eNOS controls angiogenic sprouting and retinal neovascularization through the regulation of endothelial cell polarity

AbstractThe roles of nitric oxide (NO) and endothelial NO synthase (eNOS) in the regulation of angiogenesis are well documented. However, the involvement of eNOS in the sprouting of endothelial tip-cells at the vascular front during sprouting angiogenesis remains poorly defined. In this study, we show that downregulation of eNOS markedly inhibits VEGF-stimulated migration of endothelial cells but increases their polarization, as evidenced by the reorientation of the Golgi in migrating monolayers and by the fewer filopodia on tip cells at ends of sprouts in endothelial cell spheroids. The effect of eNOS inhibition on EC polarization was prevented in Par3-depleted cells. Importantly, downregulation of eNOS increased the expression of polarity genes, such as PARD3B, PARD6A, PARD6B, PKCΖ, TJP3, and CRB1 in endothelial cells. In retinas of eNOS knockout mice, vascular development is retarded with decreased vessel density and vascular branching. Furthermore, tip cells at the extremities of the vascular front have a marked reduction in the number of filopodia per cell and are more oriented. In a model of oxygen-induced retinopathy (OIR), eNOS deficient mice are protected during the initial vaso-obliterative phase, have reduced pathological neovascularization, and retinal endothelial tip cells have fewer filopodia. Single-cell RNA sequencing of endothelial cells from OIR retinas revealed enrichment of genes related to cell polarity in the endothelial tip-cell subtype of eNOS deficient mice. These results indicate that inhibition of eNOS alters the polarity program of endothelial cells, which increases cell polarization, regulates sprouting angiogenesis and normalizes pathological neovascularization during retinopathy.

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Control of endothelial cell polarity and sprouting angiogenesis by non-centrosomal microtubules

eLife ◽

10.7554/elife.33864 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 20

Author(s):

Maud Martin ◽

Alexandra Veloso ◽

Jingchao Wu ◽

Eugene A Katrukha ◽

Anna Akhmanova

Keyword(s):

Endothelial Cell ◽

Cell Polarity ◽

Cell Polarization ◽

Sprouting Angiogenesis ◽

Cell Protrusion ◽

Radial Microtubule System ◽

3D Environments ◽

Cell Asymmetry ◽

Vessel Development

Microtubules control different aspects of cell polarization. In cells with a radial microtubule system, a pivotal role in setting up asymmetry is attributed to the relative positioning of the centrosome and the nucleus. Here, we show that centrosome loss had no effect on the ability of endothelial cells to polarize and move in 2D and 3D environments. In contrast, non-centrosomal microtubules stabilized by the microtubule minus-end-binding protein CAMSAP2 were required for directional migration on 2D substrates and for the establishment of polarized cell morphology in soft 3D matrices. CAMSAP2 was also important for persistent endothelial cell sprouting during in vivo zebrafish vessel development. In the absence of CAMSAP2, cell polarization in 3D could be partly rescued by centrosome depletion, indicating that in these conditions the centrosome inhibited cell polarity. We propose that CAMSAP2-protected non-centrosomal microtubules are needed for establishing cell asymmetry by enabling microtubule enrichment in a single-cell protrusion.

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Excess centrosomes disrupt vascular lumenization and endothelial cell adherens junctions

Angiogenesis ◽

10.1007/s10456-020-09737-7 ◽

2020 ◽

Vol 23 (4) ◽

pp. 567-575

Author(s):

Danielle B. Buglak ◽

Erich J. Kushner ◽

Allison P. Marvin ◽

Katy L. Davis ◽

Victoria L. Bautch

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Polarity ◽

Adherens Junctions ◽

Cell Junctions ◽

Microtubule Organizing Center ◽

Sprouting Angiogenesis ◽

Organizing Center ◽

Important Regulator ◽

Cell Cell

Abstract Proper blood vessel formation requires coordinated changes in endothelial cell polarity and rearrangement of cell–cell junctions to form a functional lumen. One important regulator of cell polarity is the centrosome, which acts as a microtubule organizing center. Excess centrosomes perturb aspects of endothelial cell polarity linked to migration, but whether centrosome number influences apical–basal polarity and cell–cell junctions is unknown. Here, we show that excess centrosomes alter the apical–basal polarity of endothelial cells in angiogenic sprouts and disrupt endothelial cell–cell adherens junctions. Endothelial cells with excess centrosomes had narrower lumens in a 3D sprouting angiogenesis model, and zebrafish intersegmental vessels had reduced perfusion following centrosome overduplication. These results indicate that endothelial cell centrosome number regulates proper lumenization downstream of effects on apical–basal polarity and cell–cell junctions. Endothelial cells with excess centrosomes are prevalent in tumor vessels, suggesting how centrosomes may contribute to tumor vessel dysfunction.

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The Transcription Factor Complex LMO2/TAL1 Regulates Branching and Endothelial Cell Migration in Sprouting Angiogenesis

10.21203/rs.3.rs-384580/v1 ◽

2021 ◽

Author(s):

Yoshihiro Yamada ◽

Yi Zhong ◽

Shiho Miki ◽

Akiko Taura ◽

Terence Rabbitts

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Endothelial Cell ◽

Endothelial Cell Migration ◽

Angiogenic Switch ◽

Sprouting Angiogenesis ◽

Relative Paucity ◽

Angiopoietin 2 ◽

Tip Cells ◽

Transcription Factor Complex

Abstract The transcription factor complex, consisting of LMO2, TAL1/LYL1, and GATA2, plays an important role in capillary sprouting by regulating VEGFR2, DLL4, and angiopoietin 2 in tip cells. Overexpression of the basic helix-loophelix transcription factor LYL1 in transgenic mice results in shortened tails. This phenotype is associated with vessel hyperbranching and a relative paucity of straight vessels due to DLL4 downregulation in tip cells by forming aberrant complex consisting of LMO2 and LYL1. Knockdown of LMO2 or TAL1 inhibits capillary sprouting in spheroid-based angiogenesis assays, which is associated with decreased angiopoietin 2 secretion. In the same assay using mixed TAL1- and LYL1-expressing endothelial cells, TAL1 was found to be primarily located in tip cells, while LYL1-expressing cells tended to occupy the stalk position in sprouts by upregulating VEGFR1 than TAL1. Thus, the interaction between LMO2 and TAL1 in tip cells plays a key role in angiogenic switch of sprouting angiogenesis.

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Excess Centrosomes Disrupt Vascular Lumenization and Endothelial Cell Adherens Junctions

10.1101/863787 ◽

2019 ◽

Author(s):

Danielle B Buglak ◽

Erich J Kushner ◽

Allison P Marvin ◽

Katy L Davis ◽

Victoria L Bautch

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Polarity ◽

Adherens Junctions ◽

Cell Junctions ◽

Microtubule Organizing Center ◽

Sprouting Angiogenesis ◽

Organizing Center ◽

Important Regulator ◽

Cell Cell

ABSTRACTProper blood vessel formation requires coordinated changes in endothelial cell polarity and rearrangement of cell-cell junctions to form a functional lumen. One important regulator of cell polarity is the centrosome, which acts as a microtubule organizing center. Excess centrosomes perturb aspects of endothelial cell polarity linked to migration, but whether centrosome number influences apical-basal polarity and cell-cell junctions is unknown. Here, we show that excess centrosomes alter the apical-basal polarity of endothelial cells in angiogenic sprouts and disrupt endothelial cell-cell adherens junctions. Endothelial cells with excess centrosomes had narrower lumens in a 3D sprouting angiogenesis model, and zebrafish intersegmental vessels had reduced perfusion following centrosome overduplication. These results indicate that endothelial cell centrosome number regulates proper lumenization downstream of effects on apical-basal polarity and cell-cell junctions. Endothelial cells with excess centrosomes are prevalent in tumor vessels, suggesting how centrosomes may contribute to tumor vessel dysfunction.

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Role of thromboxane in retinal microvascular degeneration in oxygen-induced retinopathy

Journal of Applied Physiology ◽

10.1152/jappl.2001.90.6.2279 ◽

2001 ◽

Vol 90 (6) ◽

pp. 2279-2288 ◽

Cited By ~ 37

Author(s):

Martin H. Beauchamp ◽

Ana Katherine Martinez-Bermudez ◽

Fernand Gobeil ◽

Anne Marilise Marrache ◽

Xin Hou ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Death ◽

Endothelial Cell ◽

Oxidant Stress ◽

Microvascular Endothelial Cell ◽

Oxygen Induced Retinopathy ◽

Rat Pups ◽

Endothelial Cell Death ◽

Peroxidation Product ◽

Synthase Inhibitor

Microvascular degeneration is an important event in oxygen-induced retinopathy (OIR), a model of retinopathy of prematurity. Because oxidant stress abundantly generates thromboxane A2(TxA2), we tested whether TxA2plays a role in retinal vasoobliteration of OIR and contributes to such vascular degeneration by direct endothelial cytotoxicity. Hyperoxia-induced retinal vasoobliteration in rat pups (80% O2exposure from postnatal days 5–14) was associated with increased TxB2generation and was significantly prevented by TxA2synthase inhibitor CGS-12970 (10 mg · kg−1· day−1) or TxA2-receptor antagonist CGS-22652 (10 mg · kg−1· day−1). TxA2mimetics U-46619 (EC5050 nM) and I-BOP (EC505 nM) caused a time- and concentration-dependent cell death of neuroretinovascular endothelial cells from rats as well as newborn pigs but not of smooth muscle and astroglial cells; other prostanoids did not cause cell death. The peroxidation product 8-iso-PGF2, which is generated in OIR, stimulated TxA2formation by endothelial cells and triggered cell death; these effects were markedly diminished by CGS-12970. TxA2-dependent neuroretinovascular endothelial cell death was mostly by necrosis and to a lesser extent by apoptosis. The data identify an important role for TxA2in vasoobliteration of OIR and unveil a so far unknown function for TxA2in directly triggering neuroretinal microvascular endothelial cell death. These effects of TxA2might participate in other ischemic neurovascular injuries.

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Abstract 48: Deletion of the Neuronal Guidance Molecule Epha2 Reduces Inflammation in Experimental Atherosclerosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a48 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Steven D Funk ◽

Arif Yurdagul ◽

Jonette Green ◽

Patrick Albert ◽

Marshall McInnis ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Activation ◽

Experimental Atherosclerosis ◽

Marker Genes ◽

Endothelial Cell Activation ◽

Enhanced Expression ◽

Guidance Molecule ◽

Neuronal Guidance ◽

Deficient Mice

Neuronal guidance molecules are increasingly implicated in inflammatory responses. Recently, our group demonstrated enhanced expression of the neuronal guidance molecule EphA2 and its ephrinA1 ligand in mouse and human atherosclerotic plaques, and elucidated a novel proinflammatory function for EphA2 perpetuating proinflammatory gene expression during endothelial cell activation. However, a direct role for Eph/ephrins in atherosclerosis has never been demonstrated. We now show that knocking out the EphA2 gene in Western diet-fed ApoE mice blunts atherosclerotic plaque location at multiple sites. This reduction in atherosclerosis is associated with decreased monocyte infiltration and diminished expression of proinflammatory genes. EphA2 reduction may affect monocyte homing through multiple mechanisms, since reducing EphA2 expression in cytokine-activated endothelial cells does not affect endothelial adhesion molecule expression or monocyte rolling but significantly decreases firm adhesion in primary human monocytes. Like endothelial cells, plaque macrophages also express EphA2, and macrophages derived from EphA2 deficient mice show diminished expression of M1 marker genes and enhanced expression of M2 marker genes compared to their ApoE counterparts. Surprisingly, EphA2 deficient mice show significantly elevated plasma cholesterol. However, this elevation does not involve increased LDL levels but instead occurs due to elevations in plasma HDL levels. Taken together, the current data suggest EphA2 inhibition results in a multifaceted protective effect on experimental atherosclerosis characterized by reduced endothelial cell activation, monocyte recruitment, and M1/M2 polarization and enhanced circulating HDL levels.

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Abstract 3640: Mitochondrial Uncoupling Protein 2 Facilitates Endothelial Cell Growth and Angiogenesis via Reduction of Mitochondrial Superoxide

Circulation ◽

10.1161/circ.118.suppl_18.s_456-b ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Yukio Shimasaki ◽

Kai Chen ◽

John F Keaney

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Growth ◽

Mitochondrial Function ◽

Endothelial Cell Proliferation ◽

Wild Type ◽

Sprouting Angiogenesis ◽

Mitochondrial Superoxide ◽

Endothelial Cell Growth ◽

Aortic Explants

Background: Growing evidence suggests that mitochondrial function contributes to cell phenotype. One important component of mitochondrial function is the membrane potential that is controlled, in part, by uncoupling proteins (UCPs). Based on our previous data, the UCP2 is predominantly expressed in cultured endothelial cells. Therefore, we sought to examine the role of UCP2 in endothelial cell growth and angiogenesis. Methods and Results: Murine lung endothelial cells (MLECs) were isolated from UCP2-null and wild-type mice. UCP2-null cells were found less proliferative than wild-type cells (P<0.02, UCP2-null cells vs. wild-type cells, n=4). This defect of UCP2-null cells was rescued by UCP2 adenovirus transfection (19% increase, p<0.02 vs. LacZ adenovirus treated cells, n=3), and also rescued by transfection with manganese superoxide dismutase (MnSOD) adenovirus (53% increase, P<0.002 vs. LacZ adenovirus treated cells, n=3). We found a reciprocal relation such as no UCP2 expression and higher mitochondrial superoxide level in the MLECs (P<0.005, UCP2-null cells vs. wild-type cells, n=3), suggesting that mitochondrial superoxide may regulate endothelial cell growth. Then, we prepared murine aortic rings from UCP2-null and wild-type mice and embedded in rat tail collagen gel. The sprouting angiogenesis of UCP2-null explants was significantly less than wild-type explants (P<0.02, UCP2-null explants vs. wild-type explants, n=3– 4). Furthermore, MLECs from MnSOD-heterozygous mice showed less proliferation with lower expression of UCP2 protein and higher mitochondrial superoxide level compared to the MLECs from wild-type littermates (P<0.02, MnSOD-heterozygous cells vs. wild-type cells, n=4 – 8). We also observed less sprouting angiogenesis in MnSOD-heterozygous aortic explants than wild-type aortic explants (P<0.05, MnSOD-heterozygous explants vs. wild-type explants, n=3– 6). Conclusions: These data indicate that mitochondrial superoxide controls endothelial cell proliferation and angiogenesis, suggesting that mitochondrial metabolism modulates the endothelial cell growth and angiogenesis.

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Endothelial NO Synthase-Dependent S-Nitrosylation of β-Catenin Prevents Its Association with TCF4 and Inhibits Proliferation of Endothelial Cells Stimulated by Wnt3a

Molecular and Cellular Biology ◽

10.1128/mcb.00089-17 ◽

2017 ◽

Vol 37 (12) ◽

Cited By ~ 6

Author(s):

Ying Zhang ◽

Rony Chidiac ◽

Chantal Delisle ◽

Jean-Philippe Gratton

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Endothelial Cell ◽

Transcriptional Activity ◽

No Synthase ◽

Endothelial Cell Proliferation ◽

Dependent Manner ◽

Endothelial No Synthase ◽

Binding Interface ◽

Critical Residue

ABSTRACT Nitric oxide (NO) produced by endothelial NO synthase (eNOS) modulates many functions in endothelial cells. S-nitrosylation (SNO) of cysteine residues on β-catenin by eNOS-derived NO has been shown to influence intercellular contacts between endothelial cells. However, the implication of SNO in the regulation of β-catenin transcriptional activity is ill defined. Here, we report that NO inhibits the transcriptional activity of β-catenin and endothelial cell proliferation induced by activation of Wnt/β-catenin signaling. Interestingly, induction by Wnt3a of β-catenin target genes, such as the axin2 gene, is repressed in an eNOS-dependent manner by vascular endothelial growth factor (VEGF). We identified Cys466 of β-catenin as a target for SNO by eNOS-derived NO and as the critical residue for the repressive effects of NO on β-catenin transcriptional activity. Furthermore, we observed that Cys466 of β-catenin, located at the binding interface of the β-catenin–TCF4 transcriptional complex, is essential for disruption of this complex by NO. Importantly, Cys466 of β-catenin is necessary for the inhibitory effects of NO on Wnt3a-stimulated proliferation of endothelial cells. Thus, our data define the mechanism responsible for the repressive effects of NO on the transcriptional activity of β-catenin and link eNOS-derived NO to the modulation by VEGF of Wnt/β-catenin-induced endothelial cell proliferation.

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Nitric oxide degradation of heparin and heparan sulphate

Biochemical Journal ◽

10.1042/bj3240473 ◽

1997 ◽

Vol 324 (2) ◽

pp. 473-479 ◽

Cited By ~ 40

Author(s):

Rolando E. VILAR ◽

Dineshchandra GHAEL ◽

Min LI ◽

Devan D. BHAGAT ◽

Lisa M. ARRIGO ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Molecular Mass ◽

Inflammatory Responses ◽

Degradation Products ◽

Bone Development ◽

Heparan Sulphate ◽

Gel Filtration ◽

No Synthase ◽

Strong Anion Exchange

NO is a bioactive free radical produced by NO synthase in various tissues including vascular endothelium. One of the degradation products of NO is HNO2, an agent known to degrade heparin and heparan sulphate. This report documents degradation of heparin by cultured endothelial-cell-derived as well as exogenous NO. An exogenous narrow molecular-mass preparation of heparin was recovered from the medium of cultured endothelial cells using strong-anion exchange. In addition, another narrow molecular-mass preparation of heparin was gassed with exogenous NO under argon. Degradation was evaluated by gel-filtration chromatography. Since HNO2 degrades heparin under acidic conditions, the reaction with NO gas was studied under various pH conditions. The results show that the degradation of exogenous heparin by endothelial cells is inhibited by NO synthase inhibitors. Exogenous NO gas at concentrations as low as 400 p.p.m. degrades heparin and heparan sulphate. Exogenous NO degrades heparin at neutral as well as acidic pH. Endothelial-cell-derived NO, as well as exogenous NO gas, did not degrade hyaluronan, an unrelated glycosaminoglycan that resists HNO2 degradation. Peroxynitrite, a metabolic product of the reaction of NO with superoxide, is an agent that degrades hyaluronan; however, peroxynitrite did not degrade heparin. Thus endothelial-cell-derived NO is capable of degrading heparin and heparan sulphate via HNO2 rather than peroxynitrite. These observations may be relevant to various pathophysiological processes in which extracellular matrix is degraded, such as bone development, apoptosis, tissue damage from inflammatory responses and possible release of growth factors and cytokines.

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PKM2 regulates endothelial cell junction dynamics and angiogenesis via ATP production

Scientific Reports ◽

10.1038/s41598-019-50866-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Jesús Gómez-Escudero ◽

Cristina Clemente ◽

Diego García-Weber ◽

Rebeca Acín-Pérez ◽

Jaime Millán ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Inflammatory Disease ◽

Chronic Inflammatory Disease ◽

Cell Junctions ◽

Cell Junction ◽

Collective Migration ◽

Sprouting Angiogenesis ◽

Cell Cell

Abstract Angiogenesis, the formation of new blood vessels from pre-existing ones, occurs in pathophysiological contexts such as wound healing, cancer, and chronic inflammatory disease. During sprouting angiogenesis, endothelial tip and stalk cells coordinately remodel their cell-cell junctions to allow collective migration and extension of the sprout while maintaining barrier integrity. All these processes require energy, and the predominant ATP generation route in endothelial cells is glycolysis. However, it remains unclear how ATP reaches the plasma membrane and intercellular junctions. In this study, we demonstrate that the glycolytic enzyme pyruvate kinase 2 (PKM2) is required for sprouting angiogenesis in vitro and in vivo through the regulation of endothelial cell-junction dynamics and collective migration. We show that PKM2-silencing decreases ATP required for proper VE-cadherin internalization/traffic at endothelial cell-cell junctions. Our study provides fresh insight into the role of ATP subcellular compartmentalization in endothelial cells during angiogenesis. Since manipulation of EC glycolysis constitutes a potential therapeutic intervention route, particularly in tumors and chronic inflammatory disease, these findings may help to refine the targeting of endothelial glycolytic activity in disease.

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