SummaryTherapeutic targeting of tumor angiogenesis with VEGF inhibitors results in demonstrable, but transitory efficacy in certain human tumors and mouse models of cancer, limited by unconventional forms of adaptive/evasive resistance. In one such mouse model, potent angiogenesis inhibitors elicit compartmental reorganization of cancer cells around remaining blood vessels. The glucose and lactate transporters GLUT1 and MCT4 are induced in distal hypoxic cells in a HIF1α-dependent fashion, indicative of glycolysis. Tumor cells proximal to blood vessels instead express the lactate transporter MCT1, and p-S6, the latter reflecting mTOR signaling. Normoxic cancer cells import and metabolize lactate, resulting in upregulation of mTOR signaling via glutamine metabolism enhanced by lactate catabolism. Thus, metabolic symbiosis is established in the face of angiogenesis inhibition, whereby hypoxic cancer cells import glucose and export lactate, while normoxic cells import and catabolize lactate. mTOR signaling inhibition disrupts this metabolic symbiosis, associated with upregulation of the glucose transporter GLUT2.

Endothelial cells and macrophages are known to engage in tight and specific interactions that contribute to the modulation of vascular function. Here we show that adult endothelial cells provide critical signals for the selective growth and differentiation of macrophages from several hematopoietic progenitors. The process features the formation of wellorganized colonies that exhibit progressive differentiation from the center to the periphery and toward an M2-like phenotype, characterized by enhanced expression of Tie2 and CD206/Mrc1. These colonies are long-lived depending on the contact with the endothelium; removal of the endothelial monolayer results in rapid colony dissolution. We further found that Csf1 produced by the endothelium is critical for the expansion of the macrophage colonies and that blockade of Csf1 receptor impairs colony growth. Functional analyses indicate that these macrophages are capable of accelerating angiogenesis, promoting tumor growth, and effectively engaging in tight associations with endothelial cells in vivo. These findings uncover a critical role of endothelial cells in the induction of macrophage differentiation and their ability to promote further polarization toward a proangiogenic phenotype. This work also highlights some of the molecules underlying the M2-like differentiation, a process that is relevant to the progression of both developmental and pathologic angiogenesis. (Blood. 2012;120(15):3152-3162) IntroductionThe link between the hematopoietic and the endothelial cell lineages is rooted early in development. In fact, definitive hematopoietic stem cells (HSCs) first emerge in the embryo from a specialized endothelial intermediate that holds hemogenic capacity. [1][2][3][4] Although the process of hematopoietic cells (HCs) budding from hemogenic endothelium is no longer present in the adult, the interactions between HCs and the endothelium continue to be critical for the trafficking and homing of HCs, as well as for activation and recruitment of inflammatory cells to specific tissue sites. 5 More recently, sinusoidal endothelial cells were shown to be essential for the self-renewal capacity of hematopoietic stem/ progenitor cells (HSPCs) through the production of specific angiocrine factors. 6,7 Intriguingly, bone marrow sinusoidal endothelial cells can also constitute a platform for the differentiation of HSPCs. This dual role of endothelial cells has been best exemplified by findings communicated by Kobayashi and colleagues, where the coculture of genetically modified human umbilical vein endothelial cells (HUVECs) with HSPCs supported both selfrenewal and lineage-specific differentiation of HSPCs. 8 Notably, the mechanisms by which endothelial cells mediate regeneration or differentiation of HCs depend largely on organ-specific determinants. Overall, mounting evidence supports the concept that the crosstalk between endothelial cells and HCs impacts the differentiation and stem cell properties of hematopoietic progenitors.The consequences of endothelial-hematopoietic...

Despite the clear major contribution of hyperlipidemia to the prevalence of cardiovascular disease in the developed world, the direct effects of lipoproteins on endothelial cells have remained obscure and are under debate. Here we report a previously uncharacterized mechanism of vessel growth modulation by lipoprotein availability. Using a genetic screen for vascular defects in zebrafish, we initially identified a mutation, stalactite (stl), in the gene encoding microsomal triglyceride transfer protein (mtp), which is involved in the biosynthesis of apolipoprotein B (ApoB)-containing lipoproteins. By manipulating lipoprotein concentrations in zebrafish, we found that ApoB negatively regulates angiogenesis and that it is the ApoB protein particle, rather than lipid moieties within ApoB-containing lipoproteins, that is primarily responsible for this effect. Mechanistically, we identified downregulation of vascular endothelial growth factor receptor 1 (VEGFR1), which acts as a decoy receptor for VEGF, as a key mediator of the endothelial response to lipoproteins, and we observed VEGFR1 downregulation in hyperlipidemic mice. These findings may open new avenues for the treatment of lipoprotein-related vascular disorders.

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.

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.

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Mutations in myosin-VIIa are responsible for the deaf-blindness, Usher disease. Myosin-VIIa is also highly expressed in testis, where it is associated with specialized adhesion plaques termed ectoplasmic specializations (ES) that form between Sertoli cells and germ cells. To identify new roles for myosin-VIIa, we undertook a yeast two-hybrid screen to identify proteins associated with myosin-VIIa in the ES. We identified Keap1, a human homologue of the Drosophila ring canal protein, kelch. The kelch-repeats in the C-terminus of human Keap1 associate with the SH3 domain of myosin-VIIa. Immunolocalization studies revealed that Keap1 is present with myosin-VIIa in the actin bundles of the ES. Myosin-VIIa and Keap1 copurify with ES and colocate with each other and with F-actin at the electron microscopy level. Interestingly, in many epithelial cell types including cells derived from retina and inner ear, Keap1 is a component of focal adhesions and zipper junctions. Keap1 can target to the ES in the absence of myosin-VIIa, suggesting that Keap1 associates with other molecules in the adhesion plaque. Keap1 and myosin-VIIa overlapped in expression in the inner hair cells of the cochlea, suggesting that Keap1 may be a part of a family of actin-binding proteins that could be important for myosin-VIIa function in testis and inner ear.

Objective Perivascular 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 non-pathological conditions. Approach and Results We 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. Conclusions This 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.