Activated Notch4 Inhibits Angiogenesis: Role of β1-Integrin Activation

ABSTRACT Notch4 is a member of the Notch family of transmembrane receptors that is expressed primarily on endothelial cells. Activation of Notch in various cell systems has been shown to regulate cell fate decisions. The sprouting of endothelial cells from microvessels, or angiogenesis, involves the modulation of the endothelial cell phenotype. Based on the function of other Notch family members and the expression pattern of Notch4, we postulated that Notch4 activation would modulate angiogenesis. Using an in vitro endothelial-sprouting assay, we show that expression of constitutively active Notch4 in human dermal microvascular endothelial cells (HMEC-1) inhibits endothelial sprouting. We also show that activated Notch4 inhibits vascular endothelial growth factor (VEGF)-induced angiogenesis in the chick chorioallantoic membrane in vivo. Activated Notch4 does not inhibit HMEC-1 proliferation or migration through fibrinogen. However, migration through collagen is inhibited. Our data show that Notch4 cells exhibit increased β1-integrin-mediated adhesion to collagen. HMEC-1 expressing activated Notch4 do not have increased surface expression of β1-integrins. Rather, we demonstrate that Notch4-expressing cells display β1-integrin in an active, high-affinity conformation. Furthermore, using function-activating β1-integrin antibodies, we demonstrate that activation of β1-integrins is sufficient to inhibit VEGF-induced endothelial sprouting in vitro and angiogenesis in vivo. Our findings suggest that constitutive Notch4 activation in endothelial cells inhibits angiogenesis in part by promoting β1-integrin-mediated adhesion to the underlying matrix.

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The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation

10.1101/2020.05.03.075085 ◽

2020 ◽

Cited By ~ 1

Author(s):

Emma Carley ◽

Rachel K. Stewart ◽

Abigail Zieman ◽

Iman Jalilian ◽

Diane. E. King ◽

...

Keyword(s):

Cell Fate ◽

Control Cell ◽

Nuclear Lamina ◽

Epidermal Differentiation ◽

Keratinocyte Differentiation ◽

Linc Complex ◽

Force Transmission ◽

Cell Fate Decisions

AbstractWhile the mechanisms by which chemical signals control cell fate have been well studied, how mechanical inputs impact cell fate decisions are not well understood. Here, using the well-defined system of keratinocyte differentiation in the skin, we examine whether and how direct force transmission to the nucleus regulates epidermal cell fate. Using a molecular biosensor, we find that tension on the nucleus through Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes requires integrin engagement in undifferentiated epidermal stem cells, and is released during differentiation concomitant with decreased tension on A-type lamins. LINC complex ablation in mice reveals that LINC complexes are required to repress epidermal differentiation in vivo and in vitro and influence accessibility of epidermal differentiation genes, suggesting that force transduction from engaged integrins to the nucleus plays a role in maintaining keratinocyte progenitors. This work reveals a direct mechanotransduction pathway capable of relaying adhesion-specific signals to regulate cell fate.

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Runx1 Is Required for Notch-Induced Specification of Megakaryocyte Development from Early Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v112.11.1370.1370 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1370-1370

Author(s):

Melanie G Cornejo ◽

Thomas Mercher ◽

Joseph D. Growney ◽

Jonathan Jesneck ◽

Ivan Maillard ◽

...

Keyword(s):

Notch Signaling ◽

Cell Fate ◽

Stromal Cells ◽

Gfp Expression ◽

Hematopoietic Stem ◽

Cell Fate Decisions ◽

Insight Into ◽

Megakaryocyte Differentiation

Abstract The Notch signaling pathway is involved in a broad spectrum of cell fate decisions during development, and in the hematopoietic system, it is known to favor T cell- vs B cell lineage commitment. However, its role in myeloid lineage development is less well understood. We have shown, using heterotypic co-cultures of murine primary hematopoietic stem cells (Lin-Sca-1+ckit+ HSCs) and OP9 stromal cells expressing the Notch ligand Delta1 (OP9-DL1), that Notch signaling derived from cell non-autonomous cues acts as a positive regulator of megakaryocyte fate from LSK cells. Bone marrow transplantation experiments with a constitutively active Notch mutant resulted in enhanced megakaryopoiesis in vivo, with increased MEP numbers and megakaryocyte colony formation. In contrast, expression of dnMAML using a conditional ROSA26 knock-in mouse model significantly impaired megakaryopoiesis in vivo, with a marked decrease in megakaryocyte progenitors. In order to understand the cellular differentiation pathways controlled by Notch, we first examined the ability of various purified progenitor populations to differentiate toward megakaryocytes upon Notch stimulation in vitro. We observed that CMP and MEP, but not GMP, can engage megakaryopoiesis upon Notch stimulation. Our results were consistent with expression analysis of Notch signaling genes in these purified progenitors and were supported by the observation that transgenic Notch reporter mice display higher levels of reporter (i.e. GFP) expression in HSC and MEP, vs. CMP and GMP in vivo. Furthermore, purified progenitors with high GFP expression gave rise to increased numbers of megakarocyte-containing colonies when plated in vitro compared to GFP-negative progenitors. In addition, further purification of the HSC population into long-term (LT), short-term (ST), and lymphoid-primed myeloid progenitors (LMPP) before plating on OP9-DL1 stroma showed that LMPP have a reduced ability to give rise to megakaryocytes compared to the other two populations. These data support the hypothesis that there is an early commitment to erythro/megakaryocytic fate from HSC prior to lymphoid commitment. To gain insight into the molecular mechanism underlying Notch-induced megakaryopoiesis, we performed global gene expression analysis that demonstrated the engagement of a megakaryopoietic transcriptional program when HSC were co-cultured with OP9-DL1 vs. OP9 stroma or OP9-DL1 treated with gamma-secretase inhibitor. Of interest, Runx1 was among the most upregulated genes in HSC co-cultured on OP9-DL1 stroma. To assess whether Notch signaling engages megakaryocytic fate through induction of Runx1, we plated HSC from Runx1 −/− mice on OP9-DL1 stroma. Compared to WT cells, Runx1 −/− HSC had a severely reduced ability to develop into CD41+ cells. In contrast, overexpression of Runx1 in WT HSC was sufficient to induce megakaryocyte fate on OP9 stroma without Notch stimulation. Together, our results indicate that Notch pathway activation induced by stromal cells is an important regulator of cell fate decisions in early progenitors. We show that Notch signaling is upstream of Runx1 during Notch-induced megakaryocyte differentiation and that Runx1 is an essential target of Notch signaling. We believe that these results provide important insight into the pathways controlling megakaryocyte differentiation, and may have important therapeutic potential for megakaryocyte lineage-related disorders.

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I-309 binds to and activates endothelial cell functions and acts as an angiogenic molecule in vivo

Blood ◽

10.1182/blood.v96.13.4039.h8004039_4039_4045 ◽

2000 ◽

Vol 96 (13) ◽

pp. 4039-4045

Author(s):

Giovanni Bernardini ◽

Gaia Spinetti ◽

Domenico Ribatti ◽

Grazia Camarda ◽

Lucia Morbidelli ◽

...

Keyword(s):

Endothelial Cells ◽

Growth Factor ◽

Umbilical Vein ◽

Rnase Protection Assay ◽

Chorioallantoic Membrane ◽

Rnase Protection ◽

Cc Chemokine ◽

Cell Functions

Several chemokines have been shown to act as angiogenic molecules or to modulate the activity of growth factors such as fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor (VEGF). The detection of the CC chemokine receptor (CCR) 8 message in human umbilical vein endothelial cells (HUVECs) by reverse transcription– polymerase chain reaction (RT-PCR) and RNase protection assay (RPA), prompted us to investigate the potential role exerted by the CC chemokine I-309, a known ligand of such receptor, in both in vitro and in vivo angiogenesis assays. We show here that I-309 binds to endothelial cells, stimulates chemotaxis and invasion of these cells, and enhances HUVEC differentiation into capillary-like structures in an in vitro Matrigel assay. Furthermore, I-309 is an inducer of angiogenesis in vivo in both the rabbit cornea and the chick chorioallantoic membrane assay (CAM).

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Preclinical Testing of Living Tissue-Engineered Heart Valves for Pediatric Patients, Challenges and Opportunities

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2021.707892 ◽

2021 ◽

Vol 8 ◽

Author(s):

Ionela Movileanu ◽

Marius Harpa ◽

Hussam Al Hussein ◽

Lucian Harceaga ◽

Alexandru Chertes ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Cell Fate ◽

Heart Valve ◽

Pediatric Patients ◽

Living Tissue ◽

Congenital Diseases ◽

Challenges And Opportunities

Introduction: Pediatric patients with cardiac congenital diseases require heart valve implants that can grow with their natural somatic increase in size. Current artificial valves perform poorly in children and cannot grow; thus, living-tissue-engineered valves capable of sustaining matrix homeostasis could overcome the current drawbacks of artificial prostheses and minimize the need for repeat surgeries.Materials and Methods: To prepare living-tissue-engineered valves, we produced completely acellular ovine pulmonary valves by perfusion. We then collected autologous adipose tissue, isolated stem cells, and differentiated them into fibroblasts and separately into endothelial cells. We seeded the fibroblasts in the cusp interstitium and onto the root adventitia and the endothelial cells inside the lumen, conditioned the living valves in dedicated pulmonary heart valve bioreactors, and pursued orthotopic implantation of autologous cell-seeded valves with 6 months follow-up. Unseeded valves served as controls.Results: Perfusion decellularization yielded acellular pulmonary valves that were stable, no degradable in vivo, cell friendly and biocompatible, had excellent hemodynamics, were not immunogenic or inflammatory, non thrombogenic, did not calcify in juvenile sheep, and served as substrates for cell repopulation. Autologous adipose-derived stem cells were easy to isolate and differentiate into fibroblasts and endothelial-like cells. Cell-seeded valves exhibited preserved viability after progressive bioreactor conditioning and functioned well in vivo for 6 months. At explantation, the implants and anastomoses were intact, and the valve root was well integrated into host tissues; valve leaflets were unchanged in size, non fibrotic, supple, and functional. Numerous cells positive for a-smooth muscle cell actin were found mostly in the sinus, base, and the fibrosa of the leaflets, and most surfaces were covered by endothelial cells, indicating a strong potential for repopulation of the scaffold.Conclusions: Tissue-engineered living valves can be generated in vitro using the approach described here. The technology is not trivial and can provide numerous challenges and opportunities, which are discussed in detail in this paper. Overall, we concluded that cell seeding did not negatively affect tissue-engineered heart valve (TEHV) performance as they exhibited as good hemodynamic performance as acellular valves in this model. Further understanding of cell fate after implantation and the timeline of repopulation of acellular scaffolds will help us evaluate the translational potential of this technology.

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Methylglyoxal Scavengers Attenuate Angiogenesis Dysfunction Induced by Methylglyoxal and Oxygen-Glucose Deprivation

Oxidative Medicine and Cellular Longevity ◽

10.1155/2022/8854457 ◽

2022 ◽

Vol 2022 ◽

pp. 1-18

Author(s):

Wei Chen ◽

Wenhui Huang ◽

Yu Yang ◽

Keshen Li

Keyword(s):

Endothelial Cells ◽

Chorioallantoic Membrane ◽

Glucose Deprivation ◽

Oxygen Glucose Deprivation ◽

Ros Generation ◽

Advanced Glycation ◽

Related Factors ◽

Cellular Apoptosis

Cerebral endothelial cells play an essential role in brain angiogenesis, and their function has been found to be impaired in diabetes. Methylglyoxal (MG) is a highly reactive dicarbonyl metabolite of glucose formed mainly during glycolysis, and its levels can be elevated in hyperglycemic conditions. MG is a potent precursor of AGEs (advanced glycation end-products). In this study, we investigated if MG can induce angiogenesis dysfunction and whether MG scavengers can ameliorate angiogenesis dysfunction induced by MG. Here, we used cultured human brain microvascular endothelial cells (HBMECs) treated with MG and oxygen-glucose deprivation (OGD) to mimic diabetic stroke in vitro. We also used the MG challenged chicken embryo chorioallantoic membrane (CAM) to study angiogenesis in vivo. Interestingly, administration of MG significantly impaired cell proliferation, cell migration, and tube formation and decreased protein expression of angiogenesis-related factors, which was rescued by three different MG scavengers, glyoxalase 1 (GLO1), aminoguanidine (AG), and N-acetyl cysteine (NAC). In cultured CAM, MG exposure significantly reduced angiogenesis and the angiogenesis-related dysfunction could be attenuated by pretreatment with AG or NAC. Treatment of cultured HBMECs with MG plus OGD increased cellular apoptosis significantly, which could be prevented by exposure to GLO1, AG, or NAC. We also noted that administration of MG increased cellular oxidative stress as measured by reactive oxygen species (ROS) generation, enhanced AGE accumulation, and receptor for advanced glycation end-product (RAGE) expression in the cultured HBMECs, which were partially reversed by GLO1, AG, or NAC. Taken together, our findings demonstrated that GLO1, AG, or NAC administration can ameliorate MG-induced angiogenesis dysfunction, and this can be mainly attributed to attenuated ROS production, reduced cellular apoptosis, and increased levels of angiogenic factors. Overall, this study suggested that GLO1, AG, or NAC may be promising candidate compounds for the treatment of angiogenesis dysfunction caused by hyperglycemia in diabetic ischemic stroke.

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Exploring the Antiangiogenic Potential of Solomonamide A Bioactive Precursors: In Vitro and in Vivo Evidences of the Inhibitory Activity of Solo F-OH During Angiogenesis

Marine Drugs ◽

10.3390/md17040228 ◽

2019 ◽

Vol 17 (4) ◽

pp. 228 ◽

Cited By ~ 4

Author(s):

Carrillo ◽

Martínez-Poveda ◽

Cheng-Sánchez ◽

Guerra ◽

Tobia ◽

...

Keyword(s):

Endothelial Cells ◽

Inhibitory Activity ◽

Cyclic Peptide ◽

Chorioallantoic Membrane ◽

Tube Formation ◽

Marine Sponges ◽

Migration And Invasion ◽

In Vivo Models

Marine sponges are a prolific source of bioactive compounds. In this work, the putative antiangiogenic potential of a series of synthetic precursors of Solomonamide A, a cyclic peptide isolated from a marine sponge, was evaluated. By means of an in vitro screening, based on the inhibitory activity of endothelial tube formation, the compound Solo F–OH was selected for a deeper characterization of its antiangiogenic potential. Our results indicate that Solo F–OH is able to inhibit some key steps of the angiogenic process, including the proliferation, migration, and invasion of endothelial cells, as well as diminish their capability to degrade the extracellular matrix proteins. The antiangiogenic potential of Solo F–OH was confirmed by means of two different in vivo models: the chorioallantoic membrane (CAM) and the zebrafish yolk membrane (ZFYM) assays. The reduction in ERK1/2 and Akt phosphorylation in endothelial cells treated with Solo F–OH denotes that this compound could target the upstream components that are common to both pathways. Taken together, our results show a new and interesting biological activity of Solo F–OH as an inhibitor of the persistent and deregulated angiogenesis that characterizes cancer and other pathologies.

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Postmitotic cells fated to become rod photoreceptors can be respecified by CNTF treatment of the retina

Development ◽

10.1242/dev.124.5.1055 ◽

1997 ◽

Vol 124 (5) ◽

pp. 1055-1067 ◽

Cited By ~ 8

Author(s):

Z.D. Ezzeddine ◽

X. Yang ◽

T. DeChiara ◽

G. Yancopoulos ◽

C.L. Cepko

Keyword(s):

Cell Fate ◽

Amacrine Cells ◽

Bipolar Cells ◽

Extrinsic Factors ◽

Rod Photoreceptors ◽

Cell Fate Decisions ◽

Retinal Explants ◽

A Cell

Lineage analyses of vertebrate retinae have led to the suggestions that cell fate decisions are made during or after the terminal cell division and that extrinsic factors can influence fate choices. The evidence for a role of extrinsic factors is strongest for development of rodent rod photoreceptors ('rods'). In an effort to identify molecules that may regulate rod development, a number of known factors were assayed in vitro. Ciliary neurotrophic factor (CNTF) was found to have a range of effects on retinal cells. Addition of CNTF to postnatal rat retinal explants resulted in a dramatic reduction in the number of differentiating rods. Conversly, the number of cells expressing markers of bipolar cell differentiation was increased to a level not normally seen in vivo or in vitro. In addition, a small increase in the percentage of cells expressing either a marker of amacrine cells or a marker of Muller glia was noted. It was determined that many of the cells that would normally differentiate into rods were the cells that differentiated as bipolar cells in the presence of CNTF. Prospective rod photoreceptors could make this change even when they were postmitotic, indicating that at least a subset of cells fated to be rods were not committed to this fate at the time they were born. These findings highlight the distinction between cell fate and commitment. Resistance to the effect of CNTF on rod differentiation occurred at about the time that a cell began to express opsin. The time of commitment to terminal rod differentiation may thus coincide with the initiation of opsin expression. In agreement with the hypothesis that CNTF plays a role in rod differentiation in vivo, a greater percentage of cells were observed differentiating as rod photoreceptors in mouse retinal explants lacking a functional CNTF receptor, relative to wild-type littermates.

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Angiogenic oligosaccharides of hyaluronan enhance the production of collagens by endothelial cells

Journal of Cell Science ◽

10.1242/jcs.105.1.213 ◽

1993 ◽

Vol 105 (1) ◽

pp. 213-218

Author(s):

P. Rooney ◽

M. Wang ◽

P. Kumar ◽

S. Kumar

Keyword(s):

Endothelial Cells ◽

Polyacrylamide Gel Electrophoresis ◽

Chorioallantoic Membrane ◽

Type I ◽

Aortic Endothelial Cells ◽

Type Viii ◽

High Degree ◽

Bovine Type

The present study demonstrates a relationship between angiogenic oligosaccharides of hyaluronan (HA) and the production of collagens during the process of angiogenesis in vivo and in vitro. The addition of angiogenic oligosaccharides of HA to the chorioallantoic membrane of the chick embryo induced a deposition of collagen fibrils. The treatment of sub-confluent cultures of bovine aortic endothelial cells with the same oligosaccharides (1 microgram/ml) increased the uptake of [3H]proline by approximately 60%. SDS-polyacrylamide gel electrophoresis of treated cultures demonstrated the enhanced synthesis of type I and type VIII collagens. The production of type VIII collagen was confirmed by western blotting and immunocytochemistry using antibodies to sheep and bovine type VIII collagen. Type VIII collagen is a short chain collagen that has a high degree of homology to cartilage-specific type X collagen. The biological functions of type VIII and type X collagens are unknown. We have suggested that the two collagens play a role in the process of angiogenesis.

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Human Erythropoietin Induces a Pro-Angiogenic Phenotype in Cultured Endothelial Cells and Stimulates Neovascularization In Vivo

Blood ◽

10.1182/blood.v93.8.2627 ◽

1999 ◽

Vol 93 (8) ◽

pp. 2627-2636 ◽

Cited By ~ 376

Author(s):

Domenico Ribatti ◽

Marco Presta ◽

Angelo Vacca ◽

Roberto Ria ◽

Roberta Giuliani ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Chorioallantoic Membrane ◽

Janus Kinase ◽

Angiogenic Factor ◽

Matrix Metalloproteinase 2 ◽

Angiogenic Potential ◽

Angiogenic Response

Abstract Hematopoietic and endothelial cell lineages share common progenitors. Accordingly, cytokines formerly thought to be specific for the hematopoietic system have been shown to affect several functions in endothelial cells, including angiogenesis. In this study, we investigated the angiogenic potential of erythropoietin (Epo), the main hormone regulating proliferation, differentiation, and survival of erythroid cells. Epo receptors (EpoRs) have been identified in the human EA.hy926 endothelial cell line by Western blot analysis. Also, recombinant human Epo (rHuEpo) stimulates Janus Kinase-2 (JAK-2) phosphorylation, cell proliferation, and matrix metalloproteinase-2 (MMP-2) production in EA.hy926 cells and significantly enhances their differentiation into vascular structures when seeded on Matrigel. In vivo, rHuEpo induces a potent angiogenic response in the chick embryo chorioallantoic membrane (CAM). Accordingly, endothelial cells of the CAM vasculature express EpoRs, as shown by immunostaining with an anti-EpoR antibody. The angiogenic response of CAM blood vessels to rHuEpo was comparable to that elicited by the prototypic angiogenic cytokine basic fibroblast growth factor (FGF2), it occurred in the absence of a significant mononuclear cell infiltrate, and it was not mimicked by endothelin-1 (ET-1) treatment. Taken together, these data demonstrate the ability of Epo to interact directly with endothelial cells and to elicit an angiogenic response in vitro and in vivo and thus act as a bona fide direct angiogenic factor.

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Notch Inhibits Osteoblast Differentiation and Causes Osteopenia

Endocrinology ◽

10.1210/en.2008-0140 ◽

2008 ◽

Vol 149 (8) ◽

pp. 3890-3899 ◽

Cited By ~ 134

Author(s):

Stefano Zanotti ◽

Anna Smerdel-Ramoya ◽

Lisa Stadmeyer ◽

Deena Durant ◽

Freddy Radtke ◽

...

Keyword(s):

Cell Fate ◽

Type I Collagen ◽

Cre Recombinase ◽

Low Fertility ◽

Type I ◽

Cmv Promoter ◽

Cell Fate Decisions ◽

Skeletal Phenotype

Notch receptors are determinants of cell fate decisions. To define the role of Notch in the adult skeleton, we created transgenic mice overexpressing the Notch intracellular domain (NICD) under the control of the type I collagen promoter. First-generation transgenics were small and osteopenic. Bone histomorphometry revealed that NICD caused a decrease in bone volume, secondary to a reduction in trabecular number; osteoblast and osteoclast number were decreased. Low fertility of founder mice and lethality of young pups did not allow the complete establishment of transgenic lines. To characterize the effect of Notch overexpression in vitro, NICD was induced in osteoblasts and stromal cells from Rosanotch mice, in which a STOP cassette flanked by loxP sites is upstream of NICD, by transduction with an adenoviral vector expressing Cre recombinase (Cre) under the control of the cytomegalovirus (CMV) promoter (Ad-CMV-Cre). NICD impaired osteoblastogenesis and inhibited Wnt/β-catenin signaling. To determine the effects of notch1 deletion in vivo, mice in which notch1 was flanked by loxP sequences (notch1loxP/loxP) were mated with mice expressing Cre recombinase under the control of the osteocalcin promoter. Conditional null notch1 mice had no obvious skeletal phenotype, possibly because of rescue by notch2; however, 1-month-old females exhibited a modest increase in osteoclast surface and eroded surface. Osteoblasts from notch1loxP/loxP mice, transduced with Ad-CMV-Cre and transfected with Notch2 small interfering RNA, displayed increased alkaline phosphatase activity. In conclusion, Notch signaling in osteoblasts causes osteopenia and impairs osteo-blastogenesis by inhibiting the Wnt/β-catenin pathway.

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