Notch-mediated cellular interactions between vascular cells.

Vessel formation and differentiation to a proper hierarchical vasculature requires a coordinated effort from endothelial and mural cells. Over the last decade Notch was identified as a key player in this process by promoting vascular arterialization and modulating endothelial tip-stalk phenotypes. Recent work has identified that Notch fine-tunes the diverse endothelial phenotypes through regulation of canonical cell-cycle and metabolism regulators, such as ERK and Myc. During arterialization, Notch signaling inhibits the cell-cycle and metabolism of endothelial cells which coincides with the acquisition of arterial identity. During angiogenesis, the same molecular machinery prevents the hypermitogenic arrest and excessive sprouting of vessels. Notch also signals in pericytes and smooth muscle cells promoting vascular coverage and maturation. Here, we will review the latest findings on how Notch signals regulate the differentiation and interactions among vascular cells during organ development and homeostasis.

Pericytes and vascular smooth muscle cells in central nervous system arteriovenous malformations.

Previously considered passive support cells, mural cells-pericytes and vascular smooth muscle cells-have started to garner more attention in disease research, as more subclassifications, based on morphology, gene expression, and function, have been discovered. Central nervous system (CNS) arteriovenous malformations (AVMs) represent a neurovascular disorder in which mural cells have been shown to be affected, both in animal models and in human patients. To study consequences to mural cells in the context of AVMs, various animal models have been developed to mimic and predict human AVM pathologies. A key takeaway from recently published work is that AVMs and mural cells are heterogeneous in their molecular, cellular, and functional characteristics. In this review, we summarize the observed perturbations to mural cells in human CNS AVM samples and CNS AVM animal models, and we discuss various potential mechanisms relating mural cell pathologies to AVMs.

BMPing up endocardial angiogenesis to generate coronary vessels.

Understanding how coronary vessels develop is important for designing better strategies to repair ischemic hearts. In this issue of Developmental Cell, D'Amato et al. report that BMP2 and CXCL12/CXCR4 act sequentially on endocardial cells to drive coronary angiogenesis and artery morphogenesis.

PDGFRß+ cells play a dual role as hematopoietic precursors and niche cells during mouse ontogeny.

Hematopoietic stem cell (HSC) generation in the aorta-gonad-mesonephros region requires HSC specification signals from the surrounding microenvironment. In zebrafish, PDGF-B/PDGFRß signaling controls hematopoietic stem/progenitor cell (HSPC) generation and is required in the HSC specification niche. Little is known about murine HSPC specification in vivo and whether PDGF-B/PDGFRß is involved. Here, we show that PDGFRß is expressed in distinct perivascular stromal cell layers surrounding the mid-gestation dorsal aorta, and its deletion impairs hematopoiesis. We demonstrate that PDGFRß+ cells play a dual role in murine hematopoiesis. They act in the aortic niche to support HSPCs, and in addition, PDGFRß+ embryonic precursors give rise to a subset of HSPCs that persist into adulthood. These findings provide crucial information for the controlled production of HSPCs in vitro.

Pericyte dynamics in the mouse germinal matrix angiogenesis.

Germinal matrix-intraventricular hemorrhage (GM-IVH) is the most devastating neurological complication in premature infants. GM-IVH usually begins in the GM, a highly vascularized region of the developing brain where glial and neuronal precursors reside underneath the lateral ventricular ependyma. Previous studies using human fetal tissue have suggested increased angiogenesis and paucity of pericytes as key factors contributing to GM-IVH pathogenesis. Yet, despite its relevance, the mechanisms underlying the GM vasculature's susceptibility to hemorrhage remain poorly understood. To gain better understanding on the vascular dynamics of the GM, we performed a comprehensive analysis of the mouse GM vascular endothelium and pericytes during development. We hypothesize that vascular development of the mouse GM will provide a good model for studies of human GM vascularization and provide insights into the role of pericytes in GM-IVH pathogenesis. Our findings show that the mouse GM presents significantly greater vascular area and vascular branching compared to the developing cortex (CTX). Analysis of pericyte coverage showed abundance in PDGFRß-positive and NG2-positive pericyte coverage in the GM similar to the developing CTX. However, we found a paucity in Desmin-positive pericyte coverage of the GM vasculature. Our results underscore the highly angiogenic nature of the GM and reveal that pericytes in the developing mouse GM exhibit distinct phenotypical and likely functional characteristics compared to other brain regions which might contribute to the high susceptibility of the GM vasculature to hemorrhage.

Release of stem cells from quiescence reveals gliogenic domains in the adult mouse brain.

Quiescent neural stem cells (NSCs) in the adult mouse ventricular-subventricular zone (V-SVZ) undergo activation to generate neurons and some glia. Here we show that platelet-derived growth factor receptor beta (PDGFRß) is expressed by adult V-SVZ NSCs that generate olfactory bulb interneurons and glia. Selective deletion of PDGFRß in adult V-SVZ NSCs leads to their release from quiescence, uncovering gliogenic domains for different glial cell types. These domains are also recruited upon injury. We identify an intraventricular oligodendrocyte progenitor derived from NSCs inside the brain ventricles that contacts supraependymal axons. Together, our findings reveal that the adult V-SVZ contains spatial domains for gliogenesis, in addition to those for neurogenesis. These gliogenic NSC domains tend to be quiescent under homeostasis and may contribute to brain plasticity.

Deficiency of Notch signaling in pericytes results in arteriovenous malformations.

Arteriovenous malformations (AVMs) are high-flow lesions directly connecting arteries and veins. In the brain, AVM rupture can cause seizures, stroke, and death. Patients with AVMs exhibit reduced coverage of the vessels by pericytes, the mural cells of microvascular capillaries; however, the mechanism underlying this pericyte reduction and its association with AVM pathogenesis remains unknown. Notch signaling has been proposed to regulate critical pericyte functions. We hypothesized that Notch signaling in pericytes is crucial to maintain pericyte homeostasis and prevent AVM formation. We inhibited Notch signaling specifically in perivascular cells and analyzed the vasculature of these mice. The retinal vessels of mice with deficient perivascular Notch signaling developed severe AVMs, together with a significant reduction in pericytes and vascular smooth muscle cells (vSMC) in the arteries, while vSMCs were increased in the veins. Vascular malformations and pericyte loss were also observed in the forebrain of embryonic mice deficient for perivascular Notch signaling. Moreover, the loss of Notch signaling in pericytes downregulated Pdgfrb levels and increased pericyte apoptosis, pointing to a critical role for Notch in pericyte survival. Overall, our findings reveal a mechanism of AVM formation and highlight the Notch signaling pathway as an essential mediator in this process.

A Tie2-Notch1 signaling axis regulates regeneration of the endothelial bone marrow niche.

Loss-of-function studies have determined that Notch signaling is essential for hematopoietic and endothelial development. By deleting a single allele of the Notch1 transcriptional activation domain we generated viable, post-natal mice exhibiting hypomorphic Notch signaling. These heterozygous mice, which lack only one copy of the transcriptional activation domain, appear normal and have no endothelial or hematopoietic phenotype, apart from an inherent, cell-autonomous defect in T-cell lineage development. Following chemotherapy, these hypomorphs exhibited severe pancytopenia, weight loss and morbidity. This phenotype was confirmed in an endothelial-specific, loss-of-function Notch1 model system. Ang1, secreted by hematopoietic progenitors after damage, activated endothelial Tie2 signaling, which in turn enhanced expression of Notch ligands and potentiated Notch1 receptor activation. In our heterozygous, hypomorphic model system, the mutant protein that lacks the Notch1 transcriptional activation domain accumulated in endothelial cells and interfered with optimal activity of the wildtype Notch1 transcriptional complex. Failure of the hypomorphic mutant to efficiently drive transcription of key gene targets such as Hes1 and Myc prolonged apoptosis and limited regeneration of the bone marrow niche. Thus, basal Notch1 signaling is sufficient for niche development, but robust Notch activity is required for regeneration of the bone marrow endothelial niche and hematopoietic recovery.

PDGFRß-P2A-CreERT2 mice: a genetic tool to target pericytes in angiogenesis.

Pericytes are essential mural cells distinguished by their association with small caliber blood vessels and the presence of a basement membrane shared with endothelial cells. Pericyte interaction with the endothelium plays an important role in angiogenesis; however, very few tools are currently available that allow for the targeting of pericytes in mouse models, limiting our ability to understand their biology. We have generated a novel mouse line expressing tamoxifen-inducible Cre-recombinase under the control of the platelet-derived growth factor receptor ß promoter: PDGFRß-P2A-CreER T2 . We evaluated the expression of the PDGFRß-P2A-CreER T2 line by crossing it with fluorescent reporter lines and analyzed reporter signal in the angiogenic retina and brain at different time points after tamoxifen administration. Reporter lines showed labeling of NG2+, desmin+, PDGFRß+ perivascular cells in the retina and the brain, indicating successful targeting of pericytes; however, signal from reporter lines was also observed in a small subset of glial cells both in the retina and the brain. We also evaluated recombination in tumors and found efficient recombination in perivascular cells associated with tumor vasculature. As a proof of principle, we used our newly generated driver to delete Notch signaling in perivascular cells and observed a loss of smooth muscle cells in retinal arteries, consistent with previously published studies evaluating Notch3 null mice. We conclude that the PDGFRß-P2A-CreER T2 line is a powerful new tool to target pericytes and will aid the field in gaining a deeper understanding of the role of these cells in physiological and pathological settings.

Endothelial notch signaling is essential to prevent hepatic vascular malformations in mice.

UNLABELLED: Liver vasculature is crucial for adequate hepatic functions. Global deletion of Notch signaling in mice results in liver vascular pathologies. However, whether Notch in endothelium is essential for hepatic vascular structure and function remains unknown. To uncover the function of endothelial Notch in the liver, we deleted Rbpj, a transcription factor mediating all canonical Notch signaling, or Notch1 from the endothelium of postnatal mice. We investigated the hepatic vascular defects in these mutants. The liver was severely affected within 2 weeks of endothelial deletion of Rbpj from birth. Two-week old mutant mice had enlarged vessels on the liver surface, abnormal vascular architecture, and dilated sinusoids. Vascular casting and fluorosphere passage experiments indicated the presence of porto-systemic shunts. These mutant mice presented with severely necrotic liver parenchyma and significantly larger hypoxic areas, likely resulting from vascular shunts. We also found elevated levels of VEGF receptor 3 together with reduced levels of ephrin-B2, suggesting a possible contribution of these factors to the generation of hepatic vascular abnormalities. Deletion of Rbpj from the adult endothelium also led to dilated sinusoids, vascular shunts, and necrosis, albeit milder than that observed in mice with deletion from birth. Similar to deletion of Rbpj, loss of endothelial Notch1 from birth led to similar hepatic vascular malformations within 2 weeks. CONCLUSIONS: Endothelial Notch signaling is essential for the development and maintenance of proper hepatic vascular architecture and function. These findings may elucidate the molecular pathogenesis of hepatic vascular malformation and the safety of therapeutics inhibiting Notch. (Hepatology 2016;64:1302-1316).

In vitro modeling of endothelial interaction with macrophages and pericytes demonstrates Notch signaling function in the vascular microenvironment.

Angiogenesis is regulated by complex interactions between endothelial cells and support cells of the vascular microenvironment, such as tissue myeloid cells and vascular mural cells. Multicellular interactions during angiogenesis are difficult to study in animals and challenging in a reductive setting. We incorporated stromal cells into an established bead-based capillary sprouting assay to develop assays that faithfully reproduce major steps of vessel sprouting and maturation. We observed that macrophages enhance angiogenesis, increasing the number and length of endothelial sprouts, a property we have dubbed "angiotrophism." We found that polarizing macrophages toward a pro-inflammatory profile further increased their angiotrophic stimulation of vessel sprouting, and this increase was dependent on macrophage Notch signaling. To study endothelial/pericyte interactions, we added vascular pericytes directly to the bead-bound endothelial monolayer. These pericytes formed close associations with the endothelial sprouts, causing increased sprout number and vessel caliber. We found that Jagged1 expression and Notch signaling are essential for the growth of both endothelial cells and pericytes and may function in their interaction. We observed that combining endothelial cells with both macrophages and pericytes in the same sprouting assay has multiplicative effects on sprouting. These results significantly improve bead-capillary sprouting assays and provide an enhanced method for modeling interactions between the endothelium and the vascular microenvironment. Achieving this in a reductive in vitro setting represents a significant step toward a better understanding of the cellular elements that contribute to the formation of mature vasculature.

Combined deficiency of Notch1 and Notch3 causes pericyte dysfunction, models CADASIL, and results in arteriovenous malformations.

Pericytes regulate vessel stability and pericyte dysfunction contributes to retinopathies, stroke, and cancer. Here we define Notch as a key regulator of pericyte function during angiogenesis. In Notch1(+/-); Notch3(-/-) mice, combined deficiency of Notch1 and Notch3 altered pericyte interaction with the endothelium and reduced pericyte coverage of the retinal vasculature. Notch1 and Notch3 were shown to cooperate to promote proper vascular basement membrane formation and contribute to endothelial cell quiescence. Accordingly, loss of pericyte function due to Notch deficiency exacerbates endothelial cell activation caused by Notch1 haploinsufficiency. Mice mutant for Notch1 and Notch3 develop arteriovenous malformations and display hallmarks of the ischemic stroke disease CADASIL. Thus, Notch deficiency compromises pericyte function and contributes to vascular pathologies.

The Cancer Cell Oxygen Sensor PHD2 Promotes Metastasis via Activation of Cancer-Associated Fibroblasts.

Several questions about the role of the oxygen sensor prolyl-hydroxylase 2 (PHD2) in cancer have not been addressed. First, the role of PHD2 in metastasis has not been studied in a spontaneous tumor model. Here, we show that global PHD2 haplodeficiency reduced metastasis without affecting tumor growth. Second, it is unknown whether PHD2 regulates cancer by affecting cancer-associated fibroblasts (CAFs). We show that PHD2 haplodeficiency reduced metastasis via two mechanisms: (1) by decreasing CAF activation, matrix production, and contraction by CAFs, an effect that surprisingly relied on PHD2 deletion in cancer cells, but not in CAFs; and (2) by improving tumor vessel normalization. Third, the effect of concomitant PHD2 inhibition in malignant and stromal cells (mimicking PHD2 inhibitor treatment) is unknown. We show that global PHD2 haplodeficiency, induced not only before but also after tumor onset, impaired metastasis. These findings warrant investigation of PHD2's therapeutic potential.

Cyclooxygenase-2 and Prostaglandin E2 Signaling through Prostaglandin Receptor EP-2 Favor the Development of Myocarditis during Acute Trypanosoma cruzi Infection.

Inflammation plays an important role in the pathophysiology of Chagas disease, caused by Trypanosoma cruzi. Prostanoids are regulators of homeostasis and inflammation and are produced mainly by myeloid cells, being cyclooxygenases, COX-1 and COX-2, the key enzymes in their biosynthesis from arachidonic acid (AA). Here, we have investigated the expression of enzymes involved in AA metabolism during T. cruzi infection. Our results show an increase in the expression of several of these enzymes in acute T. cruzi infected heart. Interestingly, COX-2 was expressed by CD68+ myeloid heart-infiltrating cells. In addition, infiltrating myeloid CD11b+Ly6G- cells purified from infected heart tissue express COX-2 and produce prostaglandin E2 (PGE2) ex vivo. T. cruzi infections in COX-2 or PGE2-dependent prostaglandin receptor EP-2 deficient mice indicate that both, COX-2 and EP-2 signaling contribute significantly to the heart leukocyte infiltration and to the release of chemokines and inflammatory cytokines in the heart of T. cruzi infected mice. In conclusion, COX-2 plays a detrimental role in acute Chagas disease myocarditis and points to COX-2 as a potential target for immune intervention.

Lack of Galectin-3 Prevents Cardiac Fibrosis and Effective Immune Responses in a Murine Model of Trypanosoma cruzi Infection.

BACKGROUND: Chagas disease is caused by the protozoan Trypanosoma cruzi, affecting millions of people worldwide. One of the major causes of mortality in the disease is the cardiomyopathy observed in chronic patients, despite the low number of parasites detected in cardiac tissue. Galectin-3, a carbohydrate-binding protein with affinity for ß-galactoside-containing glycoconjugates, is upregulated upon infection, and it has been recently involved in the pathophysiology of heart failure. METHODS: We investigated the role of galectin-3 in systemic and local responses in a murine model of T. cruzi infection, using knockout animals. Molecular mechanisms underlying galectin-3-dependent inflammatory responses were further assessed in cultured dendritic cells in vitro. RESULTS: Mice deficient for galectin-3 have elevated blood parasitemia levels and impaired cytokine production during infection. Remarkably, galectin-3 promotes cellular infiltration in the heart of infected mice and subsequent collagen deposition and cardiac fibrosis. Furthermore, we show that an unbalanced Toll-like receptor expression on antigen-presenting cells may be the cause of the impaired immune response observed in galectin-3-deficient mice in vivo. CONCLUSIONS: These results suggest that galectin-3 is strongly involved in Chagas disease, not only in the immune response against T. cruzi, but also in mediating cardiac tissue damage.

NOTCH decoys that selectively block DLL/NOTCH or JAG/NOTCH disrupt angiogenesis by unique mechanisms to inhibit tumor growth.

UNLABELLED: A proangiogenic role for Jagged (JAG)-dependent activation of NOTCH signaling in the endothelium has yet to be described. Using proteins that encoded different NOTCH1 EGF-like repeats, we identified unique regions of Delta-like ligand (DLL)-class and JAG-class ligand-receptor interactions, and developed NOTCH decoys that function as ligand-specific NOTCH inhibitors. N110-24 decoy blocked JAG1/JAG2-mediated NOTCH1 signaling, angiogenic sprouting in vitro, and retinal angiogenesis, demonstrating that JAG-dependent NOTCH signal activation promotes angiogenesis. In tumors, N110-24 decoy reduced angiogenic sprouting, vessel perfusion, pericyte coverage, and tumor growth. JAG-NOTCH signaling uniquely inhibited expression of antiangiogenic soluble (s) VEGFR1/sFLT1. N11-13 decoy interfered with DLL1-DLL4-mediated NOTCH1 signaling and caused endothelial hypersprouting in vitro, in retinal angiogenesis, and in tumors. Thus, blockade of JAG- or DLL-mediated NOTCH signaling inhibits angiogenesis by distinct mechanisms. JAG-NOTCH signaling positively regulates angiogenesis by suppressing sVEGFR1-sFLT1 and promoting mural-endothelial cell interactions. Blockade of JAG-class ligands represents a novel, viable therapeutic approach to block tumor angiogenesis and growth. SIGNIFICANCE: This is the first report identifying unique regions of the NOTCH1 extracellular domain that interact with JAG-class and DLL-class ligands. Using this knowledge, we developed therapeutic agents that block JAG-dependent NOTCH signaling and demonstrate for the first time that JAG blockade inhibits experimental tumor growth by targeting tumor angiogenesis.

Deletion of Rbpj from postnatal endothelium leads to abnormal arteriovenous shunting in mice.

Arteriovenous malformations (AVMs) are tortuous vessels characterized by arteriovenous (AV) shunts, which displace capillaries and shunt blood directly from artery to vein. Notch signaling regulates embryonic AV specification by promoting arterial, as opposed to venous, endothelial cell (EC) fate. To understand the essential role of endothelial Notch signaling in postnatal AV organization, we used inducible Cre-loxP recombination to delete Rbpj, a mediator of canonical Notch signaling, from postnatal ECs in mice. Deletion of endothelial Rbpj from birth resulted in features of AVMs by P14, including abnormal AV shunting and tortuous vessels in the brain, intestine and heart. We further analyzed brain AVMs, as they pose particular health risks. Consistent with AVM pathology, we found cerebral hemorrhage, hypoxia and necrosis, and neurological deficits. AV shunts originated from capillaries (and possibly venules), with the earliest detectable morphological abnormalities in AV connections by P8. Prior to AV shunt formation, alterations in EC gene expression were detected, including decreased Efnb2 and increased Pai1, which encodes a downstream effector of TGFß signaling. After AV shunts had formed, whole-mount immunostaining showed decreased Efnb2 and increased Ephb4 expression within AV shunts, suggesting that ECs were reprogrammed from arterial to venous identity. Deletion of Rbpj from adult ECs led to tortuosities in gastrointestinal, uterine and skin vascular beds, but had mild effects in the brain. Our results demonstrate a temporal requirement for Rbpj in postnatal ECs to maintain proper artery, capillary and vein organization and to prevent abnormal AV shunting and AVM pathogenesis.

Vascular biology 2014 in Monterey, California: celebrating 20 years of NAVBO.

A meeting report for Vascular Biology 2014, held in Monterey, California and organized by the North American Vascular Biology Organization (NAVBO).

Notch4 is required for tumor onset and perfusion.

BACKGROUND: Notch4 is a member of the Notch family of receptors that is primarily expressed in the vascular endothelial cells. Genetic deletion of Notch4 does not result in an overt phenotype in mice, thus the function of Notch4 remains poorly understood. METHODS: We examined the requirement for Notch4 in the development of breast cancer vasculature. Orthotopic transplantation of mouse mammary tumor cells wild type for Notch4 into Notch4 deficient hosts enabled us to delineate the contribution of host Notch4 independent of its function in the tumor cell compartment. RESULTS: Here, we show that Notch4 expression is required for tumor onset and early tumor perfusion in a mouse model of breast cancer. We found that Notch4 expression is upregulated in mouse and human mammary tumor vasculature. Moreover, host Notch4 deficiency delayed the onset of MMTV-PyMT tumors, wild type for Notch4, after transplantation. Vessel perfusion was decreased in tumors established in Notch4-deficient hosts. Unlike in inhibition of Notch1 or Dll4, vessel density and branching in tumors developed in Notch4-deficient mice were unchanged. However, final tumor size was similar between tumors grown in wild type and Notch4 null hosts. CONCLUSION: Our results suggest a novel role for Notch4 in the establishment of tumor colonies and vessel perfusion of transplanted mammary tumors.