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Through multiple cell-cell and cell-matrix interactions, epithelial and endothelial sheets form tight barriers. Modulators of the cytoskeleton contribute to barrier stability and act as rheostats of vascular permeability. In this study, we sought to identify cytoskeletal regulators that underlie barrier diversity across vessels. To achieve this, we correlated functional and structural barrier features to gene expression of endothelial cells (ECs) derived from different vascular beds. Within a subset of identified candidates, we found that the guanosine nucleotide exchange factor Vav3 was exclusively expressed by microvascular ECs and was closely associated with a high-resistance barrier phenotype. Ectopic expression of Vav3 in large artery and brain ECs significantly enhanced barrier resistance and cortical rearrangement of the actin cytoskeleton. Mechanistically, we found that the barrier effect of Vav3 is dependent on its Dbl homology domain and downstream activation of Rap1. Importantly, inactivation of Vav3 in vivo resulted in increased vascular leakage, highlighting its function as a key regulator of barrier stability.

Given its role as the source of definitive hematopoietic cells, we sought to determine whether mutations initiated in the hemogenic endothelium would yield hematopoietic abnormalities or malignancies. Here, we find that endothelium-specific transposon mutagenesis in mice promotes hematopoietic pathologies that are both myeloid and lymphoid in nature. Frequently mutated genes included previously recognized cancer drivers and additional candidates, such as Pi4ka, a lipid kinase whose mutation was found to promote myeloid and erythroid dysfunction. Subsequent validation experiments showed that targeted inactivation of the Pi4ka catalytic domain or reduction in mRNA expression inhibited myeloid and erythroid cell differentiation in vitro and promoted anemia in vivo through a mechanism involving deregulation of AKT, MAPK, SRC, and JAK-STAT signaling. Finally, we provide evidence linking PI4KAP2, previously considered a pseudogene, to human myeloid and erythroid leukemia.

ObjectivePerivascular cells, including pericytes, macrophages, smooth muscle cells, and other specialized cell types, like podocytes, participate in various aspects of vascular function. However, aside from the well-established roles of smooth muscle cells and pericytes, the contributions of other vascular-associated cells are poorly understood. Our goal was to ascertain the function of perivascular macrophages in adult tissues under nonpathological conditions.Approach and resultsWe combined confocal microscopy, in vivo cell depletion, and in vitro assays to investigate the contribution of perivascular macrophages to vascular function. We found that resident perivascular macrophages are associated with capillaries at a frequency similar to that of pericytes. Macrophage depletion using either clodronate liposomes or antibodies unexpectedly resulted in hyperpermeability. This effect could be rescued when M2-like macrophages, but not M1-like macrophages or dendritic cells, were reconstituted in vivo, suggesting subtype-specific roles for macrophages in the regulation of vascular permeability. Furthermore, we found that permeability-promoting agents elicit motility and eventual dissociation of macrophages from the vasculature. Finally, in vitro assays showed that M2-like macrophages attenuate the phosphorylation of VE-cadherin upon exposure to permeability-promoting agents.ConclusionsThis study points to a direct contribution of macrophages to vessel barrier integrity and provides evidence that heterotypic cell interactions with the endothelium, in addition to those of pericytes, control vascular permeability.

Autocrine VEGF is necessary for endothelial survival, although the cellular mechanisms supporting this function are unknown. Here, we show that--even after full differentiation and maturation--continuous expression of VEGF by endothelial cells is needed to sustain vascular integrity and cellular viability. Depletion of VEGF from the endothelium results in mitochondria fragmentation and suppression of glucose metabolism, leading to increased autophagy that contributes to cell death. Gene-expression profiling showed that endothelial VEGF contributes to the regulation of cell cycle and mitochondrial gene clusters, as well as several--but not all--targets of the transcription factor FOXO1. Indeed, VEGF-deficient endothelium in vitro and in vivo showed increased levels of FOXO1 protein in the nucleus and cytoplasm. Silencing of FOXO1 in VEGF-depleted cells reversed expression profiles of several of the gene clusters that were de-regulated in VEGF knockdown, and rescued both cell death and autophagy phenotypes. Our data suggest that endothelial VEGF maintains vascular homeostasis through regulation of FOXO1 levels, thereby ensuring physiological metabolism and endothelial cell survival.

The past 5 years have witnessed a significant expansion in our understanding of vascular endothelial growth factor (VEGF) signaling. In particular, the process of canonical activation of VEGF receptor tyrosine kinases by homodimeric VEGF molecules has now been broadened by the realization that heterodimeric ligands and receptors are also active participants in the signaling process. Although heterodimer receptors were described 2 decades ago, their impact, along with the effect of additional cell surface partners and novel autocrine VEGF signaling pathways, are only now starting to be clarified. Furthermore, ligand-independent signaling (noncanonical) has been identified through galectin and gremlin binding and upon rise of intracellular levels of reactive oxygen species. Activation of the VEGF receptors in the absence of ligand holds immediate implications for therapeutic approaches that exclusively target VEGF. The present review provides a concise summary of the recent developments in both canonical and noncanonical VEGF signaling and places these findings in perspective to their potential clinical and biological ramifications.

Although vascular complications are a hallmark of diabetes, the molecular mechanisms that underlie endothelial dysfunction are unclear. We showed that reactive oxygen species generated from hyperglycemia promoted ligand-independent phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2). This VEGFR2 signaling occurred within the Golgi compartment and resulted in progressively decreased availability of VEGFR2 at the cell surface. Consequently, the responses of endothelial cells to exogenous VEGF in a mouse model of diabetes were impaired because of a specific deficiency of VEGFR2 at the cell surface, despite a lack of change in transcript abundance. Hyperglycemia-induced phosphorylation of VEGFR2 did not require intrinsic receptor kinase activity and was instead mediated by Src family kinases. The reduced cell surface abundance of VEGFR2 in diabetic mice was reversed by treatment with the antioxidant N-acetyl-L-cysteine, suggesting a causative role for oxidative stress. These findings uncover a mode of ligand-independent VEGFR2 signaling that can progressively lead to continuously muted responses to exogenous VEGF and limit angiogenic events.