AIBP, Angiogenesis, Hematopoiesis, and Atherogenesis

Abstract

Purpose of review:

The goal of this manuscript is to summarize the current understanding of the secreted APOA1 binding protein (AIBP), encoded by NAXE, in angiogenesis, hematopoiesis, and inflammation. The studies on AIBP illustrate a critical connection between lipid metabolism and the aforementioned endothelial and immune cell biology.

Recent findings:

AIBP dictates both developmental processes such as angiogenesis and hematopoiesis, and pathological events such as inflammation, tumorigenesis, and atherosclerosis.

Summary:

Although cholesterol efflux dictates AIBP-mediated lipid raft disruption in many of the cell types, recent studies document cholesterol efflux-independent mechanism involving Cdc42-mediated cytoskeleton remodeling in macrophages. AIBP disrupts lipid rafts and impairs rafts-associated VEGFR2 but facilitates non-rafts associated Notch signaling. Furthermore, AIBP can induce cholesterol biosynthesis gene SREBP2 activation, which in turn transactivates NOTCH and supports specification of hematopoietic stem and progenitor cells (HSPCs). In addition, AIBP also binds TLR4 and represses TLR4-mediated inflammation. In this review, we summarize the latest research on AIBP, focusing on its role in cholesterol metabolism and the attendant effects on lipid rafts-regulated VEGFR2 and non-rafts-associated NTOCH activation in angiogenesis, SREBP2-upregulated NOTCH signaling in hematopoiesis, and TLR4 signaling in inflammation and atherogenesis. We will discuss its potential therapeutic applications in angiogenesis and inflammation due to selective targeting of activated cells.

Introduction

Cholesterol is the fundamental building block of the cell, but it is also toxic to the cell if accumulated in excess. Maintenance of cellular cholesterol homeostasis is therefore essential for normal cell function [1–6]. As such, the cell has evolved a delicate program to maintain its cholesterol content, including LDL-mediated cholesterol uptake, HDL-mediated cholesterol efflux, master cholesterol transcription factor SREBP2-regulated cholesterol biosynthesis, and conversion of free cholesterol to various oxysterols(7, 8). These mechanisms of cholesterol metabolism interact in a dynamic fashion for the maintenance of cholesterol homeostasis(9–11). Ours and other studies suggest that AIBP is a secreted protein that promotes cholesterol efflux to APOA1 and HDL(12–14). AIBP is a ubiquitously expressed protein, particularly enriched in steroidogenesis organs, e.g. breast and reproductive organs, liver, and as well as kidney(12). AIBP was previously identified to interact with APOA1via yeast two hybridization screening, and the interaction were validated using Co-Immunoprecipitation(12). Interestingly, its secretion into the media from kidney-derived 293 cells can be increased by APOA1 or HDL incubation, but the underlying mechanism is unknown yet. AIBP binds endothelial cells (ECs) in a saturable fashion(14), presumably via an unknown AIBP receptor (AIBP-R). The AIBP binding to ECs increase the overall association of HDL with ECs while reducing HDL binding affinity, thereby resulting in increased cholesterol efflux(13). AIBP per se does not accept cholesterol(13). This mechanism suggests that AIBP functions to bring HDL to the cells that express a particular AIBP-R(15), or even to a subset of microdomains on the plasma membrane where the AIBP-R is localized. Since AIBP regulates cholesterol metabolism, it plays critical roles in a variety of fundamental processes such as angiogenesis, hematopoiesis, inflammation and atherosclerosis and tumorigenesis(13, 16–18). Interestingly, a very recent paper reveals that AIBP reduces lipid raft number through the small GTPase Cdc42-dependent but cholesterol efflux-independent mechanism(19). We will discuss the latest studied on these in the following sections.

AIBP-mediated cholesterol efflux, VEGFR2 signaling, and angiogenesis

Our previous studies illustrate that AIBP accelerates cholesterol efflux from ECs in a cholesterol transporter ABCG1-dependent manner(13). Cholesterol efflux in ECs is not as extensively studies as in macrophages. ABCA1, the cholesterol transporter that delivers cholesterol to lipid poor APOA1, is expressed in ECs(20, 21). However, it does not appear to mediate cholesterol efflux to APOA1 in ECs ((22) and our unpublished observations). A further investigation reveals that the LXR agonist 22(R)-hydroxycholesterol effect on increasing ABCA1 protein expression is minimal in ECs(22), which is in sharp contrast to macrophages(10, 23). Since ABCA1 has been implicated in retrograde cholesterol transport(24), it is a likelihood that ABCA1 does not promote cholesterol efflux to APOA1 but regulates retrograde cholesterol transport from the plasma membrane to the ER in ECs. In contrast, LXR activation can markedly upregulate ABCG1 in ECs(25). Phospholipidated APOA1, however, can accept cholesterol in ECs(26); this may be dependent on ABCG1 instead of ABCA1. HDL binds EC with 6 times more molecules than its binding to vascular smooth muscle cells(27). Thus, HDL appears to play a more direct and important role in EC functions. Contrary to macrophages, in which free cholesterol accounts for 1/3 of total cholesterol, the majority of cholesterol in ECs exists as free form (~90% according to our unpublished data), but not as cholesterol esters(28). One possibility is that ACAT content/activity in ECs is lower than that in macrophages. Interestingly, the majority of free cholesterol in ECs is located on the plasma membrane, particularly in a special microdomain known as caveolae/lipid rafts, which is also enriched with sphingolipids(29). Excess free cholesterol accretion on the plasma membrane, via caveolin1 (CAV1)-enriched lipid rafts/caveolae, likely protects the ECs from the toxic effect of free cholesterol(30). In addition to the cholesterol sequestering and maintenance of rafts/caveolar structure function, CAV1 gain-of-function promotes cholesterol efflux in the liver cells(31). However, CAV1 loss-of-function studies suggest that the caveolar domains appear not to be involved in cholesterol efflux as compared with non-caveolar domains(32). Although CAV1 plays a central role in caveolae organization, CAV1 knockout mice are viable and fertile, despite the absence of caveolae in the endothelium(33, 34), which suggests a compensatory mechanism to retain free cholesterol on the plasma membrane(35). The enrichment of cholesterol on the plasma membrane implies its critical role in EC behavior. AIBP-accelerated cholesterol efflux reduces numbers of lipid rafts per cell, which perturbs rafts-associated plasmalemmal receptors and their signaling (Fig. 1A), e.g. VEGFR2 signal transduction and its downstream effectors Akt and ERK activation(13). Since AIBP achieves this effect largely via HDL, prolonged and higher levels of HDL exposure sometimes elicits an effect similar to that of AIBP treatment(13). Notably, the HDL-mediated cholesterol efflux disrupts lipid rafts in an undiscriminating fashion, which presumably exerts a broader effect on cell signaling than AIBP. Global ABCG1 knockout or endothelial ABCA1/ABCG1 deficiency increases aortic neovascularization in vitro (4)^,(13). Surprisingly, developmental retinal angiogenesis is unaffected. Given that the retinal tissues are enriched with anti-angiogenic ω−3 polyunsaturated fatty acids(36, 37), these lipid species may override the ABC cholesterol transporters-mediated restriction on angiogenesis in the retinal vascular beds. In contrast, LXR activation increases cholesterol transporter ABCG1 expression in ECs, resulting in reduced angiogenesis(38, 39). This mechanism appears to function in pathological angiogenesis, e.g. aged-related macular degeneration(40). In addition to its role in reducing macrophage lipid accumulation in aged animals, administration of LXR agonist can increase endothelial ABCG1 expression and suppresses dysregulated neovascularization in a direct manner(40). Loss of ABCA1/ABCG1 also augments vascular inflammation, i.e. greater adhesion molecules ICAM1, VCAM1, and E-selectin expression, which in turn recruits inflammatory myeloid cells and exacerbates atherosclerosis(41). At the molecular levels, absence of cholesterol transporters would increase cell cholesterol levels, contributing to enhanced lipid raft formation and the associated inflammatory signaling(42, 43). Plasma HDL levels have been reported to regulate neovascularization, in a pro- or anti-angiogenesis fashion, depending on the context(13, 44–49). It should be noted that HDL is an important carrier of angiostatic lipid S1P, which binds HDL via apoM(50–52). Thus, certain HDL-mediated effects on angiogenesis may be attributed to the presence of S1P, and an angiogenesis assay using reconstituted HDL, prepared by complexing APOA1with defined phospholipids, may differentiate S1P or non-S1P- mediated effect(53–55). In addition, there are crosstalks between cell membrane and intracellular cholesterol pool via vesicle-mediated transport, cytoskeleton rearrangement, and deregulation in these dynamic events may affect lipid raft content, thereby conferring an angiogenic regulation(15, 38). In this paper, we have used AIBP to exemplify the critical role of cholesterol metabolism in angiogenesis.

Figure 1.

A, VEGFA-engaged VEGFR2 is clustered in the endothelial lipid rafts. AIBP-mediated cholesterol efflux to HDL, which disrupts lipid rafts and attenuates rafts-associated VEGFR2 signaling. B, NOTCH1 ligand binds and activates NOTCH1 signaling in non-raft domain of the neighboring ECs. AIBP-regulated lipid raft disruption translocates γ-secretase, the enzyme that cleaves NOTCH1 for activation, from lipid rafts to non-rafts, thereby facilitating NOTCH1 activation. C, AIBP is secreted from the sclerotome in zebrafish and acts on the hemogenic endothelium (HE).AIBP-mediated cholesterol efflux activates SREBP2, which in turn upregulates NOTCH1 and dictates HSPC specification. SC: spinal cord; N: notochord; DA: dorsal aorta. ER: endoplasmic reticulum. SRE: sterol responsive elements.

By using a murine choroidal neovascularization (CNV) model, which reproduces the dysregulated angiogenesis phenotype observed in patients with wet type age-related macular degeneration (AMD), we found that AIBP and APOA1 combination achieves the same anti-angiogenic effect as that of anti-VEGF antibody therapy(56). Furthermore, the AIBP therapy for CNV overcomes anti-VEGF resistance found in the aged animals. This refractory effect to anti-VEGF treatment is ascribed to the ability of the macrophages in the old mice to enhance angiogenesis, presumably by secreting pro-angiogenic factors(57, 58). AIBP-mediated cholesterol efflux rescues the proangiogenic property of the old macrophages and reduces CNV lesions. As a matter of fact, AIBP gene expression is reduced in the retinal specimens of AMD patients(56). In addition, by limiting cell migration, metastasis, and tumor angiogenesis, AIBP-mediated cholesterol efflux to APOA1 inhibits colorectal tumorigenesis(59). Thus, AIBP can bestow angiogenic suppression via a direct effect on ECs and an indirect effect on macrophages.

AIBP-accelerated cholesterol efflux and Notch signaling.

The Notch signaling antagonizes VEGFR2 signal transduction and limits angiogenesis(60–63). Our studies show that in addition to its disruption of VEGFR2 signaling AIBP can augment NOTCH activation (14, 18). Lipid rafts have been shown to regulate γ-secretase activity, which cleaves NOTCH intracellular domain, thereby activating its signaling(14). In biochemical studies or in some cell culture models, γ-secretase appears to demonstrate greater activity in lipid rafts and enhances the cleavage of its substrate APP(14, 64, 65). Nonetheless, there are also some studies suggesting the opposite. For instance, statin-mediated reduction of brain cholesterol levels in mice unexpectedly increases amyloid production(66, 67). Furthermore, patients with genetically-determined dysregulated cellular cholesterol accumulation confers early onset of APP aggregation(68, 69). As a matter of fact, γ-secretase is reported to be present in non-lipid raft domain during embryogenesis, but in lipid rafts in adults(14, 70). These studies suggest that reduced lipid raft content may not necessarily affect γ-secretase activity to a physiologically meaningful extent. Paradoxically, a modest reduction of lipid raft content in cultured neurons instead augments APP cleavage by γ-secretase in the non-raft domains(71). Consistent with these studies, we found that AIBP/HDL₃ compound treatment elicits γ-secretase translocation from the lipid rafts to the non-rafts, which results in co-existence of γ-secretase with NOTCH1 in the non-rafts and enhances NOTCH signaling (Fig. 1B)(14).

AIBP-regulated SREBP2-dependent NOTCH program in hematopoiesis

Our recent studies suggest that AIBP-mediated cholesterol efflux activates a SREBP2-controlled NOTCH signaling program in developmental and hyperlipidemia-associated hematopoiesis (Fig. 1C)(18). We showed that Aibp2 (the zebrafish functional paralog of human AIBP) overexpression in zebrafish, by accelerating cholesterol efflux, activates Srebp2, the master regulator of cellular cholesterol biosynthesis. Subsequent analysis revealed an unexpected role in transactivating Notch. Genome scale bioinformatics analysis shows that many components of the Notch signaling pathway possess Srebp2 binding motifs in their promoters. RNA-seq analysis of paired ECs, hemogenic endothelial cells (HECs), and progenitors with lymphoid potential from E10.5 murine embryos show enrichment of Notch pathway during the endothelial to hematopoietic transition (EHT). Typical Srebp2-regulated cholesterol metabolism genes other than SCAP, however, are not highlighted in this dataset. Due to enrichment of the Notch signaling pathway in Srebp2-regulated genes, we speculate that Srebp2 functions more towards Notch signaling than cholesterol biosynthesis during EHT. Consistent with developmental hematopoiesis, suppression of Srebp2 activity using the small molecule inhibitor betulin rescues HSPC hyper-proliferation in hyperlipidemia. Similarly, plasma LDL-cholesterol (LDL-c) levels are associated positively with circulating HSPCs counts, SREBP2 activation, and NOTCH1 expression in the peripheral HSPCs of human subjects. The finding that SREBP2-NOTCH axis promotes HSPC expansion in hyperlipidemia, at a first glance, is unexpected given that hyperlipidemia paradoxically activates SREBP2. As a matter of fact, many components associated with hyperlipidemia, e.g. elevated reactive oxygen species(72), increased oxidized phospholipids(73), or augmented ER stress(74), can activate SREBP2 in a cholesterol-independent fashion. The importance of SREBP-NOTCH signaling axis is also documented in the liver (75), which can give rise to immune cells even in adults. Within the morbidly obese patient population, expression of AIBP negatively correlates with carotid intima-media thickness, leukocyte numbers, and plasma metabolites associated with atheroprotection(75). Whereas SREBP-NOTCH signaling axis, mainly SREBP1, which preferential regulates fatty acid biosynthesis(76), is associated with increased atherosclerotic burden, liver steatosis, and metabolites that increases inflammation and coronary artery disease(75). It should be noted that SREBP2 is essential for SREBP1 activity and fatty acid biosynthesis in the liver(77). The contribution of SREBP2 to NOTCH signaling and hematopoiesis in the liver await further studies in the future.

AIBP, TLR4, inflammation and atherosclerosis.

Inflammatory signaling often occurs in cholesterol-rich lipid rafts in macrophages(78, 79). AIBP similarly promotes cholesterol efflux in macrophages(80–83). In contrast to using HDL as cholesterol acceptor in ECs, AIBP facilitates the delivery of cholesterol to APOA1, HDL, or surfactant, dependent on the homing tissues for macrophage(80). AIBP was recently found to bind TLR4 in macrophages(83). TLR4 is one of the key receptors regulating innate immune response, and functions as the cognate receptor of LPS, a polysaccharide present in the bacterial wall(84). Mounting studies show that TLR4 also binds danger associated molecular patterns, e.g oxidized cholesterol esters that are abundantly present in plasma and vascular tissues in hyperlipidemia(85, 86). In line with its role as TLR4 receptor, TLR4 deletion completely eliminates AIBP binding to macrophages(83). Since MD2 also binds TLR4 and functions as a co-receptor(87, 88), it will be interesting to determine the AIBP binding site on TLR4 and assess its influence on TLR4/MD2 interaction. AIBP-accelerated cholesterol efflux disrupts TLR4 dimerization in lipid rafts, impedes the LPS-elicited TLR4-dependent signaling, i.e. NFκB nuclear translocation and phosphorylation of its downstream effectors including ERK, p38 and c-Jun(80, 81, 83, 89). LPS stimulation induces rapid (~15 min) recombinant AIBP binding to macrophages as assessed by FACS analysis(80). This rapid increase of cell surface AIBP binding suggests a subcellular translocation and/or conformational changes, e.g., TLR4 clustering in lipid rafts and their accessibility for AIBP binding. Furthermore, the selective AIBP binding to LPS-activated macrophages but not to unstimulated ones hold great promise for using AIBP to specifically target inflammatory cells, which will be elaborated further below. In addition, AIBP treatment also increases the stability of cholesterol transporter ABCA1 by preventing its thiol protease-mediated degradation(82). Deletion of the APOA1 binding site negates this protective effect, suggesting the concerted effects of AIBP and APOA1(89). AIBP overexpression in the liver of hyperlipidemic mice markedly reduces atherosclerosis burden, which may result from mitigated systemic inflammation, e.g. reduced white blood cells and reduced endothelium inflammation, or from enhanced reverse cholesterol transport(90). EC-specific AIBP receptor and myeloid-specific TLR4 knockout will differentiate the individual contributions of the vascular and immune system to AIBP-regulated atherogenesis.

Interestingly, a recent study shows that AIBP also disrupts lipid rafts in macrophages via binding phosphatidylinositol 3-phosphate (PI3P)(19), the lipid enriched in the endosome. Through this binding, AIBP activates Cdc42, thereby triggering the associated cytoskeleton rearrangement. Supplement with NAD(P)H, the reported AIBP substrate identified from metabolite screening (91), impairs the Cdc42/PI3P binding and abolishes effect on lipid raft and cholesterol efflux. The mechanism by which AIBP binds PI3P is unknown yet; this is likely occurred via a cell surface receptor that mediates AIBP endocytosis.

Consistent with the critical importance of AIBP in inflammation, AIBP reduces neuroinflammation(83), ameliorate acute respiratory distress syndrome (ARDS)(80), and inhibit HIV replication(92). AIBP expression is high in the central nerve system, and has been implicated in neurometabolic degeneration(83). As with peripheral macrophages, AIBP increases cholesterol efflux in activated microglial cells(83), a type of neuronal cells characteristic of macrophage properties, but not in control microglial cells. In the retina, AIBP protects elevated intraocular pressure-induced glaucomatous neurodegeneration by reducing inflammatory TLR4 signaling and maintaining mitochondrial network and function(93). Thus, AIBP demonstrates a selectivity towards the inflamed cells while sparing the intact ones, which offers an opportunity of applying AIBP to target diseased cells. The lung is a metabolically active tissue in cholesterol metabolism(71, 94). AIBP expression is significantly increased in the lungs of ARDS patients, which may be a compensatory self-defense mechanism to reduce inflammation(80). Recombinant AIBP delivery diminishes the presence of immune cells in the LPS-challenged murine lungs and maintains the barrier functions of lung vasculature(80). The lower presence of immune cells in the LPS-treated lungs by AIBP treatment suggest that AIBP impedes their recruitment (i.e. migration, retention, and/or local proliferation) or reduces mobilization or differentiation of hematopoietic progenitors.

Conclusions

As evidenced by merely ~30 publications in the PubMed, study of AIBP is in its infancy. The AIBP research represents a unique therapeutic direction, by targeting this secreted protein that regulates cholesterol efflux, to modulate EC and macrophage functions. On the other hand, the studies on extracellular AIBP function are confounded by the fact that AIBP appears to have a metabolic role in the mitochondria(95), which is implicated in human neurometabolic degeneration and lethality(96). As evidence on both sides accumulate, it is challenging to reconcile the two sided stories at this stage(97). Furthermore, a moonlighting function of AIBP in B6 biosynthesis, independent of its NA(P)DHX epimerase role, has been reported(98). Nonetheless, it may be worth of testing whether AIBP gain-of-function, with or without mitochondria targeting signal, corrects metabolic functions associated with intracellular AIBP deficiency. The differences of pathophysiological phenotype penetrance between patients and animal models further confound the mechanistic studies on the intracellular AIBP function as a NAD(P)HX epimerase. Regardless, targeting extracellular AIBP function may be a promising strategy to modulate angiogenesis, hematopoiesis, inflammation, and their associated diseases, including atherosclerosis. In particular, the remarkable selectivity of AIBP towards activated cell type makes AIBP-based therapy tantalizing.