Postnatal Retinal Angiogenesis Research Articles

Endothelial Zmiz1 modulates physiological and pathophysiological angiogenesis during retinal development.

Angiogenesis is a highly coordinated process involving the control of various endothelial cell behaviors. Mechanisms for transcription factor involvement in the regulation of endothelial cell dynamics and angiogenesis have become better understood, however much remains unknown, especially the role of non-DNA binding transcriptional cofactors. Here, we show that Zmiz1, a transcription cofactor, is enriched in the endothelium and critical for embryonic vascular development, postnatal retinal angiogenesis, and pathological angiogenesis in oxygen induced retinopathy (OIR). In mice, endothelial cell-specific deletion of Zmiz1 during embryogenesis led to lethality due to abnormal angiogenesis and vascular defects. Inducible endothelial cell-specific ablation of Zmiz1 postnatally resulted in impaired retinal vascular outgrowth, decreased vascular density, and increased vessel regression. In addition, angiogenic sprouting in the superficial and deep layers of the retina was markedly reduced. Correspondingly, vascular sprouting in fibrin bead assays was significantly reduced in the absence of Zmiz1, while further in vitro and in vivo evidence also suggested deficits in EC migration. In agreement with the defective sprouting angiogenesis phenotype, gene expression analysis of isolated retinal endothelial cells revealed downregulation of tip-cell enriched genes upon inactivation of Zmiz1. Lastly, our study suggested that endothelial Zmiz1 is critical for intraretinal revascularization following hypoxia exposure in the OIR model. Taken together, these findings begin to define the previously unspecified role of endothelial Zmiz1 in physiological and pathological angiogenesis.

Null mutation of exocyst complex component 3-like does not affect vascular development in mice.

Exocyst is an octameric protein complex implicated in exocytosis. The exocyst complex is highly conserved among mammalian species, but the physiological function of each subunit in exocyst remains unclear. Previously, we identified exocyst complex component 3-like (Exoc3l) as a gene abundantly expressed in embryonic endothelial cells and implicated in the process of angiogenesis in human umbilical cord endothelial cells. Here, to reveal the physiological roles of Exoc3l during development, we generated Exoc3l knockout (KO) mice by genome editing with CRISPR/Cas9. Exoc3l KO mice were viable and showed no significant phenotype in embryonic angiogenesis or postnatal retinal angiogenesis. Exoc3l KO mice also showed no significant alteration in cholesterol homeostasis or insulin secretion, although several reports suggest an association of Exoc3l with these processes. Despite the implied roles, Exoc3l KO mice exhibited no apparent phenotype in vascular development, cholesterol homeostasis, or insulin secretion.

The vascular gene Apold1 is dispensable for normal development but controls angiogenesis under pathological conditions

The molecular mechanisms of angiogenesis have been intensely studied, but many genes that control endothelial behavior and fate still need to be described. Here, we characterize the role of Apold1 (Apolipoprotein L domain containing 1) in angiogenesis in vivo and in vitro. Single-cell analyses reveal that - across tissues - the expression of Apold1 is restricted to the vasculature and that Apold1 expression in endothelial cells (ECs) is highly sensitive to environmental factors. Using Apold1−/− mice, we find that Apold1 is dispensable for development and does not affect postnatal retinal angiogenesis nor alters the vascular network in adult brain and muscle. However, when exposed to ischemic conditions following photothrombotic stroke as well as femoral artery ligation, Apold1−/− mice display dramatic impairments in recovery and revascularization. We also find that human tumor endothelial cells express strikingly higher levels of Apold1 and that Apold1 deletion in mice stunts the growth of subcutaneous B16 melanoma tumors, which have smaller and poorly perfused vessels. Mechanistically, Apold1 is activated in ECs upon growth factor stimulation as well as in hypoxia, and Apold1 intrinsically controls EC proliferation but not migration. Our data demonstrate that Apold1 is a key regulator of angiogenesis in pathological settings, whereas it does not affect developmental angiogenesis, thus making it a promising candidate for clinical investigation.

The TRPM7 channel reprograms cellular glycolysis to drive tumorigenesis and angiogenesis

Cancer or endothelial cells preferably catabolize glucose through aerobic glycolysis rather than oxidative phosphorylation. Intracellular ionic signaling has been shown to regulate glucose metabolism, but the underlying ion channel has yet to be identified. RNA-seq, metabolomics and genetic assay revealed that the TRPM7 channel regulated cellular glycolysis. Deletion of TRPM7 suppressed cancer cell glycolysis and reduced the xenograft tumor burden. Deficiency of endothelial TRPM7 inhibited postnatal retinal angiogenesis in mice. Mechanistically, TRPM7 transcriptionally regulated the solute carrier family 2 member 3 (SLC2A3, also known as GLUT3) via Ca2+ influx-induced calcineurin activation. Furthermore, CREB-regulated transcription coactivator 2 (CRTC2) and CREB act downstream of calcineurin to relay Ca2+ signal to SLC2A3 transcription. Expression of the constitutively active CRTC2 or CREB in TRPM7 knockout cell normalized glycolytic metabolism and cell growth. The TRPM7 channel represents a novel regulator of glycolytic reprogramming. Inhibition of the TRPM7-dependent glycolysis could be harnessed for cancer therapy.

Astrocyte-intrinsic and -extrinsic Fat1 activities regulate astrocyte development and angiogenesis in the retina.

Angiogenesis is a stepwise process leading to blood vessel formation. In the vertebrate retina, endothelial cells are guided by astrocytes migrating along the inner surface, and the two processes are coupled by a tightly regulated cross-talks between the two cell types. Here, I have investigated how the FAT1 cadherin, a regulator of tissue morphogenesis that governs tissue cross-talk, influences retinal vascular development. Late-onset Fat1 inactivation in the neural lineage in mice, by interfering with astrocyte progenitor migration polarity and maturation, delayed postnatal retinal angiogenesis, leading to persistent vascular abnormalities in adult retinas. Impaired astrocyte migration and polarity were not associated with alterations of retinal ganglion cell axonal trajectories or of the inner limiting membrane. In contrast, inducible Fat1 ablation in postnatal astrocytes was sufficient to alter their migration polarity and proliferation. Altogether, this study uncovers astrocyte-intrinsic and -extrinsic Fat1 activities that influence astrocyte migration polarity, proliferation and maturation, disruption of which impacts retinal vascular development and maintenance.

Developmental Perspectives on Arterial Fate Specification.

Blood vessel acquisition of arterial or venous fate is an adaptive phenomenon in response to increasing blood circulation during vascular morphogenesis. The past two decades of effort in this field led to development of a widely accepted paradigm of molecular regulators centering on VEGF and Notch signaling. More recent findings focused on shear stress-induced cell cycle arrest as a prerequisite for arterial specification substantially modify this traditional understanding. This review aims to summarize key molecular mechanisms that work in concert to drive the acquisition of arterial fate in two distinct developmental settings of vascular morphogenesis: de novo vasculogenesis of the dorsal aorta and postnatal retinal angiogenesis. We will also discuss the questions and conceptual controversies that potentially point to novel directions of investigation and possible clinical relevance.

Alternatively Spliced Form of Angiopoietin-2 as a New Vascular Rheostat.

Angiopoietin (ANPGT)-TIE signaling serves as a critical regulator of vessel maturation controlling vascular quiescence, maintenance, and homeostasis (primarily through ANGPT1-TIE2 signaling), as well as enabling vascular plasticity and responsiveness to exogenous cytokines (primarily through antagonistically acting ANGPT2). An alternatively spliced form of ANGPT2 (ANGPT2443) was first reported 20 years ago. Yet, little is known to this day about its biological functions. In this issue of Cancer Research, Kapiainen and colleagues report an elegant series of experiments adding to the complexity and contextuality of ANGPT-TIE signaling. The authors studied the function of ANGPT2443 in cellular experiments as well as in a genetic model in vivo, revealing that it is proteolytically cleaved into a lower molecular weight isoform (termed ANGPT2DAP) that lacks the superclustering domain necessary for multimer formation. When compared with full-length ANGPT2, ANGPT2443 and ANGPT2DAP showed lower binding affinity to α5β1 integrin, but were more potent inhibitors of ANGPT1-TIE2 signaling. Functionally, ANGPT2443 impaired vessel enlargement and vein morphogenesis during postnatal retinal angiogenesis. Tumor experiments in Angpt2443-expressing mice showed enhanced destabilization of the lung vasculature, with varying effects on metastasis. Taken together, the study provides important insight into the significance of ANGPT2 alternative splicing and identifies ANGPT2443 and ANGPT2DAP as a biological rheostat of ANGPT1-TIE2 signaling. Future work will need to characterize the relative ratios and functional contributions of the ANGPT2 variants in different pathophysiologic settings.See related article by Kapiainen et al., p. 129.

Preclinical validation of a novel metastasis-inhibiting Tie1 function-blocking antibody.

The angiopoietin (Ang)–Tie pathway has been intensely pursued as candidate second‐generation anti‐angiogenic target. While much of the translational work has focused on the ligand Ang2, the clinical efficacy of Ang2‐targeting drugs is limited and failed to improve patient survival. In turn, the orphan receptor Tie1 remains therapeutically unexplored, although its endothelial‐specific genetic deletion has previously been shown to result in a strong reduction in metastatic growth. Here, we report a novel Tie1 function‐blocking antibody (AB‐Tie1‐39), which suppressed postnatal retinal angiogenesis. During primary tumor growth, neoadjuvant administration of AB‐Tie1‐39 strongly impeded systemic metastasis. Furthermore, the administration of AB‐Tie1‐39 in a perioperative therapeutic window led to a significant survival advantage as compared to control‐IgG‐treated mice. Additional in vivo experimental metastasis and in vitro transmigration assays concurrently revealed that AB‐Tie1‐39 treatment suppressed tumor cell extravasation at secondary sites. Taken together, the data phenocopy previous genetic work in endothelial Tie1 KO mice and thereby validate AB‐Tie1‐39 as a Tie1 function‐blocking antibody. The study establishes Tie1 as a therapeutic target for metastasis in a perioperative or neoadjuvant setting.

P3489Endothelial adenosine-to-inosine RNA editing is indispensable for vascular integrity

Abstract Background Adenosine (A)-to-inosine (I) RNA editing is a powerful upstream regulator of double-stranded RNA (dsRNA) metabolism, its role however in tissue homeostasis remains poorly understood. We have recently reported that RNA editing is dysregulated in human atherosclerotic heart disease, although its role in the maintenance of endothelial cell (EC) function is unknown. Purpose The goal of the present study is to evaluate the role of EC-specific ADAR1 in vascular homeostasis in vivo. Methods Constitutional or inducible EC restricted ADAR1 ablation was achieved by crossing mice carrying a conditional (floxed) ADAR1 allele with either a Tie2-Cre or with a tamoxifen-inducible Cdh5-CreERT2 mouse line. EC function was assessed by the postnatal retinal angiogenesis model and barrier function assays and immunohistochemistry of lung and liver tissues. Cell culture assays, gene-silencing techniques, RT-qPCR, western blot and confocal microscopy were utilized to assess the ADAR1 effects in intracellular endothelial signalling. Results Constitutive endothelial ADAR1 ablation caused prenatal lethality at embryonic stage E13.5, demonstrating an essential role for endothelial ADAR1 in development. Postnatal ADAR1 ablation resulted in reduced vascular outgrowth, reduced vessel branching in the central vascular plexus and decreased filopodial protrusions from ECs at the angiogenic front of the vascular plexus compared with littermate control mice at P5. Furthermore, endothelial ADAR1 ablation in adult mice resulted in sudden death within 6–8 days after activation of Cre recombinase due to development of severe pleural effusions caused by widespread vascular leakage, indicating a disturbance of EC barrier function in lung microvasculature. Mechanistically, ADAR1-mediated RNA editing is essential for the metabolism of endogenous long-to-short dsRNAs, while silencing of endothelial ADAR1 resulted in accumulation of cytoplasmic long dsRNAs. Long dsRNAs are recognised as a danger-associated molecular pattern by the cytosolic innate immune sensing and signalling sensors. Consequently, the cytoplasmic accumulation of endogenous long dsRNAs resulted in activation of innate immune system in ECs, as assessed by the induction of interferon-β. Concomitantly, activation of the cytoplasmic dsRNA sensors resulted in dissociation of beta-catenin from VE-cadherin in EC junctions and endocytosis of VE-cadherin. Conclusion ADAR1-mediated RNA editing of long dsRNAs is essential for the long-to-short dsRNA metabolism and thus suppression of endogenous innate immune sensing and signalling. EC-restricted ADAR1 ablation results in embryonic lethality, impaired retinal angiogenesis at P5 and sudden death in adult mice due to pleural effusions, which all indicate that ADAR1-mediated RNA editing is indispensable for maintenance of vascular barrier integrity.

Inhibition of Sphingosine Phosphate Receptor 1 Signaling Enhances the Efficacy of VEGF Receptor Inhibition.

Inhibition of VEGFR signaling is an effective treatment for renal cell carcinoma, but resistance continues to be a major problem. Recently, the sphingosine phosphate (S1P) signaling pathway has been implicated in tumor growth, angiogenesis, and resistance to antiangiogenic therapy. S1P is a bioactive lipid that serves an essential role in developmental and pathologic angiogenesis via activation of the S1P receptor 1 (S1P1). S1P1 signaling counteracts VEGF signaling and is required for vascular stabilization. We used in vivo and in vitro angiogenesis models including a postnatal retinal angiogenesis model and a renal cell carcinoma murine tumor model to test whether simultaneous inhibition of S1P1 and VEGF leads to improved angiogenic inhibition. Here, we show that inhibition of S1P signaling reduces the endothelial cell barrier and leads to excessive angiogenic sprouting. Simultaneous inhibition of S1P and VEGF signaling further disrupts the tumor vascular beds, decreases tumor volume, and increases tumor cell death compared with monotherapies. These studies suggest that inhibition of angiogenesis at two stages of the multistep process may maximize the effects of antiangiogenic therapy. Together, these data suggest that combination of S1P1 and VEGFR-targeted therapy may be a useful therapeutic strategy for the treatment of renal cell carcinoma and other tumor types.

In This Issue

HomeCirculation ResearchVol. 122, No. 5In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published2 Mar 2018https://doi.org/10.1161/RES.0000000000000201Circulation Research. 2018;122:639Clonal Expansion of Endothelial Cells (p 670)How blood vessels grow depends on the physiological context, say Manavski et al.Download figureDownload PowerPointThe requirement for new blood vessels is minimal, once human development is complete. However, after tissue ischemia, such as a myocardial infarction, new capillaries grow to restore oxygen to the hypoxic tissue. Manavski and colleagues now show that in confetti mice—animals engineered to have different colored cells for the purposes of lineage tracing—the process of neovascularization differs depending on whether it is a normal part of development or induced via hypoxia. The team found that during normal postnatal retinal angiogenesis in the mice, new blood vessels were formed from the random integration, or mixing, of endothelial cells—evinced by an assortment of different colored cells comprising the vessels. When the animals’ retinas were temporarily exposed to hypoxia, however, neovascularization resulted largely from the clonal expansion of endothelial cells (cells of one color dominated). Endothelial cell clonal expansion also drove neovascularization after myocardial infarction and hind limb ischemia in the mice. Knowing how endothelial cells behave during hypoxia could help guide strategies for treating ischemic diseases, as well as prevention of pathological vascularization.Improved Survival Upon Suppression of FOXO TFs in Laminopathies (p 678)FOXO transcription factors contribute to cardiac problems in laminopathies, report Auguste et al.Download figureDownload PowerPointClassical laminopathies, such as Emery-Dreifuss muscular dystrophy and Hutchinson-Gilford progeria syndrome, are a diverse collection of disorders caused by mutations to LMNA—a nuclear lamina protein. The wide range of phenotypes associated with laminopathies is thought to reflect both the ubiquitous expression of LMNA and its interactions with chromatin across the genome, which could disturb gene expression in a variety of ways. Despite the diverse manifestations of LMNA mutations, most patients die of dilated cardiomyopathy (DCM), a progressive cardiac dysfunction that leads to heart failure. To examine the molecular mechanisms linking LMNA mutation to DCM, Auguste and colleagues studied gene expression in the hearts of LMNA-deficient mice before the animals showed symptoms of cardiac problems. Of the large number of genes aberrantly expressed, transcription factors of the FOXO family were among the most dysregulated. Moreover, knockdown of FOXO 1 and 3 in the hearts of LMNA-lacking mice almost doubled the animals’ survival rate. These results indicate that FOXO transcription factors might be key players in DCM and thus potential therapeutic targets for patients with laminopathy-associated heart failure.TRAF-1 Dissects Inflammation and Metabolism (p 693)Chronic inflammation may protect against metabolic pathology in obesity, say Michel et al.Download figureDownload PowerPointDiet-induced obesity is often associated with chronic inflammation and dysregulated metabolic states—such as hyperlipidemia and insulin resistance. This association suggests that metabolic pathologies, that accompany diabetes, may be in part due to chronic low-grade inflammation. However, obesity-related inflammation may not be solely pathogenic, as inhibition of some inflammatory pathways in mice has been found to aggravate obesity or metabolic dysregulation. To examine the role of inflammation in obesity in greater detail, Michel et al studied mice engineered to lack the anti-inflammatory factor TRAF-1 (tumor-necrosis receptor–associated factor 1), which the authors found was upregulated in obese mice and humans. While the TRAF-1 null mice exhibited increased inflammatory cytokine release from adipose tissue with increased immune cell recruitment, the animals did not gain weight on par with wild-type mice when fed a high-fat diet. In comparison with wild-type mice, TRAF-1 null mice also showed improved insulin resistance and greater lipid breakdown. Furthermore, this increased lipolysis was shown to require inflammatory signaling. Together, these results add to the growing body of evidence that in the setting of obesity inflammation is not purely pathogenic, and requires further investigation. Previous Back to top Next FiguresReferencesRelatedDetails March 2, 2018Vol 122, Issue 5 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000201 Originally publishedMarch 2, 2018 PDF download Advertisement

Pericyte-expressed Tie2 controls angiogenesis and vessel maturation

The Tie receptors with their Angiopoietin ligands act as regulators of angiogenesis and vessel maturation. Tie2 exerts its functions through its supposed endothelial-specific expression. Yet, Tie2 is also expressed at lower levels by pericytes and it has not been unravelled through which mechanisms pericyte Angiopoietin/Tie signalling affects angiogenesis. Here we show that human and murine pericytes express functional Tie2 receptor. Silencing of Tie2 in pericytes results in a pro-migratory phenotype. Pericyte Tie2 controls sprouting angiogenesis in in vitro sprouting and in vivo spheroid assays. Tie2 downstream signalling in pericytes involves Calpain, Akt and FOXO3A. Ng2-Cre-driven deletion of pericyte-expressed Tie2 in mice transiently delays postnatal retinal angiogenesis. Yet, Tie2 deletion in pericytes results in a pronounced pro-angiogenic effect leading to enhanced tumour growth. Together, the data expand and revise the current concepts on vascular Angiopoietin/Tie signalling and propose a bidirectional, reciprocal EC-pericyte model of Tie2 signalling.

Role of Müller cell cytochrome P450 2c44 in murine retinal angiogenesis

Polyunsaturated fatty acids (PUFA) and their cytochrome P450 (CYP450) metabolites have been linked to angiogenesis and vessel homeostasis. However, the role of individual CYP isoforms and their endogenous metabolites in those processes are not clear. Here, we focused on the role of Cyp2c44 in postnatal retinal angiogenesis and report that Cyp2c44 is highly expressed in Müller glial cells in the retina. The constitutive as well as inducible postnatal genetic deletion of Cyp2c44 resulted in an increased vessel network density without affecting vessel radial expansion during the first postnatal week. This phenotype was associated with an increased endothelial cell proliferation and attenuated Notch signaling. LC–MS/MS analyses revealed that levels of hydroxydocosahexaenoic acids (HDHA), i.e., 10-, 17- and 20-HDHA were significantly elevated in retinas from 5day old Cyp2c44−/− mice compared to their wild-type littermates. Enzymatic activity assays revealed that HDHAs were potential substrates for Cyp2c44 which could account for the increased levels of HDHAs in retinas from Cyp2c44−/− mice. These data indicate that Cyp2c44 is expressed in the murine retina and, like the soluble epoxide hydrolase, is expressed in Müller glia cells. The enhanced endothelial cell proliferation and Notch inhibition seen in retinas from Cyp2c44-deficient mice indicate a role for Cyp2c44-derived lipid mediators in physiological angiogenesis.

Abscisic Acid/Dormin, a Plant Hormone that Inhibits Angiogenesis in vitro and Neovascular Growth in vivo

Vessel development and regeneration by angiogenesis is critical for adequate oxygen and nutrient delivery to every organ in the body. However, the concept of anti‐angiogenesis therapy has previously been proposed due to the important role of blood vessels in solid tumor growth and dissemination. Non‐neoplastic disorders associated with numerous proliferative diseases (e.g. diabetes or chronic inflammation) also are promoted by pathological angiogenesis that leads to blinding diseases. Current anti‐angiogenic treatments are based on the use of synthetic drugs, many of which, not only cause serious side effects without correcting the underlying pathology, but also have an unfavorable cost to benefit ratio. The objective of this study is to explore the anti‐angiogenic potential of abscisic acid (ABA), a natural plant hormone that inhibits germination from occurring when the environmental conditions are not favorable and contributes to the acquisition of stress tolerance. Using a fibrin beads‐based sprouting assay, we show that ABA effectively inhibits in a dose‐dependent manner, migration, growth and expansion of endothelial tubes without affecting cell viability. In the in vivo model of postnatal retinal angiogenesis, administration of ABA affects neither the differentiation and normal development of the retinal vasculature nor the growth of the animals although it incidentally increases fetal hyaloid vessel persistence. In the mouse model of oxygen‐induced retinopathy (OIR) characterized by a hyperoxia‐induced vaso‐obliteration phase and a subsequent ischemia‐induced neovascular growth, administration of ABA reduces aberrant growth of retinal vessels during the ischemic phase of OIR without altering the normal vascularization of the retina. Thus, as a natural vascular stabilization signal restricting neovascular growth, ABA is an anti‐angiogenic molecule with potential translational implications in vascular diseases of the eye.

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

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. 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. 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. 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.

A mouse model of hereditary hemorrhagic telangiectasia generated by transmammary-delivered immunoblocking of BMP9 and BMP10.

Hereditary hemorrhagic telangiectasia (HHT) is a potentially life-threatening genetic vascular disorder caused by loss-of-function mutations in the genes encoding activin receptor-like kinase 1 (ALK1), endoglin, Smad4, and bone morphogenetic protein 9 (BMP9). Injections of mouse neonates with BMP9/10 blocking antibodies lead to HHT-like vascular defects in the postnatal retinal angiogenesis model. Mothers and their newborns share the same immunity through the transfer of maternal antibodies during lactation. Here, we investigated whether the transmammary delivery route could improve the ease and consistency of administering anti-BMP9/10 antibodies in the postnatal retinal angiogenesis model. We found that anti-BMP9/10 antibodies, when intraperitoneally injected into lactating dams, are efficiently transferred into the blood circulation of lactationally-exposed neonatal pups. Strikingly, pups receiving anti-BMP9/10 antibodies via lactation displayed consistent and robust vascular pathology in the retina, which included hypervascularization and defects in arteriovenous specification, as well as the presence of multiple and massive arteriovenous malformations. Furthermore, RNA-Seq analyses of neonatal retinas identified an increase in the key pro-angiogenic factor, angiopoietin-2, as the most significant change in gene expression triggered by the transmammary delivery of anti-BMP9/10 antibodies. Transmammary-delivered BMP9/10 immunoblocking in the mouse neonatal retina is therefore a practical, noninvasive, reliable, and robust model of HHT vascular pathology.

Abstract 304: Selective Targeting of a Novel Epsin-VEGFR2 Interaction Promotes VEGF-mediated Angiogenesis

Background: VEGF-induced binding of VEGFR2 to epsins 1 and 2 triggers VEGFR2 degradation and attenuates VEGF signaling. The epsin ubiquitin interacting motif (UIM) was shown to be required for the interaction with VEGFR2, however the molecular determinants that govern how epsin specifically interacts with and regulates VEGFR2 were unknown. The goals for the present study were (1) to identify critical molecular determinants that drive the specificity of the epsin and VEGFR2 interaction and (2) to ascertain if such determinants were critical for physiological angiogenesis in vivo . Methods and Results: Structural modeling uncovered two novel binding surfaces within VEGFR2 that mediate specific interactions with epsin UIM. Three glutamic acid residues in epsin UIM were found to interact with residues in VEGFR2. Further, we found that the VEGF-induced VEGFR2-epsin interaction promoted c-Cbl-mediated ubiquitination of epsin, and uncovered a previously unappreciated ubiquitin-binding surface within VEGFR2. Mutational analysis revealed that the VEGFR2-epsin interaction is supported by VEGFR2 interacting specifically with the UIM and with ubiquitinated epsin. An epsin UIM peptide, but not a mutant UIM peptide, potentiated endothelial cell proliferation, migration and angiogenic properties in vitro , increased postnatal retinal angiogenesis, and enhanced VEGF-induced physiological angiogenesis and wound healing. Conclusions: Distinct residues in the epsin UIM and VEGFR2 mediate specific interactions between epsin and VEGFR2, in addition to UIM recognition of ubiquitin moieties on VEGFR2. These novel interactions are critical for pathophysiological angiogenesis, suggesting that these sites could be selectively targeted by therapeutics to modulate angiogenesis.

Abstract 257: Selective Targeting of a Novel Epsin-VEGFR2 Interaction Promotes VEGF-mediated Angiogenesis

Background: VEGF-induced binding of VEGFR2 to epsins 1 and 2 triggers VEGFR2 degradation and attenuates VEGF signaling. The epsin ubiquitin interacting motif (UIM) was shown to be required for the interaction with VEGFR2, however the molecular determinants that govern how epsin specifically interacts with and regulates VEGFR2 were unknown. The goals for the present study were (1) to identify critical molecular determinants that drive the specificity of the epsin and VEGFR2 interaction and (2) to ascertain if such determinants were critical for physiological angiogenesis in vivo . Methods and Results: Structural modeling uncovered two novel binding surfaces within VEGFR2 that mediate specific interactions with epsin UIM. Three glutamic acid residues in epsin UIM were found to interact with residues in VEGFR2. Further, we found that the VEGF-induced VEGFR2-epsin interaction promoted c-Cbl-mediated ubiquitination of epsin, and uncovered a previously unappreciated ubiquitin-binding surface within VEGFR2. Mutational analysis revealed that the VEGFR2-epsin interaction is supported by VEGFR2 interacting specifically with the UIM and with ubiquitinated epsin. An epsin UIM peptide, but not a mutant UIM peptide, potentiated endothelial cell proliferation, migration and angiogenic properties in vitro , increased postnatal retinal angiogenesis, and enhanced VEGF-induced physiological angiogenesis and wound healing. Conclusions: Distinct residues in the epsin UIM and VEGFR2 mediate specific interactions between epsin and VEGFR2, in addition to UIM recognition of ubiquitin moieties on VEGFR2. These novel interactions are critical for pathophysiological angiogenesis, suggesting that these sites could be selectively targeted by therapeutics to modulate angiogenesis.

Selective Targeting of a Novel Epsin-VEGFR2 Interaction Promotes VEGF-Mediated Angiogenesis.

We previously reported that vascular endothelial growth factor (VEGF)-induced binding of VEGF receptor 2 (VEGFR2) to epsins 1 and 2 triggers VEGFR2 degradation and attenuates VEGF signaling. The epsin ubiquitin interacting motif (UIM) was shown to be required for the interaction with VEGFR2. However, the molecular determinants that govern how epsin specifically interacts with and regulates VEGFR2 were unknown. The goals for the present study were as follows: (1) to identify critical molecular determinants that drive the specificity of the epsin and VEGFR2 interaction and (2) to ascertain whether such determinants were critical for physiological angiogenesis in vivo. Structural modeling uncovered 2 novel binding surfaces within VEGFR2 that mediate specific interactions with epsin UIM. Three glutamic acid residues in epsin UIM were found to interact with residues in VEGFR2. Furthermore, we found that the VEGF-induced VEGFR2-epsin interaction promoted casitas B-lineage lymphoma-mediated ubiquitination of epsin, and uncovered a previously unappreciated ubiquitin-binding surface within VEGFR2. Mutational analysis revealed that the VEGFR2-epsin interaction is supported by VEGFR2 interacting specifically with the UIM and with ubiquitinated epsin. An epsin UIM peptide, but not a mutant UIM peptide, potentiated endothelial cell proliferation, migration and angiogenic properties in vitro, increased postnatal retinal angiogenesis, and enhanced VEGF-induced physiological angiogenesis and wound healing. Distinct residues in the epsin UIM and VEGFR2 mediate specific interactions between epsin and VEGFR2, in addition to UIM recognition of ubiquitin moieties on VEGFR2. These novel interactions are critical for pathophysiological angiogenesis, suggesting that these sites could be selectively targeted by therapeutics to modulate angiogenesis.

Role of secreted modular calcium-binding protein 1 (SMOC1) in transforming growth factor β signalling and angiogenesis.

Secreted modular calcium-binding protein 1 (SMOC1) is a matricellular protein that potentially interferes with growth factor receptor signalling. The aim of this study was to determine how its expression is regulated in endothelial cells and its role in the regulation of endothelial cell function. SMOC1 was expressed by native murine endothelial cells as well as by cultured human, porcine, and murine endothelial cells. SMOC1 expression in cultured cells was increased by hypoxia via the down-regulation of miR-223, and SMOC1 expression was increased in lungs from miR-223-deficient mice. Silencing SMOC1 (small interfering RNA) attenuated endothelial cell proliferation, migration, and sprouting in in vitro angiogenesis assays. Similarly endothelial cell sprouting from aortic rings ex vivo as well as postnatal retinal angiogenesis in vivo was attenuated in SMOC1(+/-) mice. In endothelial cells, transforming growth factor (TGF)-β signalling via activin-like kinase (ALK) 5 leads to quiescence, whereas TGF-β signalling via ALK1 results in endothelial cell activation. SMOC1 acted as a negative regulator of ALK5/SMAD2 signalling, resulting in altered α2 integrin levels. Mechanistically, SMOC1 associated (immunohistochemistry, proximity ligation assay, and co-immunoprecipitation) with endoglin; an endothelium-specific type III auxiliary receptor for the TGF-β super family and the effects of SMOC1 down-regulation on SMAD2 phosphorylation were abolished by the down-regulation of endoglin. These results indicate that SMOC1 is an ALK5 antagonist produced by endothelial cells that tips TGF-β signalling towards ALK1 activation, thus promoting endothelial cell proliferation and angiogenesis.