Epigenetic landscape reveals MECOM as an endothelial lineage regulator.

A comprehensive understanding of endothelial cell lineage specification will advance cardiovascular regenerative medicine. Recent studies found that unique epigenetic signatures preferentially regulate cell identity genes. We thus systematically investigate the epigenetic landscape of endothelial cell lineage and identify MECOM to be the leading candidate as an endothelial cell lineage regulator. Single-cell RNA-Seq analysis verifies that MECOM-positive cells are exclusively enriched in the cell cluster of bona fide endothelial cells derived from induced pluripotent stem cells. Our experiments demonstrate that MECOM depletion impairs human endothelial cell differentiation, functions, and Zebrafish angiogenesis. Through integrative analysis of Hi-C, DNase-Seq, ChIP-Seq, and RNA-Seq data, we find MECOM binds enhancers that form chromatin loops to regulate endothelial cell identity genes. Further, we identify and verify the VEGF signaling pathway to be a key target of MECOM. Our work provides important insights into epigenetic regulation of cell identity and uncovered MECOM as an endothelial cell lineage regulator.

AIBP Regulates Metabolism of Ketone and Lipids but Not Mitochondrial Respiration.

Accumulating evidence indicates that the APOA1 binding protein (AIBP)-a secreted protein-plays a profound role in lipid metabolism. Interestingly, AIBP also functions as an NAD(P)H-hydrate epimerase to catalyze the interconversion of NAD(P)H hydrate [NAD(P)HX] epimers and is renamed as NAXE. Thus, we call it NAXE hereafter. We investigated its role in NAD(P)H-involved metabolism in murine cardiomyocytes, focusing on the metabolism of hexose, lipids, and amino acids as well as mitochondrial redox function. Unbiased metabolite profiling of cardiac tissue shows that NAXE knockout markedly upregulates the ketone body 3-hydroxybutyric acid (3-HB) and increases or trends increasing lipid-associated metabolites cholesterol, α-linolenic acid and deoxycholic acid. Paralleling greater ketone levels, ChemRICH analysis of the NAXE-regulated metabolites shows reduced abundance of hexose despite similar glucose levels in control and NAXE-deficient blood. NAXE knockout reduces cardiac lactic acid but has no effect on the content of other NAD(P)H-regulated metabolites, including those associated with glucose metabolism, the pentose phosphate pathway, or Krebs cycle flux. Although NAXE is present in mitochondria, it has no apparent effect on mitochondrial oxidative phosphorylation. Instead, we detected more metabolites that can potentially improve cardiac function (3-HB, adenosine, and α-linolenic acid) in the Naxe-/- heart; these mice also perform better in aerobic exercise. Our data reveal a new role of NAXE in cardiac ketone and lipid metabolism.

Free Cholesterol Bioavailability and Atherosclerosis.

PURPOSE OF REVIEW: As both a cholesterol acceptor and carrier in the reverse cholesterol transport (RCT) pathway, high-density lipoprotein (HDL) is putatively atheroprotective. However, current pharmacological therapies to increase plasma HDL cholesterol (HDL-c) concentration have paradoxically failed to prevent or reduce atherosclerosis and cardiovascular disease (CVD). Given that free cholesterol (FC) transfer between surfaces of lipoproteins and cells is reversible, excess plasma FC can be transferred to the cells of peripheral tissue sites resulting in atherosclerosis. Here, we summarize potential mechanisms contributing to this paradox and highlight the role of excess free cholesterol (FC) bioavailability in atherosclerosis vs. atheroprotection. RECENT FINDINGS: Recent findings have established a complex relationship between HDL-c concentration and atherosclerosis. Systemic scavenger receptor class B type 1 (SR-B1) knock out (KO) mice exhibit with increased diet-induced atherosclerosis despite having an elevated plasma HDL-c concentration compared to wild type (WT) mice. The greater bioavailability of HDL-FC in SR-B1 vs. WT mice is associated with a higher FC content in multiple cell types and tissue sites. These results suggest that dysfunctional HDL with high FC bioavailability is atheroprone despite high HDL-c concentration. Past oversimplification of HDL-c involvement in cholesterol transport has led to the failures in HDL targeted therapy. Evidence suggests that FC-mediated functionality of HDL is of higher importance than its quantity; as a result, deciphering the regulatory mechanisms by which HDL-FC bioavailability can induce atherosclerosis can have far-reaching clinical implications.

Metabolic flexibility maintains proliferation and migration of FGFR signaling-deficient lymphatic endothelial cells.

Metabolic flexibility is the capacity of cells to alter fuel metabolism in response to changes in metabolic demand or nutrient availability. It is critical for maintaining cellular bioenergetics and is involved in the pathogenesis of cardiovascular disease and metabolic disorders. However, the regulation and function of metabolic flexibility in lymphatic endothelial cells (LECs) remain unclear. We have previously shown that glycolysis is the predominant metabolic pathway to generate ATP in LECs and that fibroblast growth factor receptor (FGFR) signaling controls lymphatic vessel formation by promoting glycolysis. Here, we found that chemical inhibition of FGFR activity or knockdown of FGFR1 induces substantial upregulation of fatty acid ß-oxidation (FAO) while reducing glycolysis and cellular ATP generation in LECs. Interestingly, such compensatory elevation was not observed in glucose oxidation and glutamine oxidation. Mechanistic studies show that FGFR blockade promotes the expression of carnitine palmitoyltransferase 1A (CPT1A), a rate-limiting enzyme of FAO; this is achieved by dampened extracellular signal-regulated protein kinase activation, which in turn upregulates the expression of the peroxisome proliferator-activated receptor alpha. Metabolic analysis further demonstrates that CPT1A depletion decreases total cellular ATP levels in FGFR1-deficient rather than wildtype LECs. This result suggests that FAO, which makes a negligible contribution to cellular energy under normal conditions, can partially compensate for energy deficiency caused by FGFR inhibition. Consequently, CPT1A silencing potentiates the effect of FGFR1 knockdown on impeding LEC proliferation and migration. Collectively, our study identified a key role of metabolic flexibility in modulating the effect of FGFR signaling on LEC growth.

Systemic metabolite profiling reveals sexual dimorphism of AIBP control of metabolism in mice.

Emerging studies indicate that APOA-I binding protein (AIBP) is a secreted protein and functions extracellularly to promote cellular cholesterol efflux, thereby disrupting lipid rafts on the plasma membrane. AIBP is also present in the mitochondria and acts as an epimerase, facilitating the repair of dysfunctional hydrated NAD(P)H, known as NAD(P)H(X). Importantly, AIBP deficiency contributes to lethal neurometabolic disorder, reminiscent of the Leigh syndrome in humans. Whereas cyclic NADPHX production is proposed to be the underlying cause, we hypothesize that an unbiased metabolic profiling may: 1) reveal new clues for the lethality, e.g., changes of mitochondrial metabolites., and 2) identify metabolites associated with new AIBP functions. To this end, we performed unbiased and profound metabolic studies of plasma obtained from adult AIBP knockout mice and control littermates of both genders. Our systemic metabolite profiling, encompassing 9 super pathways, identified a total of 640 compounds. Our studies demonstrate a surprising sexual dimorphism of metabolites affected by AIBP deletion, with more statistically significant changes in the AIBP knockout female vs male when compared with the corresponding controls. AIBP knockout trends to reduce cholesterol but increase the bile acid precursor 7-HOCA in female but not male. Complex lipids, phospholipids, sphingomyelin and plasmalogens were reduced, while monoacylglycerol, fatty acids and the lipid soluble vitamins E and carotene diol were elevated in AIBP knockout female but not male. NAD metabolites were not significantly different in AIBP knockout vs control mice but differed for male vs female mice. Metabolites associated with glycolysis and the Krebs cycle were unchanged by AIBP knockout. Importantly, polyamine spermidine, critical for many cellular functions including cerebral cortex synapses, was reduced in male but not female AIBP knockout. This is the first report of a systemic metabolite profile of plasma samples from AIBP knockout mice, and provides a metabolic basis for future studies of AIBP regulation of cellular metabolism and the pathophysiological presentation of AIBP deficiency in patients.

AIBP, Angiogenesis, Hematopoiesis, and Atherogenesis.

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. 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 raft-associated VEGFR2 but facilitates non-raft-associated NOTCH1 signaling. Furthermore, AIBP can induce cholesterol biosynthesis gene SREBP2 activation, which in turn transactivates NOTCH1 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 raft-regulated VEGFR2 and non-raft-associated NOTCH1 activation in angiogenesis, SREBP2-upregulated NOTCH1 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.

Combination of apolipoprotein-A-I/apolipoprotein-A-I binding protein and anti-VEGF treatment overcomes anti-VEGF resistance in choroidal neovascularization in mice.

Many patients of choroidal neovascularization (CNV) are unresponsive to the current anti-VEGF treatment. The mechanisms for anti-VEGF resistance are poorly understood. We explore the unique property of the apolipoprotein A-I (apoA-I) binding protein (AIBP) that enhances cholesterol efflux from endothelial cells and macrophages to thereby limit angiogenesis and inflammation to tackle anti-VEGF resistance in CNV. We show that laser-induced CNV in mice with increased age showed increased resistance to anti-VEGF treatment, which correlates with increased lipid accumulation in macrophages. The combination of AIBP/apoA-I and anti-VEGF treatment overcomes anti-VEGF resistance and effectively suppresses CNV. Furthermore, macrophage depletion in old mice restores CNV sensitivity to anti-VEGF treatment and blunts the synergistic effect of combination therapy. These results suggest that cholesterol-laden macrophages play a critical role in inducing anti-VEGF resistance in CNV. Combination therapy by neutralizing VEGF and enhancing cholesterol removal from macrophages is a promising strategy to combat anti-VEGF resistance in CNV.

Hematopoiesis is regulated by cholesterol efflux pathways and lipid rafts: connections with cardiovascular diseases.

Lipid rafts are highly ordered regions of the plasma membrane that are enriched in cholesterol and sphingolipids and play important roles in many cells. In hematopoietic stem and progenitor cells (HSPCs), lipid rafts house receptors critical for normal hematopoiesis. Lipid rafts also can bind and sequester kinases that induce negative feedback pathways to limit proliferative cytokine receptor cycling back to the cell membrane. Modulation of lipid rafts occurs through an array of mechanisms, with optimal cholesterol efflux one of the major regulators. As such, cholesterol homeostasis also regulates hematopoiesis. Increased lipid raft content, which occurs in response to changes in cholesterol efflux in the membrane, can result in prolonged receptor occupancy in the cell membrane and enhanced signaling. In addition, certain diseases, like diabetes, may contribute to lipid raft formation and affect cholesterol retention in rafts. In this review, we explore the role of lipid raft-related mechanisms in hematopoiesis and CVD (specifically, atherosclerosis) and discuss how defective cholesterol efflux pathways in HSPCs contribute to expansion of lipid rafts, thereby promoting myelopoiesis and thrombopoiesis. We also discuss the utility of cholesterol acceptors in contributing to lipid raft regulation and disruption, and highlight the potential to manipulate these pathways for therapeutic gain in CVD as well as other disorders with aberrant hematopoiesis.jlr;61/5/667/F1F1f1.

Regulation of lipid rafts, angiogenesis and inflammation by AIBP.

PURPOSE OF REVIEW: Recent studies demonstrate an important role of the secreted apolipoprotein A-I binding protein (AIBP) in regulation of cholesterol efflux and lipid rafts. The article discusses these findings in the context of angiogenesis and inflammation. RECENT FINDINGS: Lipid rafts are cholesterol-rich and sphingomyelin-rich membrane domains in which many receptor complexes assemble upon activation. AIBP mediates selective cholesterol efflux, in part via binding to toll-like receptor-4 (TLR4) in activated macrophages and microglia, and thus reverses lipid raft increases in activated cells. Recent articles report AIBP regulation of vascular endothelial growth factor receptor-2, Notch1 and TLR4 function. In zebrafish and mouse animal models, AIBP deficiency results in accelerated angiogenesis, increased inflammation and exacerbated atherosclerosis. Spinal delivery of recombinant AIBP reduces neuraxial inflammation and reverses persistent pain state in a mouse model of chemotherapy-induced polyneuropathy. Inhalation of recombinant AIBP reduces lipopolysaccharide-induced acute lung injury in mice. These findings are discussed in the perspective of AIBP's proposed other function, as an NAD(P)H hydrate epimerase, evolving into a regulator of cholesterol trafficking and lipid rafts. SUMMARY: Novel findings of AIBP regulatory circuitry affecting lipid rafts and related cellular processes may provide new therapeutic avenues for angiogenic and inflammatory diseases.

AIBP-mediated cholesterol efflux instructs hematopoietic stem and progenitor cell fate.

Hypercholesterolemia, the driving force of atherosclerosis, accelerates the expansion and mobilization of hematopoietic stem and progenitor cells (HSPCs). The molecular determinants connecting hypercholesterolemia with hematopoiesis are unclear. Here, we report that a somite-derived prohematopoietic cue, AIBP, orchestrates HSPC emergence from the hemogenic endothelium, a type of specialized endothelium manifesting hematopoietic potential. Mechanistically, AIBP-mediated cholesterol efflux activates endothelial Srebp2, the master transcription factor for cholesterol biosynthesis, which in turn transactivates Notch and promotes HSPC emergence. Srebp2 inhibition impairs hypercholesterolemia-induced HSPC expansion. Srebp2 activation and Notch up-regulation are associated with HSPC expansion in hypercholesterolemic human subjects. Genome-wide chromatin immunoprecipitation followed by sequencing (ChIP-seq), RNA sequencing (RNA-seq), and assay for transposase-accessible chromatin using sequencing (ATAC-seq) indicate that Srebp2 transregulates Notch pathway genes required for hematopoiesis. Our studies outline an AIBP-regulated Srebp2-dependent paradigm for HSPC emergence in development and HPSC expansion in atherosclerotic cardiovascular disease.

A minimally invasive approach to induce myocardial infarction in mice without thoracotomy.

Acute myocardial infarction (MI) is a leading cause of morbidity and mortality in the world. Traditional method to induce MI by left coronary artery (LCA) ligation is typically performed by an invasive approach that requires ventilation and thoracotomy, causing serious injuries in animals undergoing this surgery. We attempted to develop a minimally invasive method (MIM) to induce MI in mice. Under the guide of ultrasound, LCA ligation was performed in mice without ventilation and chest-opening. Compared to sham mice, MIM induced MI in mice as determined by triphenyltetrazolium chloride staining and Masson staining. Mice with MIM surgery revealed the reductions of LVEF, LVFS, E/A and ascending aorta (AAO) blood flow, and the elevations of S-T segment and serum cTn-I levels at 24 post-operative hours. The effects of MI induced by MIM were comparable to the effects of MI produced by traditional method in mice. Importantly, MIM increased the survival rates and caused less inflammation after the surgery of LCA ligation, compared to the surgery of traditional method. Further, MIM induced angiogenesis and apoptosis in ischaemic hearts from mice at postoperative 28 days as similarly as traditional method did. Finally, the MIM model was able to develop into the myocardial ischaemia/reperfusion model by using a balloon catheter with minor modifications. The MI model is able to be efficiently induced by a minimally invasive approach in mice without ventilation and chest-opening. This new model is potentially to be used in studying ischaemia-related heart diseases.

AIBP augments cholesterol efflux from alveolar macrophages to surfactant and reduces acute lung inflammation.

Acute respiratory distress syndrome (ARDS) is characterized by an excessive pulmonary inflammatory response. Removal of excess cholesterol from the plasma membrane of inflammatory cells helps reduce their activation. The secreted apolipoprotein A-I binding protein (AIBP) has been shown to augment cholesterol efflux from endothelial cells to the plasma lipoprotein HDL. Here, we find that AIBP was expressed in inflammatory cells in the human lung and was secreted into the bronchoalveolar space in mice subjected to inhalation of LPS. AIBP bound surfactant protein B and increased cholesterol efflux from alveolar macrophages to calfactant, a therapeutic surfactant formulation. In vitro, AIBP in the presence of surfactant reduced LPS-induced p65, ERK1/2 and p38 phosphorylation, and IL-6 secretion by alveolar macrophages. In vivo, inhalation of AIBP significantly reduced LPS-induced airspace neutrophilia, alveolar capillary leak, and secretion of IL-6. These results suggest that, similar to HDL in plasma, surfactant serves as a cholesterol acceptor in the lung. Furthermore, lung injury increases pulmonary AIBP expression, which likely serves to promote cholesterol efflux to surfactant and reduce inflammation.

AIBP protects against metabolic abnormalities and atherosclerosis.

Apolipoprotein A-I binding protein (AIBP) has been shown to augment cholesterol efflux from endothelial cells and macrophages. In zebrafish and mice, AIBP-mediated regulation of cholesterol levels in the plasma membrane of endothelial cells controls angiogenesis. The goal of this work was to evaluate metabolic changes and atherosclerosis in AIBP loss-of-function and gain-of-function animal studies. Here, we show that Apoa1bp-/-Ldlr-/- mice fed a high-cholesterol, high-fat diet had exacerbated weight gain, liver steatosis, glucose intolerance, hypercholesterolemia, hypertriglyceridemia, and larger atherosclerotic lesions compared with Ldlr-/- mice. Feeding Apoa1bp-/-Ldlr-/- mice a high-cholesterol, normal-fat diet did not result in significant differences in lipid levels or size of atherosclerotic lesions from Ldlr-/- mice. Conversely, adeno-associated virus-mediated overexpression of AIBP reduced hyperlipidemia and atherosclerosis in high-cholesterol, high-fat diet-fed Ldlr-/- mice. Injections of recombinant AIBP reduced aortic inflammation in Ldlr-/- mice fed a short high-cholesterol, high-fat diet. Conditional overexpression of AIBP in zebrafish also reduced diet-induced vascular lipid accumulation. In experiments with isolated macrophages, AIBP facilitated cholesterol efflux to HDL, reduced lipid rafts content, and inhibited inflammatory responses to lipopolysaccharide.jlr Our data demonstrate that AIBP confers protection against diet-induced metabolic abnormalities and atherosclerosis.

TBX20 Regulates Angiogenesis Through the Prokineticin 2-Prokineticin Receptor 1 Pathway.

BACKGROUND: Angiogenesis is integral for embryogenesis, and targeting angiogenesis improves the outcome of many pathological conditions in patients. TBX20 is a crucial transcription factor for embryonic development, and its deficiency is associated with congenital heart disease. However, the role of TBX20 in angiogenesis has not been described. METHODS: Loss- and gain-of-function approaches were used to explore the role of TBX20 in angiogenesis both in vitro and in vivo. Angiogenesis gene array was used to identify key downstream targets of TBX20. RESULTS: Unbiased gene array survey showed that TBX20 knockdown profoundly reduced angiogenesis-associated PROK2 (prokineticin 2) gene expression. Indeed, loss of TBX20 hindered endothelial cell migration and in vitro angiogenesis. In a murine angiogenesis model using subcutaneously implanted Matrigel plugs, we observed that TBX20 deficiency markedly reduced PROK2 expression and restricted intraplug angiogenesis. Furthermore, recombinant PROK2 administration enhanced angiogenesis and blood flow recovery in murine hind-limb ischemia. In zebrafish, transient knockdown of tbx20 by morpholino antisense oligos or genetic disruption of tbx20 by CRISPR/Cas9 impaired angiogenesis. Furthermore, loss of prok2 or its cognate receptor prokr1a also limited angiogenesis. In contrast, overexpression of prok2 or prokr1a rescued the impaired angiogenesis in tbx20-deficient animals. CONCLUSIONS: Our study identifies TBX20 as a novel transcription factor regulating angiogenesis through the PROK2-PROKR1 (prokineticin receptor 1) pathway in both development and disease and reveals a novel mode of angiogenic regulation whereby the TBX20-PROK2-PROKR1 signaling cascade may act as a "biological capacitor" to relay and sustain the proangiogenic effect of vascular endothelial growth factor. This pathway may be a therapeutic target in the treatment of diseases with dysregulated angiogenesis.

Lmo2 (LIM-Domain-Only 2) Modulates Sphk1 (Sphingosine Kinase) and Promotes Endothelial Cell Migration.

OBJECTIVE: Lmo (LIM-domain-only)2 transcription factor is involved in hematopoiesis and vascular remodeling. Sphk (sphingosine kinase)1 phosphorylates sphingosine to S1P (sphingosine-1-phosphate). We hypothesized that Lmo2 regulates Sphk1 to promote endothelial cell (EC) migration and vascular development. APPROACH AND RESULTS: Lmo2 and Sphk1 knockdown (KD) were performed in Tg(fli1:EGFP) y1 zebrafish and in human umbilical vein EC. Rescue of phenotypes or overexpression of these factors were achieved using mRNA encoding Lmo2 or Sphk1. EC proliferation in vivo was assessed by BrdU (bromodeoxyuridine) immunostaining and fluorescence-activated cell sorter analysis of dissociated Tg(fli1:EGFP) y1 embryos. Cell migration was assessed by scratch assay in human umbilical vein EC and mouse aortic rings. Lmo2 interactions with Sphk1 promoter were assessed by ChIP-PCR (chromatin immunoprecipitation-polymerase chain reaction). Lmo2 or Sphk1 KD reduced number and length of intersegmental vessels. There was no reduction in the numbers of GFP+ (green fluorescent protein) ECs after Lmo2 KD. However, reduced numbers of BrdU+GFP+ nuclei were observed along the dysmorphic intersegmental vessels, accumulating instead at the sprouting origin of the intersegmental vessels. This anomaly was likely because of impaired EC migration, which was confirmed in migration assays using Lmo2 KD human umbilical vein ECs and mouse aortic rings. Both in vivo and in vitro, Lmo2 KD reduced Sphk1 gene expression, associated with less Lmo2 binding to the Sphk1 promoter as assessed by ChIP-PCR. Sphk1 mRNA rescued the Lmo2 KD phenotype. CONCLUSIONS: Our data showed that Lmo2 is necessary for Sphk1 gene expression in ECs. Lmo2 KD reduced Lmo2-Sphk1 gene interaction, impaired intersegmental vessels formation, and reduced cell migration. We identified for the first time Sphk1 as downstream effector of Lmo2.

AIBP Limits Angiogenesis Through γ-Secretase-Mediated Upregulation of Notch Signaling.

RATIONALE: Angiogenesis improves perfusion to the ischemic tissue after acute vascular obstruction. Angiogenesis in pathophysiological settings reactivates signaling pathways involved in developmental angiogenesis. We showed previously that AIBP (apolipoprotein A-I [apoA-I]-binding protein)-regulated cholesterol efflux in endothelial cells controls zebra fish embryonic angiogenesis. OBJECTIVE: This study is to determine whether loss of AIBP affects angiogenesis in mice during development and under pathological conditions and to explore the underlying molecular mechanism. METHODS AND RESULTS: In this article, we report the generation of AIBP knockout (Apoa1bp-/-) mice, which are characterized of accelerated postnatal retinal angiogenesis. Mechanistically, AIBP triggered relocalization of γ-secretase from lipid rafts to nonlipid rafts where it cleaved Notch. Consistently, AIBP treatment enhanced DLL4 (delta-like ligand 4)-stimulated Notch activation in human retinal endothelial cells. Increasing high-density lipoprotein levels in Apoa1bp-/- mice by crossing them with apoA-I transgenic mice rescued Notch activation and corrected dysregulated retinal angiogenesis. Notably, the retinal vessels in Apoa1bp-/- mice manifested normal pericyte coverage and vascular integrity. Similarly, in the subcutaneous Matrigel plug assay, which mimics ischemic/inflammatory neovascularization, angiogenesis was dramatically upregulated in Apoa1bp-/- mice and associated with a profound inhibition of Notch activation and reduced expression of downstream targets. Furthermore, loss of AIBP increased vascular density and facilitated the recovery of blood vessel perfusion function in a murine hindlimb ischemia model. In addition, AIBP expression was significantly increased in human patients with ischemic cardiomyopathy. CONCLUSIONS: Our data reveal a novel mechanistic connection between AIBP-mediated cholesterol metabolism and Notch signaling, implicating AIBP as a possible druggable target to modulate angiogenesis under pathological conditions.

AIBP: A Novel Molecule at the Interface of Cholesterol Transport, Angiogenesis, and Atherosclerosis.

Cardiovascular disease, which is often driven by hypercholesterolemia and subsequent coronary atherosclerosis, is the number-one cause of morbidity and mortality in the United States. Based on long-term epidemiological studies, high-density lipoprotein cholesterol (HDL-C) levels are inversely correlated with risk for coronary artery disease (CAD). HDL-mediated reverse cholesterol transport (RCT) is responsible for cholesterol removal from the peripheral tissues and return to the liver for final elimination.1 In atherosclerosis, intraplaque angiogenesis promotes plaque growth and increases plaque vulnerability. Conceivably, the acceleration of RCT and disruption of intraplaque angiogenesis would inhibit atherosclerosis and reduce CAD. We have identified a protein called apoA-I binding protein (AIBP) that augments HDL functionality by accelerating cholesterol efflux. Furthermore, AIBP inhibits vascular endothelial growth factor receptor 2 activation in endothelial cells and limits angiogenesis.2 The following discusses the prospect of using AIBP as a novel therapeutic approach for the treatment of CAD.

Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia.

Apolipoprotein C-II (APOC2) is an obligatory activator of lipoprotein lipase. Human patients with APOC2 deficiency display severe hypertriglyceridemia while consuming a normal diet, often manifesting xanthomas, lipemia retinalis and pancreatitis. Hypertriglyceridemia is also an important risk factor for development of cardiovascular disease. Animal models to study hypertriglyceridemia are limited, with no Apoc2-knockout mouse reported. To develop a genetic model of hypertriglyceridemia, we generated an apoc2 mutant zebrafish characterized by the loss of Apoc2 function. apoc2 mutants show decreased plasma lipase activity and display chylomicronemia and severe hypertriglyceridemia, which closely resemble the phenotype observed in human patients with APOC2 deficiency. The hypertriglyceridemia in apoc2 mutants is rescued by injection of plasma from wild-type zebrafish or by injection of a human APOC2 mimetic peptide. Consistent with a previous report of a transient apoc2 knockdown, apoc2 mutant larvae have a minor delay in yolk consumption and angiogenesis. Furthermore, apoc2 mutants fed a normal diet accumulate lipid and lipid-laden macrophages in the vasculature, which resemble early events in the development of human atherosclerotic lesions. In addition, apoc2 mutant embryos show ectopic overgrowth of pancreas. Taken together, our data suggest that the apoc2 mutant zebrafish is a robust and versatile animal model to study hypertriglyceridemia and the mechanisms involved in the pathogenesis of associated human diseases.

Polo-like kinase 2 regulates angiogenic sprouting and blood vessel development.

Angiogenesis relies on specialized endothelial tip cells to extend toward guidance cues in order to direct growing blood vessels. Although many of the signaling pathways that control this directional endothelial sprouting are well known, the specific cellular mechanisms that mediate this process remain to be fully elucidated. Here, we show that Polo-like kinase 2 (PLK2) regulates Rap1 activity to guide endothelial tip cell lamellipodia formation and subsequent angiogenic sprouting. Using a combination of high-resolution in vivo imaging of zebrafish vascular development and a human umbilical vein endothelial cell (HUVEC) in vitro cell culture system, we observed that loss of PLK2 function resulted in a reduction in endothelial cell sprouting and migration, whereas overexpression of PLK2 promoted angiogenesis. Furthermore, we discovered that PLK2 may control angiogenic sprouting by binding to PDZ-GEF to regulate RAP1 activity during endothelial cell lamellipodia formation and extracellular matrix attachment. Consistent with these findings, constitutively active RAP1 could rescue the endothelial cell sprouting defects observed in zebrafish and HUVEC PLK2 knockdowns. Overall, these findings reveal a conserved PLK2-RAP1 pathway that is crucial to regulate endothelial tip cell behavior in order to ensure proper vascular development and patterning in vertebrates.