Endothelial Notch Signaling Research Articles

Notch signaling regulates UNC5B to suppress endothelial proliferation, migration, junction activity, and retinal plexus branching

Notch signaling guides vascular development and function by regulating diverse endothelial cell behaviors, including migration, proliferation, vascular density, endothelial junctions, and polarization in response to flow. Notch proteins form transcriptional activation complexes that regulate endothelial gene expression, but few of the downstream effectors that enable these phenotypic changes have been characterized in endothelial cells, limiting our understanding of vascular Notch activities. Using an unbiased screen of translated mRNA rapidly regulated by Notch signaling, we identified novel in vivo targets of Notch signaling in neonatal mouse brain endothelium, including UNC5B, a member of the netrin family of angiogenic-regulatory receptors. Endothelial Notch signaling rapidly upregulates UNC5B in multiple endothelial cell types. Loss or gain of UNC5B recapitulated specific Notch-regulated phenotypes. UNC5B expression inhibited endothelial migration and proliferation and was required for stabilization of endothelial junctions in response to shear stress. Loss of UNC5B partially or wholly blocked the ability of Notch activation to regulate these endothelial cell behaviors. In the developing mouse retina, endothelial-specific loss of UNC5B led to excessive vascularization, including increased vascular outgrowth, density, and branchpoint count. These data indicate that Notch signaling upregulates UNC5B as an effector protein to control specific endothelial cell behaviors and inhibit angiogenic growth.

Endothelial cell Notch signaling programs cancer-associated fibroblasts to promote tumor immune evasion.

Stromal cells within the tumor tissue promote immune evasion as a critical strategy for cancer development and progression, but the underlying mechanisms remain poorly understood. In this study, we explore the role of endothelial cells (ECs) in the regulation of the immunosuppressive tumor microenvironment. Using mouse pancreatic ductal adenocarcinoma (PDAC) models, we found that canonical Notch signaling in endothelial cells suppresses the recruitment of antitumor T cells and promotes tumor progression by inhibiting the pro-inflammatory functions of cancer-associated fibroblasts (CAFs). Abrogation of endothelial Notch signaling modulates EC-derived angiocrine factors to enhance the pro-inflammatory activities of CAFs, which drive CXCL9/10-CXCR3-mediated T cell recruitment to inhibit tumor growth. Additionally, abrogation of endothelial Notch unleashed interferon gamma responses in the tumor microenvironment, upregulated PDL1 expression on tumor cells, and sensitized PDAC to PD1-based immunotherapy. Collectively, these data uncover a pivotal role of endothelial cells in shaping the immunosuppressive microenvironment, and suggest the potential of targeting EC-CAF interaction as a novel therapeutic modality to boost antitumor immunity.

Angiogenesis is uncoupled from osteogenesis during calvarial bone regeneration

Bone regeneration requires a well-orchestrated cellular and molecular response including robust vascularization and recruitment of mesenchymal and osteogenic cells. In femoral fractures, angiogenesis and osteogenesis are closely coupled during the complex healing process. Here, we show with advanced longitudinal intravital multiphoton microscopy that early vascular sprouting is not directly coupled to osteoprogenitor invasion during calvarial bone regeneration. Early osteoprogenitors emerging from the periosteum give rise to bone-forming osteoblasts at the injured calvarial bone edge. Microvessels growing inside the lesions are not associated with osteoprogenitors. Subsequently, osteogenic cells collectively invade the vascularized and perfused lesion as a multicellular layer, thereby advancing regenerative ossification. Vascular sprouting and remodeling result in dynamic blood flow alterations to accommodate the growing bone. Single cell profiling of injured calvarial bones demonstrates mesenchymal stromal cell heterogeneity comparable to femoral fractures with increase in cell types promoting bone regeneration. Expression of angiogenesis and hypoxia-related genes are slightly elevated reflecting ossification of a vascularized lesion site. Endothelial Notch and VEGF signaling alter vascular growth in calvarial bone repair without affecting the ossification progress. Our findings may have clinical implications for bone regeneration and bioengineering approaches.

Endothelial Notch Signaling Regulates the Function of the Retinal Pigment Epithelial Barrier via EC Angiocrine Signaling.

The outer blood-retina barrier (oBRB), comprises tightly connected retinal pigment epithelium (RPE) cells, Bruch's membrane, and choroid blood vessels, and is essential for retinal health and normal visual function. Disruption of the RPE barrier and its dysfunction can lead to retinal disorders such as age-related macular degeneration (AMD). In the present study, we investigated the essential role of choroid endothelial cells (ECs) in the RPE barrier formation process and its dysfunction. We discovered that ECs promoted RPE barrier formation through angiocrine signaling. Through blocking or activating endothelial Notch signaling and conducting experiments in vitro and in vivo, we confirmed that endothelial Notch signaling regulated the expression of heparin-binding epidermal growth factor (HBEGF) and consequently impacted the expression and activity of matrix metalloproteinases (MMP)-9 in RPE cells. This modulation influenced the RPE extracellular matrix deposition, tight junctions and RPE barrier function. In in vivo experiments, the intravitreal administration of recombinant HBEGF (r-HBEGF) alleviated the RPE barrier disruption induced by subretinal injection (SI) or laser treatment and also rescued RPE barrier disruption in endothelial Notch-deficient mice. Our results showed that the endothelial Notch signaling drove HBEGF expression through angiocrine signaling and effectively improved RPE barrier function by regulating the MMP-9 expression in RPE cells. It suggests that the modulation of Notch signaling in the choroidal endothelium may offer a novel therapeutic strategy for retinal degenerative diseases.

A new fasciocutaneous flap model identifies a critical role for endothelial Notch signaling in wound healing and flap survival

Flap surgery is a common treatment for severe wounds and a major determinant of surgical outcome. Flap survival and healing depends on adaptation of the local flap vasculature. Using a novel and defined model of fasciocutaneous flap surgery, we demonstrate that the Notch ligand Delta-like 1 (Dll1), expressed in vascular endothelial cells, regulates flap arteriogenesis, inflammation and flap survival. Utilizing the stereotyped anatomy of dorsal skin arteries, ligation of the major vascular pedicle induced strong collateral vessel development by end-to-end anastomosis in wildtype mice, which supported flap perfusion recovery over time. In mice with heterozygous deletion of Dll1, collateral vessel formation was strongly impaired, resulting in aberrant vascularization and subsequent necrosis of the tissue. Furthermore, Dll1 deficient mice showed severe inflammation in the flap dominated by monocytes and macrophages. This process is controlled by endothelial Dll1 in vivo, since the results were recapitulated in mice with endothelial-specific deletion of Dll1. Thus, our model provides a platform to study vascular adaptation to flap surgery and molecular and cellular regulators influencing flap healing and survival.

Abstract LB009: Notch4 inhibition by a novel neutralizing antibody reduces tumor progression and increases macrophage recruitment within the tumor microenvironment

Abstract The Notch signaling pathway, particularly endothelial Notch signaling, plays a key role in tumor formation and angiogenesis. Inhibiting Notch1 or its ligand, Delta-like ligand 4 (Dll4), can induce hypersprouting of the tumor vasculature and thus can reduce tumor perfusion and progression. However, due to the expression of Notch1 in multiple healthy cell types, pharmacologic blockade poses the challenge of inducing tissue hypoxia, vascular neoplasms, and toxicity in non-target tissues. Unlike Notch1, Notch4 expression is largely restricted to endothelium and is upregulated in models of triple negative breast cancer (TNBC). Loss of Notch4 in mice results in delayed developmental angiogenesis and reduced mammary tumor angiogenesis and perfusion. Therefore, Notch4 is an attractive potential pharmacologic target to interfere with tumorigenesis and minimize nonspecific toxicity. We determined that Notch4 is expressed primarily in tumor endothelium in both human and mouse mammary tumors, including murine syngeneic Py8119 tumors, as well as a selection of other human and syngeneic mouse tumor types such as B16F10 melanoma tumors. We explored the effects of Notch4 inhibition on the progression of murine mammary epithelial carcinoma using newly developed anti-Notch4 antibodies, 6-3-A6 and its humanized derivative E7011. Administration of E7011 or 6-3-A6 to orthotopically implanted Py8119 murine breast carcinoma and B16F10 melanoma significantly reduced tumor size in comparison to IgG-treated controls. We performed single cell RNA sequencing of Py8119 tumors treated with E7011 and found that although Notch4 transcripts were found specifically in the endothelium, the most notable effect of E7011 was a dramatic increase in the number of macrophages and cancer-associated fibroblasts, suggesting a non-cell autonomous effect. To examine the potential role of these immune cell population changes in facilitating E7011’s anti-tumor activity, we investigated treatment of Py8119 murine mammary carcinomas in the absence of macrophages. Administration of clodronate liposomes to eliminate macrophages blocked the anti-tumor effects of E7011 treatment. Taken together, our data suggests that tumor endothelial Notch4 promotes TNBC growth by altering the immune landscape and increasing anti-tumor macrophage recruitment, and that blockade via the novel anti-Notch4 E7011 antibody has potential therapeutic efficacy. Citation Format: Jason W. Eng, L.A. Naiche, Bhairavi Swaminathan, Yu Kato, Krishna Thakkar, Katherine Ann Alexander, Rahul Vadakath, James H. Herts, Rajyasree Emmadi, Junji Matsui, Jan K. Kitajewski. Notch4 inhibition by a novel neutralizing antibody reduces tumor progression and increases macrophage recruitment within the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB009.

The sinusoidal hematopoietic niche is formed by Jam1a via Notch signaling in the zebrafish kidney

The zebrafish is a unique model to understand hematopoietic niches as hematopoietic stem/progenitor cells are maintained in the kidney. However, little is known about which cell types in the kidney play a role in hematopoietic niches. Here, we demonstrate that the sinusoidal endothelium is an essential and conserved niche component in the zebrafish kidney. Histological analysis revealed that runx1:mCherry + hematopoietic cells were predominantly detected in the dorsolateral region of the kidney where sinusoids are highly developed. Loss of Junctional adhesion molecule 1a (Jam1a), which is expressed in both sinusoidal endothelial cells and hematopoietic cells, resulted in a remarkable reduction in sinusoids and a defect in hematopoietic niches. We found that Jam1a regulates jagged-1a expression in vascular endothelial cells to form a sinusoidal structure in the kidney. Collectively, these data suggest that sinusoids are formed by Jam1a via endothelial Notch signaling to provide hematopoietic niches in the zebrafish kidney.

Endothelial RBPJ Is Essential for the Education of Tumor-Associated Macrophages.

Endothelial Notch signaling favors immunosuppression by increasing CXCL2 secretion to stimulate CD44 expression in macrophages, facilitating their education by tumor cells.

Notch Signaling in the Vasculature: Angiogenesis and Angiocrine Functions.

Formation of a functional blood vessel network is a complex process tightly controlled by pro- and antiangiogenic signals released within the local microenvironment or delivered through the bloodstream. Endothelial cells precisely integrate such temporal and spatial changes in extracellular signals and generate an orchestrated response by modulating signaling transduction, gene expression, and metabolism. A key regulator in vessel formation is Notch signaling, which controls endothelial cell specification, proliferation, migration, adhesion, and arteriovenous differentiation. This review summarizes the molecular biology of endothelial Notch signaling and how it controls angiogenesis and maintenance of the established, quiescent vasculature. In addition, recent progress in the understanding of Notch signaling in endothelial cells for controlling organ homeostasis by transcriptional regulation of angiocrine factors and its relevance to disease will be discussed.

Loss of vascular endothelial notch signaling promotes spontaneous formation of tertiary lymphoid structures

Tertiary lymphoid structures (TLS) are lymph node-like immune cell clusters that emerge during chronic inflammation in non-lymphoid organs like the kidney, but their origin remains not well understood. Here we show, using conditional deletion strategies of the canonical Notch signaling mediator Rbpj, that loss of endothelial Notch signaling in adult mice induces the spontaneous formation of bona fide TLS in the kidney, liver and lung, based on molecular, cellular and structural criteria. These TLS form in a stereotypical manner around parenchymal arteries, while secondary lymphoid structures remained largely unchanged. This effect is mediated by endothelium of blood vessels, but not lymphatics, since a lymphatic endothelial-specific targeting strategy did not result in TLS formation, and involves loss of arterial specification and concomitant acquisition of a high endothelial cell phenotype, as shown by transcriptional analysis of kidney endothelial cells. This indicates a so far unrecognized role for vascular endothelial cells and Notch signaling in TLS initiation.

Endothelial Notch signaling directly regulates the small GTPase RND1 to facilitate Notch suppression of endothelial migration

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.

Abstract 15169: De Novo Variants of USP10 in Early Onset Bicuspid Aortic Valve Disease

Introduction: Bicuspid Aortic Valve (BAV), the most common congenital heart defect, is a major cause of aortic regurgitation or stenosis requiring valve replacement and thoracic aortic aneurysms predisposing to acute aortic dissections (TAD). The spectrum of BAV ranges from severe early onset valve and aortic complications to sporadic late onset disease. Hypothesis: Early onset BAV (EBAV) cases with valve or aortic complications that require intervention prior to age 30 are enriched for rare genetic variants that cause BAV and TAD. Methods: We performed whole exome sequencing of 147 EBAV cases in 141 families who were enrolled in the UTHealth Bicuspid Aortic Valve Research Registry. Candidate variants in the EBAV cohort (26% female, mean age 18, 44% with TAD) were compared to unselected controls from the Genome Aggregation Database (gnoMAD) and the Database of Genotypes and Phenotypes (dbGAP). We considered variants with minor allele frequencies (MAF) < 1%, Combined Annotation Dependent Depletion (CADD) scores > 25, and damaging (Polyphen-2) or deleterious (SIFT) functional prediction scores. Genomic copy number variants (CNVs) were detected using CoNIFER and prioritized when deletions involved genes with probability of loss intolerance (pLI) > 0.9. Variants were validated using quantitative PCR or Sanger sequencing. Results: We identified 6 rare variants of USP10 in 6 EBAV families (4% of cohort): 4 CNVs (2 duplications and 2 deletions) that are rare in dbGAP controls (4 in 15,414) and 2 deleterious rare missense variants (MAF<5x10 -5 in gnoMAD). Two of the 4 CNVs were de novo events in trios. In contrast, rare deleterious variants of the known causal BAV genes NOTCH1 (1), ROBO4 (1), GATA4 (1), GATA5 (1), and SMAD6 (4) were found in 7 total families. USP10 encodes a ubiquitin peptidase that is required for endothelial Notch signaling during vascular development. Conclusions: We identified rare and de novo variants of USP10 that implicate USP10 as a new candidate gene for BAV.

Endothelial Jagged1 Antagonizes Dll4/Notch Signaling in Decidual Angiogenesis during Early Mouse Pregnancy.

Maternal spiral arteries and newly formed decidual capillaries support embryonic development prior to placentation. Previous studies demonstrated that Notch signaling is active in endothelial cells of both decidual capillaries and spiral arteries, however the role of Notch signaling in physiologic decidual angiogenesis and maintenance of the decidual vasculature in early mouse pregnancy has not yet been fully elucidated. We used the Cdh5-CreERT2; Jagged1(Jag1)flox/flox (Jag1∆EC) mouse model to delete Notch ligand, Jag1, in maternal endothelial cells during post-implantation, pre-placentation mouse pregnancy. Loss of endothelial Jag1 leads to increased expression of Notch effectors, Hey2 and Nrarp, and increased endothelial Notch signaling activity in areas of the decidua with remodeling angiogenesis. This correlated with an increase in Dll4 expression in capillary endothelial cells, but not spiral artery endothelial cells. Consistent with increased Dll4/Notch signaling, we observed decreased VEGFR2 expression and endothelial cell proliferation in angiogenic decidual capillaries. Despite aberrant Dll4 expression and Notch activation in Jag1∆EC mutants, pregnancies were maintained and the decidual vasculature was not altered up to embryonic day 7.5. Thus, Jag1 functions in the newly formed decidual capillaries as an antagonist of endothelial Dll4/Notch signaling during angiogenesis, but Jag1 signaling is not necessary for early uterine angiogenesis.

ECM1 secreted by HER2-overexpressing breast cancer cells promotes formation of a vascular niche accelerating cancer cell migration and invasion

The tumor microenvironment is increasingly recognized as key player in cancer progression. Investigating heterotypic interactions between cancer cells and their microenvironment is important for understanding how specific cell types support cancer. Forming the vasculature, endothelial cells (ECs) are a prominent cell type in the microenvironment of both normal and neoplastic breast gland. Here, we sought out to analyze epithelial–endothelial cross talk in the breast using isogenic non-tumorigenic vs. tumorigenic breast epithelial cell lines and primary ECs. The cellular model used here consists of D492, a breast epithelial cell line with stem cell properties, and two isogenic D492-derived EMT cell lines, D492M and D492HER2. D492M was generated by endothelial-induced EMT and is non-tumorigenic while D492HER2 is tumorigenic, expressing the ErbB2/HER2 oncogene. To investigate cellular cross talk, we used both conditioned medium (CM) and 2D/3D co-culture systems. Secretome analysis of D492 cell lines was performed using mass spectrometry and candidate knockdown (KD), and overexpression (OE) was done using siRNA and CRISPRi/CRISPRa technology. D492HER2 directly enhances endothelial network formation and activates a molecular axis in ECs promoting D492HER2 migration and invasion, suggesting an endothelial feedback response. Secretome analysis identified extracellular matrix protein 1 (ECM1) as potential angiogenic inducer in D492HER2. Confirming its involvement, KD of ECM1 reduced the ability of D492HER2-CM to increase endothelial network formation and induce the endothelial feedback, while recombinant ECM1 (rECM1) increased both. Interestingly, NOTCH1 and NOTCH3 expression was upregulated in ECs upon treatment with D492HER2-CM or rECM1 but not by CM from D492HER2 with ECM1 KD. Blocking endothelial NOTCH signaling inhibited the increase in network formation and the ability of ECs to promote D492HER2 migration and invasion. In summary, our data demonstrate that cancer-secreted ECM1 induces a NOTCH-mediated endothelial feedback promoting cancer progression by enhancing migration and invasion. Targeting this interaction may provide a novel possibility to improve cancer treatment.

Endothelial Notch signaling controls insulin transport in muscle

The role of the endothelium is not just limited to acting as an inert barrier for facilitating blood transport. Endothelial cells (ECs), through expression of a repertoire of angiocrine molecules, regulate metabolic demands in an organ‐specific manner. Insulin flux across the endothelium to muscle cells is a rate‐limiting process influencing insulin‐mediated lowering of blood glucose. Here, we demonstrate that Notch signaling in ECs regulates insulin transport to muscle. Notch signaling activity was higher in ECs isolated from obese mice compared to non‐obese. Sustained Notch signaling in ECs lowered insulin sensitivity and increased blood glucose levels. On the contrary, EC‐specific inhibition of Notch signaling increased insulin sensitivity and improved glucose tolerance and glucose uptake in muscle in a high‐fat diet‐induced insulin resistance model. This was associated with increased transcription of Cav1, Cav2, and Cavin1, higher number of caveolae in ECs, and insulin uptake rates, as well as increased microvessel density. These data imply that Notch signaling in the endothelium actively controls insulin sensitivity and glucose homeostasis and may therefore represent a therapeutic target for diabetes.

MicroRNA-24-3p Targets Notch and Other Vascular Morphogens to Regulate Post-ischemic Microvascular Responses in Limb Muscles.

MicroRNAs (miRs) regulate complex processes, including angiogenesis, by targeting multiple mRNAs. miR-24-3p-3p directly represses eNOS, GATA2, and PAK4 in endothelial cells (ECs), thus inhibiting angiogenesis during development and in the infarcted heart. miR-24-3p is widely expressed in cardiovascular cells, suggesting that it could additionally regulate angiogenesis by acting on vascular mural cells. Here, we have investigated: (1) new miR-24-3p targets; (2) the expression and the function of miR-24-3p in human vascular ECs; (3) the impact of miR-24-3p inhibition in the angiogenesis reparative response to limb ischemia in mice. Using bioinformatics target prediction platforms and 3′-UTR luciferase assays, we newly identified Notch1 and its Delta-like ligand 1 (Dll1) to be directly targeted by miR-24-3p. miR-24-3p was expressed in human ECs and pericytes cultured under normal conditions. Exposure to hypoxia increased miR-24-3p in ECs but not in pericytes. Transfection with a miR-24-3p precursor (pre-miR-24-3p) increased miR-24-3p expression in ECs, reducing the cell survival, proliferation, and angiogenic capacity. Opposite effects were caused by miR-24-3p inhibition. The anti-angiogenic action of miR-24-3p overexpression could be prevented by simultaneous adenovirus (Ad)-mediated delivery of constitutively active Notch intracellular domain (NICD) into cultured ECs. We next demonstrated that reduced Notch signalling contributes to the anti-angiogenic effect of miR-24-3p in vitro. In a mouse unilateral limb ischemia model, local miR-24-3p inhibition (by adenovirus-mediated miR-24-3p decoy delivery) restored endothelial Notch signalling and increased capillary density. However, the new vessels appeared disorganised and twisted, worsening post-ischemic blood perfusion recovery. To better understand the underpinning mechanisms, we widened the search for miR-24-3p target genes, identifying several contributors to vascular morphogenesis, such as several members of the Wingless (Wnt) signalling pathway, β-catenin signalling components, and VE-cadherin, which synergise to regulate angiogenesis, pericytes recruitment to neoformed capillaries, maturation, and stabilization of newly formed vessels. Among those, we next focussed on β-catenin to demonstrate that miR-24-3p inhibition reduces β-catenin expression in hypoxic ECs, which is accompanied by reduced adhesion of pericytes to ECs. In summary, miR-24-3p differentially targets several angiogenesis modulators and contributes to autonomous and non-autonomous EC crosstalk. In ischemic limbs, miR-24-3p inhibition increases the production of dysfunctional microvessels, impairing perfusion. Caution should be observed in therapeutic targeting of miR-24-3p.

Endothelial Notch activation promotes neutrophil transmigration via downregulating endomucin to aggravate hepatic ischemia/reperfusion injury.

Inflammatory leukocytes infiltration is orchestrated by mechanisms involving chemokines, selectins, addressins and other adhesion molecules derived from endothelial cells (ECs), but how they respond to inflammatory cues and coordinate leukocyte transmigration remain elusive. In this study, using hepatic ischemia/reperfusion injury (HIRI) as a model, we identified that endothelial Notch activation was rapidly and dynamically induced in liver sinusoidal endothelial cells (LSECs) in acute inflammation. In mice with EC-specific Notch activation (NICeCA), HIRI induced exacerbated liver damage. Consistently, endothelial Notch activation enhanced neutrophil infiltration and tumor necrosis factor (TNF)-α expression in HIRI. Transcriptome analysis and further qRT-PCR as well as immunofluorescence indicated that endomucin (EMCN), a negative regulator of leukocyte adhesion, was downregulated in LSECs from NICeCA mice. EMCN was downregulated during HIRI in wild-type mice and in vitro cultured ECs insulted by hypoxia/re-oxygenation injury. Notch activation in ECs led to increased neutrophil adhesion and transendothelial migration, which was abrogated by EMCN overexpression in vitro. In mice deficient of RBPj, the integrative transcription factor of canonical Notch signaling, although overwhelming sinusoidal malformation aggravated HIRI, the expression of EMCN was upregulated; and pharmaceutical Notch blockade in vitro also upregulated EMCN and inhibited transendothelial migration of neutrophils. The Notch activation-exaggerated HIRI was compromised by blocking LFA-1, which mediated leukocyte adherence by associating with EMCN. Therefore, endothelial Notch signaling controls neutrophil transmigration via EMCN to modulate acute inflammation in HIRI.

Retinal myeloid cells regulate tip cell selection and vascular branching morphogenesis via Notch ligand Delta-like 1

During angiogenesis, single endothelial cells (EC) specialize into tip cells that guide vessel sprouting towards growth factor gradients and instruct the adjacent vessel stalk. The balance between tip and stalk cells is regulated by endothelial Notch signalling through the expression of Notch ligand Delta-like 4 (Dll4) in tip cells, which suppresses a tip cell fate in adjacent stalk cells. Here we show, using genetic reporter and conditional deletion strategies, that myeloid cells regulate tip cell numbers and Dll4 expression via the Notch ligand Dll1 during vascular development in the retina. Dll1 is selectively expressed by a subpopulation of retinal myeloid cells, which progressively localizes to the sprouting vascular network. Conditional, myeloid-specific deletion of Dll1 impairs endothelial Dll4 tip-stalk gradient resulting in an increase of endothelial tip cells and EC filopodia, accompanied by an increase in vascular density and branching. In vitro, co-culture of human EC with monocyte-derived macrophages induced Dll1 upregulation in macrophages and Dll4 upregulation and an endothelial tip cell signature in EC. Furthermore, culturing human EC on recombinant DLL1 induced endothelial Dll4 expression and a tip cell program, indicating that changes are Dll1-dependent. Thus, myeloid cells regulate tip cell fate and angiogenesis through expression of Notch ligand Dll1.

Inactivation of the serine protease HTRA1 inhibits tumor growth by deregulating angiogenesis.

The serine protease HTRA1 is involved in several vascular diseases and its expression is often deregulated in cancer. We aimed at identifying how HTRA1 in the vasculature affects tumor growth. Here we report that silencing of HTRA1 in cultured endothelial cells increased migration rate and tube formation, whereas forced HTRA1 expression impaired sprouting angiogenesis. Mechanistically, endothelial HTRA1 expression enhanced Delta/Notch signaling by reducing the amount of the weak Notch ligand JAG1. HTRA1 physically interacted with JAG1 and cleaved it within the intracellular domain, leading to protein degradation. Expression of a constitutive active Notch1 prevented the hypersprouting phenotype upon silencing of HTRA1. In HtrA1-deficient mice, endothelial Notch signaling was diminished and isolated endothelial cells had increased expression of VEGF receptor-2. Growth of syngeneic tumors was strongly impaired in HtrA1-/- mice. The tumor vasculature was much denser in HtrA1-/- mice and less covered with mural cells. This chaotic and immature vascular network was poorly functional as indicated by large hypoxic tumor areas and low tumor cell proliferation rates. In summary, inhibition of HTRA1 in the tumor stroma impaired tumor progression by deregulating angiogenesis.

Inhibition of Endothelial Notch Signaling Impairs Fatty Acid Transport and Leads to Metabolic and Vascular Remodeling of the Adult Heart.

Nutrients are transported through endothelial cells before being metabolized in muscle cells. However, little is known about the regulation of endothelial transport processes. Notch signaling is a critical regulator of metabolism and angiogenesis during development. Here, we studied how genetic and pharmacological manipulation of endothelial Notch signaling in adult mice affects endothelial fatty acid transport, cardiac angiogenesis, and heart function. Endothelial-specific Notch inhibition was achieved by conditional genetic inactivation of Rbp-jκ in adult mice to analyze fatty acid metabolism and heart function. Wild-type mice were treated with neutralizing antibodies against the Notch ligand Delta-like 4. Fatty acid transport was studied in cultured endothelial cells and transgenic mice. Treatment of wild-type mice with Delta-like 4 neutralizing antibodies for 8 weeks impaired fractional shortening and ejection fraction in the majority of mice. Inhibition of Notch signaling specifically in the endothelium of adult mice by genetic ablation of Rbp-jκ caused heart hypertrophy and failure. Impaired heart function was preceded by alterations in fatty acid metabolism and an increase in cardiac blood vessel density. Endothelial Notch signaling controlled the expression of endothelial lipase, Angptl4, CD36, and Fabp4, which are all needed for fatty acid transport across the vessel wall. In endothelial-specific Rbp-jκ-mutant mice, lipase activity and transendothelial transport of long-chain fatty acids to muscle cells were impaired. In turn, lipids accumulated in the plasma and liver. The attenuated supply of cardiomyocytes with long-chain fatty acids was accompanied by higher glucose uptake, increased concentration of glycolysis intermediates, and mTOR-S6K signaling. Treatment with the mTOR inhibitor rapamycin or displacing glucose as cardiac substrate by feeding a ketogenic diet prolonged the survival of endothelial-specific Rbp-jκ-deficient mice. This study identifies Notch signaling as a novel regulator of fatty acid transport across the endothelium and as an essential repressor of angiogenesis in the adult heart. The data imply that the endothelium controls cardiomyocyte metabolism and function.