect of titrating FGF concentration on random sheet migration against directed migration into cell-free space. While the cell velocity within the sheet was nearly constant in the absence or presence of FGF (Fig. 4A), the same increase in FGF caused a 10-fold increase in sheet migration. To investigate how FGF directs cells during sheet migration, we tracked individual cells within a migrating sheet. As expected, in regions distal to the open edge, cells moved randomly with similar speeds in both serum-starved and FGF treated cells. Near the sheet boundary, however, FGF-treated cells persistently migrate toward the cell-free space (Fig. 4B). To quantify this behavior, we counted the fraction of time cells spend migrating toward the monolayer edge (plus or minus 45°) and plotted the average value as a function of cell dis- tance from the open edge (Fig. 4C). In this analysis, a value of 0.25 reflects a random orientation since a 90° window represents one-quarter of a circle. Addition of FGF caused a significant increase in directed movement throughout the cell monolayer that was maximal near the sheet boundary, dropping off gradually at positions further from the edge. In contrast, serum-starved cells showed weaker orientation overall but maintained some directed movements near the sheet boundary (Fig. 4C). Next, we tracked cells transfected with siRNA pools against four members of the directed migration module, namely FGFR1, FRS2, GAB1, and PTEN, and compared their capacity for directed migration relative to control cells. Consistent with their role in directed migration, the first three genes, which slow sheet migration, showed a reduced orientation toward the cell-free band, while PTEN, which enhances sheet migration, showed a more pronounced orientation into cell-free space (Fig. 4D).

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Paracrine Factors of Mesenchymal Stem Cells Recruit Macrophages and Endothelial Lineage Cells and Enhance Wound Healing: ”

by Liwen Chen, Edward E. Tredget, Philip Y. G. Wu, Yaojiong Wu

Bone marrow derived mesenchymal stem cells (BM-MSCs) have been shown to enhance wound healing; however, the mechanisms involved are barely understood. In this study, we examined paracrine factors released by BM-MSCs and their effects on the cells participating in wound healing compared to those released by dermal fibroblasts. Analyses of BM-MSCs with Real-Time PCR and of BM-MSC-conditioned medium by antibody-based protein array and ELISA indicated that BM-MSCs secreted distinctively different cytokines and chemokines, such as greater amounts of VEGF-α, IGF-1, EGF, keratinocyte growth factor, angiopoietin-1, stromal derived factor-1, macrophage inflammatory protein-1alpha and beta and erythropoietin, compared to dermal fibroblasts. These molecules are known to be important in normal wound healing. BM-MSC-conditioned medium significantly enhanced migration of macrophages, keratinocytes and endothelial cells and proliferation of keratinocytes and endothelial cells compared to fibroblast-conditioned medium. Moreover, in a mouse model of excisional wound healing, where concentrated BM-MSC-conditioned medium was applied, accelerated wound healing occurred compared to administration of pre-conditioned or fibroblast-conditioned medium. Analysis of cell suspensions derived from the wound by FACS showed that wounds treated with BM-MSC-conditioned medium had increased proportions of CD4/80-postive macrophages and Flk-1-, CD34- or c-kit-positive endothelial (progenitor) cells compared to wounds treated with pre-conditioned medium or fibroblast-conditioned medium. Consistent with the above findings, immunohistochemical analysis of wound sections showed that wounds treated with BM-MSC-conditioned medium had increased abundance of macrophages. Our results suggest that factors released by BM-MSCs recruit macrophages and endothelial lineage cells into the wound thus enhancing wound healing.

Chigurupati S, Arumugam TV, Son TG, Lathia JD, Jameel S, et al (2007) Involvement of Notch Signaling in Wound Healing. PLoS ONE 2(11): e1167. doi:10.1371/journal.pone.0001167

The Notch signaling pathway is critically involved in cell fate decisions during development of many tissues and organs. In the present study we employed in vivo and cell culture models to elucidate the role of Notch signaling in wound healing. The healing of full-thickness dermal wounds was significantly delayed in Notch antisense transgenic mice and in normal mice treated with c-secretase inhibitors that block proteolytic cleavage and activation of Notch. In contrast, mice treated with a Notch ligand Jagged peptide showed significantly enhanced wound healing compared to controls. Activation or inhibition of Notch signaling altered the behaviors of cultured vascular endothelial cells, keratinocytes and fibroblasts in a scratch wound healing model in ways consistent with roles for Notch signaling in wound healing functions all three cell types. These results suggest that Notch signaling plays important roles in wound healing and tissue repair, and that targeting the Notch pathway might provide a novel strategy for treatment of wounds and for modulation of angiogenesis in other pathological conditions.