Pericyte migration and proliferation are tightly synchronized to endothelial cell sprouting dynamics.

Pericytes are critical for microvascular stability and maintenance, among other important physiological functions, yet their involvement in vessel formation processes remains poorly understood. To gain insight into pericyte behaviors during vascular remodeling, we developed two complementary tissue explant models utilizing 'double reporter' animals with fluorescently-labeled pericytes and endothelial cells (via Ng2:DsRed and Flk-1:eGFP genes, respectively). Time-lapse confocal imaging of active vessel remodeling within adult connective tissues and embryonic skin revealed a subset of pericytes detaching and migrating away from the vessel wall. Vessel-associated pericytes displayed rapid filopodial sampling near sprouting endothelial cells that emerged from parent vessels to form nascent branches. Pericytes near angiogenic sprouts were also more migratory, initiating persistent and directional movement along newly forming vessels. Pericyte cell divisions coincided more frequently with elongating endothelial sprouts, rather than sprout initiation sites, an observation confirmed with in vivo data from the developing mouse brain. Taken together, these data suggest that (i) pericyte detachment from the vessel wall may represent an important physiological process to enhance endothelial cell plasticity during vascular remodeling, and (ii) pericyte migration and proliferation are highly synchronized with endothelial cell behaviors during the coordinated expansion of a vascular network.

Flt-1 (VEGFR-1) coordinates discrete stages of blood vessel formation.

AIMS: In developing blood vessel networks, the overall level of vessel branching often correlates with angiogenic sprout initiations, but in some pathological situations, increased sprout initiations paradoxically lead to reduced vessel branching and impaired vascular function. We examine the hypothesis that defects in the discrete stages of angiogenesis can uniquely contribute to vessel branching outcomes. METHODS AND RESULTS: Time-lapse movies of mammalian blood vessel development were used to define and quantify the dynamics of angiogenic sprouting. We characterized the formation of new functional conduits by classifying discrete sequential stages-sprout initiation, extension, connection, and stability-that are differentially affected by manipulation of vascular endothelial growth factor-A (VEGF-A) signalling via genetic loss of the receptor flt-1 (vegfr1). In mouse embryonic stem cell-derived vessels genetically lacking flt-1, overall branching is significantly decreased while sprout initiations are significantly increased. Flt-1(-/-) mutant sprouts are less likely to retract, and they form increased numbers of connections with other vessels. However, loss of flt-1 also leads to vessel collapse, which reduces the number of new stable conduits. Computational simulations predict that loss of flt-1 results in ectopic Flk-1 signalling in connecting sprouts post-fusion, causing protrusion of cell processes into avascular gaps and collapse of branches. Thus, defects in stabilization of new vessel connections offset increased sprout initiations and connectivity in flt-1(-/-) vascular networks, with an overall outcome of reduced numbers of new conduits. CONCLUSIONS: These results show that VEGF-A signalling has stage-specific effects on vascular morphogenesis, and that understanding these effects on dynamic stages of angiogenesis and how they integrate to expand a vessel network may suggest new therapeutic strategies.