Dll4-Notch Signaling in Macrophage Activation

Abstract

The Notch signaling pathway regulates the development of various cell types and organs, and also contributes to disease mechanisms in adults. Accumulating evidence suggests its role in cardiovascular and metabolic diseases. Notch signaling components also control the phenotype of immune cells. Delta-like ligand 4 (Dll4) of the Notch pathway promotes pro-inflammatory activation of macrophages in vitro and in vivo. Dll4 blockade attenuates chronic atherosclerosis, vein graft disease, vascular calcification, insulin resistance and fatty liver in mice. The Dll4-Notch axis may thus participate in the shared mechanisms for cardiometabolic disorders, serving as a potential therapeutic target for ameliorating these global health problems.

Chronic inflammation contributes to the pathogenesis of vascular diseases such as atherosclerosis, stenosis after percutaneous coronary intervention (PCI), and vein graft failure^1–3. The initial steps of atherogenesis include endothelial cell (EC) activation in response to various stimuli such as dyslipidemia, leading to increased expression of adhesion molecules and chemokines. Activated EC mediates the recruitment of circulating monocytes into the vascular wall where monocytes differentiate into macrophages. By taking up lipids, these phagocytes become foam cells, a key feature of atherosclerotic plaques. Various stimuli such as oxidized lipids and cytokines may promote pro-inflammatory activation of macrophages. Factors derived from activated macrophages may further promote activation of neighboring cell types in the vessel wall, including macrophage themselves, EC and smooth muscle cells (SMC), amplifying excessive pro-inflammatory responses. The accumulating evidence from over decades of research suggests that macrophages play key roles in the initiation and the development of atherosclerosis and the onset of its acute thrombotic complications. Further, the plasticity and heterogeneity of macrophages and the balance of their phenotypes (pro-inflammatory vs. anti-inflammatory populations) may be determined by each microenvironment and then in turn, help to determine the progression and the nature of atherosclerotic lesions. Fine-tuning key regulators of macrophage activation may provide a way to effectively attenuate atherogenesis. The development of such therapies requires better understanding of the mechanism for macrophage activation in each of the specific disease contexts and stages.

The Notch signaling pathway plays various roles in the development of the cardiovascular system and of the diseases of this system in adults⁴. The Notch pathway is composed of ligands (Delta-like ligand [Dll]1, Dll3, Dll4, Jagged1, and Jagged2) and cell surface receptors (Notch1-4). Direct cell-to-cell contact via the binding of a ligand to a receptor triggers downstream responses. We previously demonstrated that Dll4-mediated Notch signaling promotes pro-inflammatory activation of human macrophages in vitro and in vivo and accelerates the development of atherosclerosis, vein graft lesions, vascular calcification and metabolic diseases in mouse models^5–7. This review briefly summarizes the role of Notch signaling in vascular diseases and then focuses on Dll4 in macrophage activation and vascular inflammation.

The Notch signaling pathway

The Notch pathway regulates aspects of embryonic development and differentiation of various cell types and organs⁴. Figure 1 illustrates the canonical Notch signaling pathway. In mammals, ligands of the Jagged (Jagged1, Jagged2) and Delta-like (Dll1, Dll3, Dll4) families interact with Notch family receptors (Notch1, Notch2, Notch3, Notch4) expressed on a neighboring cell. Notch receptors exist on the cell surface as a proteolytically cleaved heterodimer consisting of a large ectodomain and a membrane-tethered intracellular domain. Notch signaling occurs when a ligand binds to the extracellular domain of a Notch receptor. This binding triggers a cascade of enzymatic cleavages of the receptor by ADAM family members and the γ-secretase complex, resulting in the release of the Notch intracellular domain (NICD). NICD translocates to the nucleus, where it forms a complex with a DNA binding protein RBP-Jκ. With absence of Notch signaling, RBP-Jκ acts as a transcriptional repressor and interacts with co-repressor proteins. Activation of the signaling mechanism and nuclear translocation of NICD appear to replace co-repressors with co-activators such as Mastermind-like protein 1 (MAML1). The NICD/RBP-Jκ/MAML1 complex then leads to the transcriptional activation of the genes targeted by Notch. We refer readers to other review articles for more detailed descriptions for the mechanisms of canonical and non-canonical Notch pathways^{4, 8–10}.

Figure 1. The Notch signaling pathway.

Notch signaling is initiated by physical ligand receptor interaction, which induces a cleavage at site S2 mediated by ADAM family proteinases followed by a cleavage at S3 within the transmembrane domain mediated by the γ-secretase complex. These proteolytic cleavages allow the Notch intracellular domain (NICD) to translocate into the nucleus. In the nucleus, NICD associates with a transcription factor, RBP-Jκ, and activates transcription from the RBP-Jκ DNA binding site. Among five mammalian Notch ligands, the function of Dll3 remains relatively unclear. Co-A, co-activator; Co-R, co-repressor; MAML, Mastermind-like 1.

Notch signaling in cardiovascular development and congenital diseases

i) Artery vs. vein differentiation and angiogenesis

The Notch pathway regulates arteriovenous differentiation¹¹. In zebra fish, Notch signaling-deficient embryos exhibited a poorly formed dorsal aorta and posterior cardinal vein with accompanying arteriovenous malformations¹². These embryos exhibited loss of expression of arterial markers such as ephrinB2. Studies of mammalian cells in culture showed that the Notch pathway is downstream of the vascular endothelial growth factor (VEGF) pathway¹³. Dll4-mediated Notch signaling induced ephrinB2 expression in cultured EC¹⁴ and leads to the differentiation of arterial EC. In Dll1 loss of function mutant embryos, generation of the Notch1 intracellular domain and expression of arterial markers such as neuropilin1, VEGF receptor 2 and ephrinB2 were lost. Dll1 is also required as a critical Notch ligand for maintaining arterial identity of EC¹⁵.

In addition, Notch signaling was reported to have a primary role in regulating formation and function of endothelial tip cells during angiogenesis¹⁶. Dll4 signaling on the tip cells regulates VEGF receptor expression on the adjacent Notch receptor-expressing cells and differentiates them into stalk cells. Dll4-Notch signaling suppressed tip cell numbers, filopodia extension and branching of angiogenic sprouts during angiogenesis¹⁷. In contrast to Dll4, Jagged1 overexpression enhanced angiogenesis and resulted in increased number of tip cells¹⁸. The inhibition study of Jagged-type or Dll-type Notch ligands in EC using Notch decoys showed that blockade of Jagged1/Jagged2-Notch1 signaling suppressed angiogenic sprouting, while blockade of Dll1/Dll4-Notch1 signaling caused endothelial hypersprouting¹⁹. In another report, overexpression of Jagged1 in EC led to increased vessel density and maturation during wound healing, although Dll4 blockade increased vascular density but decreased mural cells²⁰. Therefore, Jagged-type and Dll-type Notch ligand may have the opposite roles in angiogenesis.

Taken together, Notch signaling mediated by Dll1 and Dll4 is essential for arterial differentiation via VEGF-A during vascular morphogenesis. Notch signaling crossregulated by Jagged-type vs. Dll-type ligands determines the balance between angiogenic growth and arterial maturation.

ii) Cardiac development

Notch signaling contributes to cardiomyocyte differentiation, valve development, ventricular trabeculation and outflow tract development. Notch activation showed to decrease myocardial gene expression within the early in Xenopus heart field²¹. Notch effector RBP-Jκ deficiency in ES cells increased cardiomyogenic differentiation in the embryoid body²². As described above, Notch inhibits cardiogenesis in early development; however, it is required for cardiomyocyte differentiation during subsequent cardiac development. Notch signaling is active in the formation of ventricular trabeculation starting in early development²³. Notch signaling regulates cardiac valve development²⁴. Endocardial cells undergo epithelial-mesenchymal transformation (EMT) and form the valve primordia²⁵. The Notch-Hey-Bmp2/4 pathway promotes EMT and subsequent completion of valve tissue development²⁶. During development of the outflow tract including the pulmonary artery and aorta, neural crest-derived tissues express the Jagged1 and Jagged2 ligands while the endothelium expresses Dll1 and Dll4²⁷. The endothelium expresses Notch1 and Notch4 receptors and neural crest-derived cells surrounding the aortic arch express Notch2 and Notch3. A complex interplay among Notch signaling components appears therefore to regulate cardiac development.

iii) Congenital diseases

Evidence has linked Notch signaling with congenital heart diseases. Notch1 mutations cause bicuspid aortic valve and aortic valve calcification^{28, 29}. Jagged 1 mutations are also related with Alagille syndrome, and tetralogy of Fallot³⁰.

Notch3 appears to play an important role in the function and survival of vascular SMC. CADSIL is an inherited condition that causes stroke and vascular dementia³¹. Degeneration of SMC with accumulation of granular osmiophilic material is a typical characteristic of CADSIL. Notch3 ectodomain accumulates in the cerebral microvasculature in CADASIL patients. Notch 3 CADASIL mutations promote SMC abnormalities³². Combined Notch1 and Notch3 mutations cause pericyte dysfunction, leading to features typical of CADASIL³³.

Mechanisms for macrophage activation

Macrophage activation plays a central role in atherosclerotic vascular diseases^{2, 3}. Activated macrophages express a variety of pro-inflammatory mediators that contribute to the initiation and development of acute complications of atherosclerosis. Macrophage expression of potent chemokines such as CCL2/MCP-1 contributes to enhancing infiltration of monocytes/macrophages into the vessel wall. Accumulation of macrophages expressing proteinases (e.g., matrix metalloproteinase (MMP)-family collagenases) may degrade extracellular matrix components in atherosclerotic plaques (such as fibrillar collagen) and reduce their mechanical strength, leading to physical disruption (“plaque rupture”) and acute thrombosis^{34, 35}.

Evidence suggests that macrophages are a heterogeneous population.³⁶ A well established concept of macrophage polarization has helped to understand that the balance of a pro-inflammatory phenotype (“M1”) and a non/anti-inflammatory phenotype (“M2”) regulates normal homeostasis of various organs³⁶. The M1/M2 imbalance (e.g., M1 dominance in atherosclerotic lesions) in a microenvironment may promote inflammatory disorders. IFNγ or LPS typically induces M1 activation in vitro as gauged by increased expression of pro-inflammatory factors such as CCL2/MCP-1, IL-1β, IL-6, IL-12, TNF-α, and iNOS. IL-4 or IL-10 promotes M2 macrophage activation with increased expression levels of arginase 1, MRC1, and IL-10. IL-10 also represents pro-resolving proteins that promote resolution of inflammation³. M2-like macrophages may also contribute to regression of atherosclerosis². The evidence suggests that M2 macrophages may further be classified into four subgroups depending on a stimulus: M2a, M2b, M2c and M2d^37–39. M2a macrophages, induced by IL-4 and IL-13, express high levels of the mannose receptor (MR or CD206), and may contribute to the tissue repair. M2b macrophages, induced by stimulation with immune complexes and Toll-like receptor (TLR) agonist or IL-1 receptor ligands, produce both anti-inflammatory (IL-10) and pro-inflammatory (IL-1β, IL-6 and TNF-α) cytokines. IL-10 and glucocorticoids induce M2c macrophages, which exhibit anti-inflammatory activities and release pentoraxin-3, IL-10 and transforming growth factor (TGF)-β. M2c macrophages also express Mer receptor kinase (MERTK) that is essential in supporting the efferocytotic function⁴⁰. M2d macrophages are induced by TLR agonists through the adenosine A2A receptor, produce IL-10 and VEGF, and provide proangiogenic property.

Accumulating evidence suggests there are additional macrophage phenotypes (e.g, M4, Mox, Mhem)^{3, 37, 38}. The existence of several subsets may indicate the multi-dimensional nature of macrophage activation rather what has been suggested under the M1/M2 dichotomy^{39, 41}. A recently proposed new nomenclature thus reflects specific stimuli, e.g., M(IFNγ) and M(LPS) rather than M1 for pro-inflammatory macrophages; M(IL-4) and M(IL-10) for anti-inflammatory or pro-resolving phenotypes instead of M2⁴¹. In vivo situations (particularly diseased organs in humans) should involve more than one instigator. Multiple downstream signaling pathways may be activated in parallel and intertwined through complex crosstalk. Combination and relative contributions of such pathways or co-existing phenotypes may vary among different disease contexts and depend on the stage or site of each disease. Overall, the balance of cross-regulated signaling mechanisms or multiple macrophage subsets may thus determine the state of inflammation. Although the significance of macrophage heterogeneity in human vascular disease remains incompletely understood, this framework of the macrophage phenotypes helps to clarify specific molecular mechanisms by which each stimulator affects mediators and downstream genes. Using M(IFNγ) and M(IL-4) in global proteomics of human and mouse macrophages, we recently identified PARP9 and PARP14 as potential regulators of the balance between pro- and anti-inflammatory subpopulations⁴².

Notch signaling in macrophage activation

In the last decade, we have tested the hypothesis that Notch signaling participates in macrophage activation. Fung et al. reported that, in response to a pro-inflammatory stimulus ─ LPS, IL-1β or minimally-modified low density-lipoprotein (LDL), primary human macrophages, derived from peripheral blood mononuclear cells, acquire the ability to express Dll4⁵, which had previously been considered as a EC-specific Notch ligand (Figure 2A). This may indicate that a pro-inflammatory subpopulation(s) of macrophages, e.g., M1, M(LPS), may contain greater amounts of Dll4 on the cell surface. The same study further demonstrated that Dll4 binding triggers Notch signaling, leading to various responses such as the induction of iNOS expression and the nuclear factor κB (NF-κB) pathway, features typical of pro-inflammatory macrophage activation⁵ (Figure 2B). Many studies have demonstrated the crosstalk between Notch and NF-κB pathways at multiple levels^43–45. We also reported that Dll4 binding to the macrophage-like cell line RAW264.7 decreased the levels of the endogenous NF-κB inhibitor IκBα, suggesting NF-κB activation⁶, and that pharmacologic NF-κB suppression abrogated Dll4-triggerd MCP-1 induction. These results indicate that Notch signaling induces pro-inflammatory macrophage activation in part via the NF-κB pathway. Other reports also showed that in macrophages, LPS and other Toll-like receptor (TLR) ligands induce Notch1 and Notch2 expression, and that Notch signaling increases the expression of TNF-α and iNOS^{46, 47}. Another study reported that Notch-RBP-J signaling controls the transcription factor IRF8 and induces M1 macrophage genes⁴⁸. Outtz et al. reported that macrophages from Notch1+/− mice showed decreased induction of IL-6, IL-12 and TNF-α in response to LPS/IFNγ⁴⁹. Interestingly, we demonstrated that Dll4 binding to human primary macrophages promotes the expression of Dll4 itself⁵, indicating that Dll4-mediated Notch signaling may accelerate a positive feedback loop of pro-inflammatory activation of macrophages. Collectively, these studies indicate that Notch signaling may promote an excessive pro-inflammatory microenvironment in cardiovascular and other tissues and contribute to disease mechanisms.

Figure 2. The role of Dll4 in pro-inflammatory macrophage activation and atherogenesis.

A, LPS or IL-1β treatment increased the expression of Dll4 protein in human primary macrophages. B, Co-culture experiments of human primary macrophages with the mouse stroma cell-line overexpressing Dll4 (MS5-Dll4) demonstrated that Dll4 binding promoted activation of the NF-κB pathway, and expression of pro-inflammatory genes such as iNOS. MS5-GFP cells served as control. C and D, The effects of Dll4 blockade on atherogenesis. H & E staining of the aortic arch (C). Immunostaining for macrophages (Mac3) in plaques (D). n=7–8 (early phase) and 14–15 (late phase). These figures were reproduced from Fung E, et al.⁵ (A,B) and Fukuda D, et al.⁶ (C,D) with permission.

We thus investigated whether suppression of Dll4-Notch signaling reduces pro-inflammatory macrophage activation. Fung et al. found that siRNA silencing of Notch1, Notch2, Notch3, or Notch4 reduces Dll4-triggered iNOS induction in human primary macrophages⁵. Fukuda et al. demonstrated that blockade of Dll4 by anti-Dll4 antibody decreased a pro-inflammatory macrophage phenotype in the epididymal fat of metabolically-challenged mice⁶. Koga et al. showed that in lesional macrophages isolated from mouse vein grafts, Dll4 antibody decreased the pro-inflammatory genes IL-1β and TNF-α and increased anti-inflammatory arginase 1⁷. This in vitro and in vivo evidence indicates that Dll4-mediated Notch signaling may skew the vascular microenvironment toward the dominance of pro-inflammatory macrophages over anti-inflammatory or pro-resolving macrophages.

Notch signaling in atherosclerosis

In atherosclerosis, pro-inflammatory activation of macrophages may play a critical role in the lesion progression from the early phase of fatty-streak formation to plaque rupture and thrombus formation⁵⁰. We tested the hypothesis that Notch signaling promotes the progression of atherosclerosis in vivo⁶. The expression of Dll4 mRNA and protein increased in the aorta of LDL receptor deficient (Ldlr^−/−) mice that were fed high cholesterol/high fat diet for 24 weeks. The blockade of Dll4-Notch signaling by neutralizing anti-Dll4 antibody administration suppressed atheroma progression in Ldlr^−/− mice (Figure 2C). The expression level of CCL2/MCP-1 and the accumulation of macrophages in the aorta of the mice treated with neutralizing anti-Dll4 antibody were lower than those in control group (Figure 2D). The expression of pro-inflammatory genes IL-1β, IL-6 and iNOS in the aorta was also lower in the neutralizing anti-antibody group. Dll4 suppression inhibited the expression of MMP-9 and MMP-13, potent proteolytic enzymes responsible for collagen degradation and possibly plaque rupture and acute thrombosis^{34, 35, 51}. Moreover, neutralizing anti-antibody tended to reduce MMP-9 expression in macrophages and overexpression of Dll4 increased MMP-9 in vitro⁷. These results indicate that the Dll4-Notch axis may promote not only the progression of atherosclerosis but also the onset of its acute thrombotic complications.

T lymphocytes contribute to atherogenesis. Notch signaling plays critical roles in maintenance and differentiation of T-lymphocytes⁵². The evidence has also implicated Notch signaling in the regulation of T helper (Th) cell differentiation. Antigen presenting cells expressing Dll1 and Dll4 promote differentiation of Th1 cells⁵³, whose cytokines (Th1 cytokines, e.g., IFNγ, TNF-α) accelerate vascular inflammation. This mirrors what we observed in the relationship between Dll4-Notch and pro-inflammatory macrophage activation. In contrast, Jagged ligands on antigen presenting cells induce the differentiation of Th2 cells⁵³ which produce anti-inflammatory molecules such as IL-4, and suppress vascular inflammation. How Notch signaling on T cells affects vascular inflammation remains unknown. T cells migrate into the artery wall, and T cell activation amplifies inflammatory response in arteries during the progression of atherosclerosis.^{50, 54} Notch signaling on T cells may thus contribute to the pathogenesis of vascular disease.

The pathogenesis of atherosclerosis involves SMC. Accumulating evidence suggests that Notch signaling regulates SMC biology⁵⁵. The evidence also suggests Notch signaling regulates SMC differentiation and proliferation, leading to vascular lesion formation⁵⁶. During the lesion development, Notch pathway regulates the differentiation of bone marrow-derived cells into SMC-like cells⁵⁷. A recent report showed that Notch2 inhibits and Notch3 promotes PDGF-B-dependent SMC proliferation in human aortic SMC⁵⁸. In the same study, overexpression of Notch2 ICD suppressed ERK phosphorylation, however, Notch3 ICD increased ERK phosphorylation and led to PDGF-B-mediated SMC proliferation. Thus, in the context of SMC biology, the functions of Notch2 and Notch3 might differ.

Notch signaling promotes pro-inflammatory responses (IL-6, IL-8, IL-1α, RANTES and ICAM-1) in EC and induces senescence of EC⁵⁹. Notch1 inhibition reduces shear stress-induced IL-1β, IL-6 and ICAM-1 expression in EC⁶⁰. EC Dll4 induces M1 macrophage activation⁶¹. A recent study identified crosstalk between Dll4-Notch and BMP9 pathways in EC homeostasis⁶². TNF-α decreased Notch4 expression, while increasing Notch2 expression in human EC, leading to caspase activation and apoptosis⁶³. The same study provided mechanistic evidence that silencing of Notch4 and overexpression of Notch2 ICD similarly induced caspase activity in human EC. TNF-α-mediated caspase-dependent apoptosis in EC may thus involve these two Notch receptors in an opposite manner. The Notch pathway regulates the integrity of endothelium by controlling EC proliferation⁶⁴ and recruiting EC precursors from the bone marrow⁶⁵. Briot et al. recently reported that high-fat diet and inflammatory lipids reduced the expression of Notch1 in EC, and decreased Notch1 expression increased monocyte attachment to EC⁶⁶. Taken together, Notch signaling in EC may regulate inflammation, apoptosis and EC proliferation and contribute to atherogenesis.

Notch signaling in calcification

Vascular calcification is an independent predictor of cardiovascular disease⁶⁷. Calcification associates with plaque instability in atheroma⁶⁸ and arterial stiffness⁶⁹. Accumulating evidence has implicated macrophages in cardiovascular calcification⁷⁰. Our study linked Dll4-Notch signaling with calcification in the aorta and aortic valves in Ldlr^−/− mice⁶ (Figures 3A and 3B). The aortic expression of osteogenic regulators Cbfa1/RUNX2, osteopontin, osteocalcin and bone morphogenetic proteins (BMPs) decreased by neutralizing anti-Dll4 antibody treatment. In mouse primary macrophages, Dll4 blockade suppressed BMP2⁶. Another group reported that Notch1 ICD overexpression in human aortic SMCs led to Msx2 gene expression, a key regulator of osteogenesis, and induced alkaline phosphatase (ALP) activity and matrix mineralization in SMCs^{71, 72}.

Figure 3. The effects of Dll4 blockade on calcification, vein graft disease, and insulin sensitivity.

A, Dll4 antibody treatment reduced calcification in atherosclerotic plaques of Ldlr^−/− mice (von Kossa staining from Dll4 antibody or IgG treated mice, n=7–8 (early phase), and 14–15 (late phase). B, Dll4 suppression reduced osteogenic activity in the aorta of Ldlr^−/− mice (ex vivo mapping by fluorescence reflectance imaging, n=4). C, Dll4 antibody treatment retarded the development of vein graft lesions in Ldlr−/− mice (n=9–10). D, Dll4 blockade reduced glucose intolerance and increased insulin sensitivity as examined by glucose tolerance test (GTT) and insulin tolerance test (ITT) in Ldlr^−/− mice (n=7). Dll4 blockade improved body weight gain and reduced genes related to insulin sensitivity. These figures were reproduced from Fukuda D, et al.⁶ (A,B, and D), and Koga J, et al.⁷ (C) with permission.

Notch signaling in vein graft disease

Peripheral artery disease (PAD) is prevalent in approximately 8 million people in the US⁷³. Due to the diabetes pandemic the incidence of PAD is projected to further increase. Autologous implantation of saphenous vein is a common surgical therapy for PAD. Approximately 50% of saphenous vein grafts for lower extremity PAD, however, become occluded or narrowed within a year⁷⁴. Despite its large impact as a global health problem, vein graft failure currently has no medical solutions. We examined whether blockade of Dll4-Notch signaling in macrophages suppresses the progression of vein graft diseases⁷. Dll4 expression and Notch signaling increased in macrophages of human and mouse vein graft lesions. Neutralizing anti-Dll4 antibody therapy suppressed lesion development, macrophage accumulation, and MMP activity in mouse vein grafts (Figure 3C). Dll4 blockade also attenuated the dominance of pro-inflammatory macrophages over anti-inflammatory ones in vein graft lesions as determined by decreased expression of IL-1β and TNF-α and increased arginase 1. Dll4 silencing using siRNA encapsulated in macrophage-targeted lipid nanoparticles suppressed lesion development, whereas EC-targeted Dll4 silencing produced no effects. Conditioned media of the macrophage cell line overexpressing Dll4 also stimulated SMC proliferation and migration. These data suggest macrophage-derived Dll4 promotes lesion development via macrophage activation and crosstalk between macrophages and SMC in vein graft disease. The evidence suggests that inhibition of Notch signaling using γ-secretase inhibitor or soluble Jagged-1 decreased SMC proliferation, increased apoptosis in SMC, redifferentiated the SMC phenotype, and led to attenuate intimal hyperplasia in vein grafts^{75, 76}. Notch1 expression, but not Notch3 expression, increased in advanced vein grafts⁷⁵. These results suggest that Notch signaling in SMC also involves in the development of vein graft disease.

Notch signaling in metabolic disease

Evidence has implicated inflammation in metabolic diseases^{77, 78}. We thus examined whether Dll4-mediated Notch signaling affects various parameters of metabolic disorders⁶. The expression of Dll4 increased in the adipose tissue of metabolically challenged Ldlr^−/− mice on a high cholesterol/high fat diet. In this model, neutralizing anti-Dll4 antibody treatment reduced accumulation of adipose tissue macrophages, increased insulin sensitivity, and decreased excessive body weight gain (Figure 3D). Dll4 blockade also increased the adipose expression of genes associated with insulin sensitivity ─ adiponectin, GLUT4, C/EBPα and IRS-1 (Figure 3D). Blockade of Dll4 suppressed macrophage accumulation and the expression of MCP-1 in adipose tissue. Interestingly, neutralizing anti-Dll4 antibody therapy also improved signs of fatty liver in the same model, e.g, fat deposition, and size of adipocytes in fat with no modifications of plasma lipid profile. Among many cellular and molecular responses accompanied by increased adiposity, CCL2/MCP-1 and its receptor CC chemokine receptor 2 (CCR2) may be the most critical pro-inflammatory mediators⁷⁹. In our model, neutralizing anti-Dll4 antibody treatment suppressed CCL2/MCP-1 expression in adipose tissue. A recent study revealed inhibition of Notch signaling in hepatocytes decreased insulin resistance⁸⁰. In another study, Notch signaling regulated the plasticity of white and beige adipocytes, increased metabolic rate and improved glucose tolerance and insulin sensitivity in adipocyte-specific Notch1 inactivation mice⁸¹. These lines of evidence suggest that Notch signaling merits evaluation as a novel therapeutic target for metabolic diseases.

The Dll4-Notch axis in macrophages as a shared mechanism for cardiometabolic disease

A cluster of cardiometabolic disorders causes a global health burden. Metabolic diseases such as dyslipidemia and diabetes accelerate the development of coronary artery disease, PAD, and vein graft disease. The events caused by these vascular disorders (e.g., acute myocardial infarction, vein graft failure) are major determinants of the clinical impact of metabolic diseases (e.g., low quality of life, death). Such intertwined causal relationships among these cardiometabolic disorders may reflect the involvement of shared mechanisms such as macrophage activation, and understanding these relationships may lead to the development of new therapies. Particularly, signaling pathways that promote a microenvironment where pro-inflammatory macrophage subsets dominate over anti-inflammatory or pro-resolving macrophages may be promising targets.

Clinical evidence has established that modifications of major risk factors of atherosclerotic vascular disease, such as the lowering of LDL lowering by statins, prevent the onset of acute myocardial infarction. Preclinical and clinical evidence has established that anti-inflammatory effects of LDL lowering contributes to reduced risk^{34, 82}. However, even potent statins do not prevent the events in all patients⁸³. The high residual risk has driven active research efforts to find other therapies. A few major clinical trials are currently testing the hypothesis that anti-inflammatory therapies improve cardiovascular outcomes⁸². In addition, statins do not substantially reduce the incidence of some other cardiovascular disorders (e.g., vein graft failure, aortic valve calcification). Overcoming these clinical challenges requires identifying novel disease mechanisms that lead to the development of new therapies.

We have therefore explored new triggering mechanisms of macrophage activation beyond lipids in the contexts of atherosclerotic vascular disorders^{5–7, 84}. Our studies have proposed that Dll4-mediated inflammation represents a shared mechanism for arterial atherosclerosis and calcification, vein graft disease, insulin resistance, obesity, and fatty liver (Figure 4).^5–7 According to our in vitro data, pro-inflammatory stimulants such as IL-1β induce macrophage expression of Dll4. In vitro and in vivo evidence identified downstream genes of Dll4-Notch signaling, including IL-1β, IL-6, CCL2/MCP-1, iNOS, NF-κB, and Dll4 itself. These lines of evidence suggest that excessive macrophage activation amplified by Dll4-Notch favors a sustained pro-inflammatory milieu in metabolically challenged organs.

Figure 4. Working hypothesis for the role of the Dll4-Notch axis as a potential shared mechanism for the pathogenesis of cardiometabolic disorders.

Dll4-mediated Notch signaling promotes pro-inflammatory macrophage activation in arterial atherosclerotic plaques, vein graft lesions, adipose tissue, artery, and the liver. Suppression of Dll4 attenuates various parameters of cardiometabolic disorders including macrophage accumulation and activation. Establishing the relative contribution of macrophage Dll4 requires further assessment. Dll4 expressed by adipocytes or hepatocytes may also plays a role in these disorders.

What is the relative contribution of macrophage Dll4 in these diseases? Dll4 siRNA treatment via macrophage-targeted lipid nanoparticles retarded the development and inflammation of vein graft lesions,⁷ suggesting an important role of macrophage-derived Dll4 in vascular diseases. Dll4 blockade also reduced macrophage burden in the fat and liver, which may suggest a underlying shared mechanism for attenuating insulin resistanie and fatty liver. It should be noted, however, that systemic Dll4 antibody treatment may also have suppressed activated Notch signaling in adopocytes and hepatocytes. Further investigations will be required but the work by our group and others to examine the role of Notch signaling in macrophage activation and cardiometabolic disease was a critical first step towards the possible development of new therapeutics.

Future perspectives

Ideal anti-inflammatory therapies may adjust the imbalance of various macrophage subpopulations and restore a relatively normal microenviroment by controlling excessive activation of pro-inflammatory programs without compromising anti-inflammatory or pro-resolving mechanisms. Discovering molecular switches that regulate seemingly complex signaling networks may provide promising therapeutic targets⁴². We provided a proof of concept that Dll4-Notch is a therapeutic target for macrophage-mediated inflammatory diseases. Although it remains preliminary, our evidence indicated that Dll4 inhibition appears to attenuate the imbalance of macrophage heterogeneity by suppressing pro-inflammatory mediators and enhancing some of anti-inflammatory molecules^5–7. To further establish the role of Dll4-Notch in regulating the balance of macrophage phenotypes or identify key molecular switches, it may be critical to establish better-defined models, use clinical samples for unbiased screening, and take more systemic approaches (e.g., network analysis). Such strategies may help to better understand crosstalk among pro-inflammatory, anti-inflammatory, and pro-resolving pathways and to predict potential effects of modulators of Notch signaling. Cardiometabolic disease is a devastating disorder. Despite all the challenges, research efforts to seek effective therapies must continue.