Integrating Inflammatory, Hemodynamic, and Metabolic Cues: Context-Dependent and Mechanosensitive Regulation of Endothelial Notch Signaling in Cardiovascular Disease.

Endothelial Notch signaling is critical for early vascular development and survival. Yet, previously described mice lacking endothelial a disintegrin and metalloproteinase 10 (ADAM10), a key regulator of Notch signaling, survived into adulthood with organ-specific vascular defects. These findings raised questions about whether these vascular defects were related to Notch signaling or other functions of ADAM10. The aims of the study are to determine whether compensatory or redundant functions of ADAM17 in Notch signaling can explain the survival of Adam10ΔEC mice, explore the contribution of different Tie2-Cre transgenes to the differences in survival, and establish whether the Adam10ΔEC vascular phenotypes can be recapitulated by inactivation of Notch receptors in endothelial cells. Mice lacking ADAM10 and ADAM17 in endothelial cells (Adam10/Adam17ΔEC), which survived postnatally with organ-specific vascular defects, resembled Adam10ΔEC mice. In contrast, Adam10ΔEC mice generated with the Tie2Cre transgene previously used to inactivate endothelial Notch (Adam10ΔEC(Flv)) died by E10.5. Quantitative polymerase chain reaction analysis demonstrated that Cre-mediated recombination occurs earlier in Adam10ΔEC(Flv) mice than in the previously described Adam10ΔEC mice. Finally, mice lacking endothelial Notch1 (Notch1ΔEC) share some organ-specific vascular defects with Adam10ΔEC mice, whereas Notch4(-/-) mice lacking endothelial Notch1 (Notch1ΔEC/Notch4(-/-)) had defects in all vascular beds affected in Adam10ΔEC mice. Our results argue against a major role for ADAM17 in endothelial Notch signaling and clarify the difference in phenotypes of previously described mice lacking ADAM10 or Notch in endothelial cells. Most notably, these findings uncover new roles for Notch signaling in the development of organ-specific vascular beds.

The Vacuolar Proton Pump, V-ATPase, Is Required for Notch Signaling and Endosomal Trafficking in Drosophila

Angiogenesis is a fundamental process during embryogenesis, inflammation and wound healing. The formation of new vessels is coordinated by proteins of the VEGF and the Notch signaling cascades. Dysfunction of the precisely balanced crosstalk between Notch and VEGF signaling entails the formation of a non-functional vascular network. These imbalances play a critical role during progression of many diseases including atherosclerosis and tumor growth. Blocking of Notch signaling, by small molecule inhibitors or DLL4-specific antibodies, perturbs tumor perfusion and inhibits tumor growth in animal models. This study aimed to gain deeper insight into the complex function of Notch signaling in the endothelium. For this purpose, small soluble Notch ligand and receptor peptides were generated, which consist of the respective interaction domains only. Furthermore, the effects of deleting endothelial Notch signaling in adult mice were investigated. Application of the designed soluble DLL1, DLL4, and JAG1 ligands, as well as, the soluble NOTCH1 receptor blocked Notch signaling in endothelial and myogenic cells. All soluble ligands consistently exerted pro-angiogenic effects in vitro. The effects of DLL1 and DLL4 were markedly stronger than that of the JAG1 ligand and could also evoke elevated sprouting angiogenesis in the retina of newborn mice. Treatment with the soluble Notch receptor reduced endothelial sprouting in vitro. However, in vivo application of soluble NOTCH1 receptor protein resulted in increased retinal sprouting with elevated numbers of tip cells. Thus, the soluble ligands suppressed Notch receptor activity by acting as competitors for endogenous membrane-bound ligands; whereas the soluble receptor acted as a decoy for the different Notch ligands. Genetic studies with adult mice after endothelial-specific deletion of Notch signaling were performed. These mice developed cardiomyopathy within a few months; whereas vascular tumors developed after one year. In an ApoE-deficient model of hyperlipidemia, the deletion of endothelial Notch signaling improved the glucose tolerance of mice, but caused development of steatohepatitis. Thus, Notch signaling in the adult vasculature could be identified as a critical regulator of organ homeostasis as well as glucose and fat metabolism.

Angiogenesis expands the vascular network during normal development and in response to angiogenic stress. Dysregulation of this dynamic process contributes to tumor progression and to the pathogenesis of many diseases. Evidence suggests that the alpha2beta1 integrin, a collagen and laminin receptor, plays an important role in angiogenesis. In the wound-healing and tumor microenvironment, deletion of the alpha2beta1 integrin has been reported to increase neoangiogenesis. In contrast, small molecule inhibitor (SMI) targeting of the alpha2 integrin blocks sprouting angiogenesis. To reconcile these divergent findings and gain a fuller understanding of alpha2beta1 integrin's role in angiogenesis we turned to the retina. The retinal model is uniquely suited for angiogenesis investigations as the retinal vasculature develops postnatally in a 2-dimensional plane in a well-characterized manner. Evaluation of the alpha2-null retina reveals a constellation of defects and delays in vascular development, including delayed vessel outgrowth, and increased vessel irregularity and decreased plexus density at the vascular front. Additionally we determined that alpha2 integrin-deletion has a protective effect in an oxygen-induced retinopathy model of retinopathy of prematurity (ROP) in mice by inhibiting both hyperoxia-induced vaso-obliteration and hypoxia-induced pathologic neovascularization. Confirming this result, our analysis of human microarray data shows, for the first time, that preterm infants with lower ITGA2 expression are less likely to suffer from ROP. This work clarifies the role of alpha2beta1 integrin in sprouting angiogenesis and raises the intriguing possibility of alpha2 integrin targeted therapies for prevention of ROP. These changes are reminiscent of changes observed in other models with dysregulated notch signaling. Recent studies reported that the alpha2beta1 integrin regulates sprouting angiogenesis by inducing DLL4 in ‘tip cells’. We show, for the first time, notch induced downregulation of alpha2beta1 integrin expression in ‘stalk cells’. Together these results suggest that the alpha2beta1 integrin coordinates endothelial notch signaling by stabilizing tip-stalk status. The apparent discrepancy between the effects of the alpha2 integrin inhibition and integrin-deletion may reflect differences between acute and chronic upregulation of notch signaling. We propose that synergistic use of notch and alpha2 integrin targeted therapies may provide enhanced anti-tumor angiogenesis. Citation Format: Aasakiran Madamanchi, Megan Capozzi, Ling Geng, Zhengzhi Li, Zhonghua Zhang, Richard Friedman, Kent Dickeson, John Penn, Mary Zutter. Alpha2beta1 integrin regulation of endothelial notch signaling in the retina. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3900. doi:10.1158/1538-7445.AM2013-3900

To control sprouting angiogenesis, endothelial Notch signaling suppresses tip cell formation, migration, and proliferation while promoting barrier formation. Each of these responses may be regulated by distinct Notch-regulated effectors. Notch activity is highly dynamic in sprouting endothelial cells, while constitutive Notch signaling drives homeostatic endothelial polarization, indicating the need for both rapid and constitutive Notch targets. In contrast to previous screens that focus on genes regulated by constitutively active Notch, we characterized the dynamic response to Notch. We examined transcriptional changes from 1.5 to 6 h after Notch signal activation via ligand-specific or EGTA induction in cultured primary human endothelial cells and neonatal mouse brain. In each combination of endothelial type and Notch manipulation, transcriptomic analysis identified distinct but overlapping sets of rapidly regulated genes and revealed many novel Notch target genes. Among the novel Notch-regulated signaling pathways identified were effectors in GPCR signaling, notably, the constitutively active GTPase RND1. In endothelial cells, RND1 was shown to be a novel direct Notch transcriptional target and required for Notch control of sprouting angiogenesis, endothelial migration, and Ras activity. We conclude that RND1 is directly regulated by endothelial Notch signaling in a rapid fashion in order to suppress endothelial migration.

Notch signalling is a key intercellular communication mechanism that is essential for cell specification and tissue patterning, and which coordinates critical steps of blood vessel growth. Although subtle alterations in Notch activity suffice to elicit profound differences in endothelial behaviour and blood vessel formation, little is known about the regulation and adaptation of endothelial Notch responses. Here we report that the NAD(+)-dependent deacetylase SIRT1 acts as an intrinsic negative modulator of Notch signalling in endothelial cells. We show that acetylation of the Notch1 intracellular domain (NICD) on conserved lysines controls the amplitude and duration of Notch responses by altering NICD protein turnover. SIRT1 associates with NICD and functions as a NICD deacetylase, which opposes the acetylation-induced NICD stabilization. Consequently, endothelial cells lacking SIRT1 activity are sensitized to Notch signalling, resulting in impaired growth, sprout elongation and enhanced Notch target gene expression in response to DLL4 stimulation, thereby promoting a non-sprouting, stalk-cell-like phenotype. In vivo, inactivation of Sirt1 in zebrafish and mice causes reduced vascular branching and density as a consequence of enhanced Notch signalling. Our findings identify reversible acetylation of the NICD as a molecular mechanism to adapt the dynamics of Notch signalling, and indicate that SIRT1 acts as rheostat to fine-tune endothelial Notch responses.

Signaling by Notch receptors is a key cell-cell communication mechanism essential for development and homeostasis of the cardiovascular system. Several critical steps of angiogenic blood vessel growth are coordinated by the Notch pathway and even subtle perturbations in Notch activity can profoundly influence endothelial cell behavior and blood vessel growth, yet very little is known about the dynamics and intrinsic regulation of endothelial Notch signaling. Here, we show that reversible acetylation regulates the amplitude and duration of Notch responses by altering protein turnover of the Notch1 intracellular domain (NICD) in endothelial cells. We report that the histone acetyltransferases (HATs) p300 and PCAF not only acetylate nucleosomal histones at Notch target promoters, but also the NICD itself. Acetylation of NICD induced by p300 and PCAF results in increased NICD stability and enhanced Notch target gene expression. Likewise, blocking histone deacetylase (HDAC) activity using the class III HDAC inhibitor nicotinamid (NAM) leads to a substantial increase in NICD acetylation while class I/II HDAC inhibition has no significant effect suggesting that class III HDACs primarily target NICD for deacetylation. Among the class III HDACs with deacetylase activity (SIRT1, 2, 3, 5), specifically SIRT1 associates with NICD through its catalytic domain and silencing of SIRT1 enhances NICD acetylation. Wild type SIRT1, but not a catalytically inactive mutant catalyzes the deacetylation of NICD in a nicotinamid-dependent manner indicating that SIRT1 acts as a direct NICD deacetylase, which counteracts HAT-induced NICD acetylation. Inactivation of SIRT1 by NAM or RNAi-mediated knock down results in enhanced NICD protein stability by blocking proteasomal degradation. Consistent with these results, knock down of SIRT1 amplifies Notch target gene expression in endothelial cells in response to NICD overexpression or treatment with the Notch ligand Delta-like 4 (Dll4). Taken together, these findings support a model, in which Notch signaling is dynamically regulated by reversible acetylation and suggest that the antagonistic activities of p300/PCAF and SIRT1 act as rheostat to fine-tune endothelial Notch responses.

The Notch receptor is a key mediator of developmental programs and cell-fate decisions. Imbalanced Notch signaling leads to developmental disorders and cancer. To fully characterize the Notch signaling pathway and exploit it in novel therapeutic interventions, a comprehensive view on the regulation and requirements of Notch signaling is needed. Notch is regulated at different levels, ranging from ligand binding, stability to endocytosis. Using an array of different techniques, including reporter gene assays, immunocytochemistry, and ChIP-qPCR we show here, to the best of our knowledge for the first time, regulation of Notch signaling at the level of the nuclear pore. We found that the nuclear pore protein Nup214 (nucleoporin 214) and its interaction partner Nup88 negatively regulate Notch signaling in vitro and in vivo in zebrafish. In mammalian cells, loss of Nup88/214 inhibited nuclear export of recombination signal-binding protein for immunoglobulin κJ region (RBP-J), the DNA-binding component of the Notch pathway. This inhibition increased binding of RBP-J to its cognate promoter regions, resulting in increased downstream Notch signaling. Interestingly, we also found that NUP214 fusion proteins, causative for certain cases of T-cell acute lymphatic leukemia, potentially contribute to tumorigenesis via a Notch-dependent mechanism. In summary, the nuclear pore components Nup88/214 suppress Notch signaling in vitro, and in zebrafish, nuclear RBP-J levels are rate-limiting factors for Notch signaling in mammalian cells, and regulation of nucleocytoplasmic transport of RBP-J may contribute to fine-tuning Notch activity in cells.

The highly conserved NOTCH (neurogenic locus notch homolog protein) signaling pathway functions as a key cell-cell interaction mechanism controlling cell fate and tissue patterning, whereas its dysregulation is implicated in a variety of developmental disorders and cancers. The pivotal role of endothelial NOTCH in regulation of angiogenesis is widely appreciated; however, little is known about what controls its signal transduction. Our previous study indicated the potential role of post-translational SUMO (small ubiquitin-like modifier) modification (SUMOylation) in vascular disorders. The aim of this study was to investigate the role of SUMOylation in endothelial NOTCH signaling and angiogenesis. Endothelial SENP1 (sentrin-specific protease 1) deletion, in newly generated endothelial SENP1 (the major protease of the SUMO system)-deficient mice, significantly delayed retinal vascularization by maintaining prolonged NOTCH1 signaling, as confirmed in cultured endothelial cells. An in vitro SUMOylation assay and immunoprecipitation revealed that when SENP1 associated with N1ICD (NOTCH1 intracellular domain), it functions as a deSUMOylase of N1ICD SUMOylation on conserved lysines. Immunoblot and immunoprecipitation analyses and dual-luciferase assays of natural and SUMO-conjugated/nonconjugated NOTCH1 forms demonstrated that SUMO conjugation facilitated NOTCH1 cleavage. This released N1ICD from the membrane and stabilized it for translocation to the nucleus where it functions as a cotranscriptional factor. Functionally, SENP1-mediated NOTCH1 deSUMOylation was required for NOTCH signal activation in response to DLL4 (Delta-like 4) stimulation. This in turn suppressed VEGF (vascular endothelial growth factor) receptor signaling and angiogenesis, as evidenced by immunoblotted signaling molecules and in vitro angiogenesis assays. These results establish reversible NOTCH1 SUMOylation as a regulatory mechanism in coordinating endothelial angiogenic signaling; SENP1 acts as a critical intrinsic mediator of this process. These findings may apply to NOTCH-regulated biological events in nonvascular tissues and provide a novel therapeutic strategy for vascular diseases and tumors.

This editorial refers to ‘Endothelial Jag1-RBPJ signalling promotes inflammatory leucocyte recruitment and atherosclerosis’ by M. Nus et al. , pp. 568–580 Notch signalling is an evolutionary conserved regulator of vascular and immune cell development and homeostasis.1,2 In this issue of Cardiovascular Research , Nus et al. 3 now report a new role of endothelial Notch signalling in regulating vascular homeostasis by regulating vascular inflammation in atherosclerosis. Activation of Notch receptors is controlled by membrane-bound Notch ligands consisting of the Jagged (Jag) and Delta-like (Dll) gene families.2 Notch signalling regulates endothelial cell proliferation, migration, and sprouting angiogenesis and thereby critically regulates vascular morphogenesis and function.1,4,5 In response to ischaemic vascular injury, Notch activation mediates vascular repair and regeneration, which ensures neovascularization of tissues, organs, and recovery of their function.6,7 In this issue of Cardiovascular Research , Nus et al. 3 now report that endothelial Notch signalling regulates homing of leucocytes to the vascular wall during atherosclerosis. They demonstrate that the Notch ligand Jag1 and Notch signalling are upregulated in atherosclerotic human and mouse arteries. Furthermore, …

Notch signaling is an evolutionarily conserved pathway that is widely used for multiple cellular events during development. Activation of the Notch pathway occurs when the ligand from a neighboring cell binds to the Notch receptor and induces cleavage of the intracellular domain of Notch, which further translocates into the nucleus to activate its downstream genes. The involvement of the Notch pathway in diverse biological events is possible due to the complexity in its regulation. In order to maintain tight spatiotemporal regulation, the Notch receptor, as well as its ligand, undergoes a series of physical and biochemical modifications that, in turn, helps in proper maintenance and fine-tuning of the signaling outcome. Ubiquitination is the post-translational addition of a ubiquitin molecule to a substrate protein, and the process is regulated by E3 ubiquitin ligases. The present review describes the involvement of different E3 ubiquitin ligases that play an important role in the regulation and maintenance of proper Notch signaling and how perturbation in ubiquitination results in abnormal Notch signaling leading to a number of human diseases.

Hypoxia, characterized by reduced oxygen concentration, is a significant stressor that affects the survival of aerobic species and plays a prominent role in cardiovascular diseases. From the research history and milestone events related to hypoxia in cardiovascular development and diseases, The "hypoxia-inducible factors (HIFs) switch" can be observed from both temporal and spatial perspectives, encompassing the occurrence and progression of hypoxia (gradual decline in oxygen concentration), the acute and chronic manifestations of hypoxia, and the geographical characteristics of hypoxia (natural selection at high altitudes). Furthermore, hypoxia signaling pathways are associated with natural rhythms, such as diurnal and hibernation processes. In addition to innate factors and natural selection, it has been found that epigenetics, as a postnatal factor, profoundly influences the hypoxic response and progression within the cardiovascular system. Within this intricate process, interactions between different tissues and organs within the cardiovascular system and other systems in the context of hypoxia signaling pathways have been established. Thus, it is the time to summarize and to construct a multi-level regulatory framework of hypoxia signaling and mechanisms in cardiovascular diseases for developing more therapeutic targets and make reasonable advancements in clinical research, including FDA-approved drugs and ongoing clinical trials, to guide future clinical practice in the field of hypoxia signaling in cardiovascular diseases.

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. 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).

The vertebrate skin is a barrier-forming organ in which keratinocytes form a highly organized, stratified epithelium protecting the organism from the outside environment. One of the major regulators of this structure is Notch signaling. Notch is a transmembrane receptor interacting with ligands expressed on the surface of neighboring keratinocytes. Keratinocyte-specific deletion of Notch signaling pathway impairs epidermal differentiation, resulting in skin-barrier defects and skin carcinogenesis. Our laboratory, by a genetic approach in mice, demonstrates that combined inactivation of Epsin1 and Epsin2 genes leads to embryonic lethality around E9.5-10. The phenotype of Epn1;Epn2 double knockout is characterized by a subversion of the three main developmental programs active at this developmental stage, i.e., cardiovascular development, somitogenesis, and neural tube differentiation. Collectively, these morphological alterations resemble the developmental defects observed in mutants of Notch genes or in genes essential for the activation of the Notch signaling pathway, suggesting a crucial role of Epsin in enabling Notch signaling during embryogenesis. Intriguingly, the apparently healthy Epn1+/-;Epn2-/- shows an high incidence of squamous papillomas on their skin. Furthermore, expression of another epsin family member originally localized exclusively to surface epithelia, Epsin3, dramatically increases in the hyperplastic lesions of the three-allele mutants and in human basal carcinomas. In order to get further insight on Epn3 function we performed morphological expression analyses during mouse development. In contrast with initial reports, both in embryos and adults, we could detect various levels of Epn3 expression in several tissues, i.e., surface epithelia, neural tissue and heart. Moreover, in vitro studies performed on human keratinocytes in culture show a prominent role of this Epsin in the regulation of Notch signaling in this cell compartment.

The regulation of Notch signaling in muscle stem cell activation and postnatal myogenesis