Notch2 Suppression Mimicking Changes in Human Pulmonary Hypertension Modulates Notch1 and Promotes Endothelial Cell Proliferation

2021 ◽  
Author(s):  
Sanghamitra Sahoo ◽  
Yao Li ◽  
Daniel de Jesus ◽  
John Charles Sembrat II ◽  
Mauricio M Rojas ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Target Genes ◽  
Pulmonary Arteries ◽  
Notch Receptor ◽  
Endothelial Cell Proliferation ◽  
Tissue Samples ◽  
Endothelial Proliferation ◽  
Human Pulmonary Artery

Pulmonary arterial hypertension (PAH) is a fatal cardiopulmonary disease characterized by increased vascular cell proliferation with resistance to apoptosis and occlusive remodeling of the small pulmonary arteries in humans. The Notch family of proteins are proximal signaling mediators of an evolutionarily conserved pathway that effect cell proliferation, fate determination, and development. In endothelial cells (ECs), Notch receptor 2 (Notch2) has been shown to promote endothelial apoptosis. However, a pro- or anti-proliferative role for Notch2 in pulmonary endothelial proliferation and ensuing PAH is unknown. Herein, we postulated that suppressed Notch2 signaling drives pulmonary endothelial proliferation in the setting of PAH. We observed that levels of Notch2 are ablated in lung and PA tissue samples from PAH patients compared to non-PAH controls. Interestingly, Notch2 expression was attenuated in human pulmonary artery endothelial cells (hPAECs) exposed to vasoactive factors including hypoxia, TGFβ, ET-1, and IGF-1. Gene silencing of Notch2 increased EC proliferation and reduced apoptosis. At the molecular level, Notch2-deficient hPAECs activated Akt, Erk1/2 and anti-apoptotic protein Bcl-2, and reduced levels of p21cip and Bax. Intriguingly, loss of Notch2 elicits a paradoxical activation of Notch1 and transcriptional upregulation of canonical Notch target genes Hes1, Hey1 and Hey2. Further, reduction in Rb and increased E2F1 binding to the Notch1 promoter appear to explain the upregulation of Notch1. In aggregate, our results demonstrate that loss of Notch2 derepresses Notch1 and elicits aberrant EC hallmarks of PAH. The data underscore a novel role for Notch in the maintenance of endothelial cell homeostasis.

Abstract 117: RhoC Regulates VEGF-induced Signaling in Endothelial Cells

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Luke Hoeppner ◽  
Sutapa Sinha ◽  
Ying Wang ◽  
Resham Bhattacharya ◽  
Shamit Dutta ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Vascular Permeability ◽  
Rho Gtpase ◽  
Endothelial Cell Migration ◽  
Calcium Flux ◽  
Endothelial Cell Proliferation ◽  
Vegf Receptor ◽  
Vegf Signaling

Vascular permeability factor/vascular endothelial growth factor A (VEGF) is a central regulator of angiogenesis and potently promotes vascular permeability. VEGF plays a key role in the pathologies of heart disease, stroke, and cancer. Therefore, understanding the molecular regulation of VEGF signaling is an important pursuit. Rho GTPase proteins play various roles in vasculogenesis and angiogenesis. While the functions of RhoA and RhoB in these processes have been well defined, little is known about the role of RhoC in VEGF-mediated signaling in endothelial cells and vascular development. Here, we describe how RhoC modulates VEGF signaling to regulate endothelial cell proliferation, migration and permeability. We found VEGF stimulation activates RhoC in human umbilical vein endothelial cells (HUVECs), which was completely blocked after VEGF receptor 2 (VEGFR-2) knockdown indicating that VEGF activates RhoC through VEGFR-2 signaling. Interestingly, RhoC knockdown delayed the degradation of VEGFR-2 compared to control siRNA treated HUVECs, thus implicating RhoC in VEGFR-2 trafficking. In light of our results suggesting VEGF activates RhoC through VEGFR-2, we sought to determine whether RhoC regulates vascular permeability through the VEGFR-2/phospholipase Cγ (PLCγ) /Ca 2+ /eNOS cascade. We found RhoC knockdown in VEGF-stimulated HUVECs significantly increased PLC-γ1 phosphorylation at tyrosine 783, promoted basal and VEGF-stimulated eNOS phophorylation at serine 1177, and increased calcium flux compared with control siRNA transfected HUVECs. Taken together, our findings suggest RhoC negatively regulates VEGF-induced vascular permeability. We confirmed this finding through a VEGF-inducible zebrafish model of vascular permeability by observing significantly greater vascular permeability in RhoC morpholino (MO)-injected zebrafish than control MO-injected zebrafish. Furthermore, we showed that RhoC promotes endothelial cell proliferation and negatively regulates endothelial cell migration. Our data suggests a scenario in which RhoC promotes proliferation by upregulating -catenin in a Wnt signaling-independent manner, which in turn, promotes Cyclin D1 expression and subsequently drives cell cycle progression.

The role of matrix metalloproteinase activity in the maturation of human capillary endothelial cells in vitro

1999 ◽  
Vol 112 (10) ◽  
pp. 1599-1609 ◽  
Author(s):  
B.M. Kraling ◽  
D.G. Wiederschain ◽  
T. Boehm ◽  
M. Rehn ◽  
J.B. Mulliken ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Proliferation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Endothelial Cell Proliferation ◽  
Protein Levels ◽  
Matrix Deposition ◽  
Capillary Endothelial Cells ◽  
Vessel Maturation

Vessel maturation during angiogenesis (the formation of new blood vessels) is characterized by the deposition of new basement membrane and the downregulation of endothelial cell proliferation in the new vessels. Matrix remodeling plays a crucial, but still poorly understood role, in angiogenesis regulation. We present here a novel assay system with which to study the maturation of human capillary endothelial cells in vitro. When human dermal microvascular endothelial cells (HDMEC) were cultured in the presence of dibutyryl cAMP (Bt2) and hydrocortisone (HC), the deposition of a fibrous lattice of matrix molecules consisting of collagens type IV, type XVIII, laminin and thrombospondin was induced. In basal medium (without Bt2 and HC), HDMEC released active matrix metalloproteinases (MMPs) into the culture medium. However, MMP protein levels were significantly reduced by treatment with Bt2 and HC, while protein levels and activity of endogenous tissue inhibitor of MMPs (TIMP) increased. This shift in the proteolytic balance and matrix deposition was inhibited by the specific protein kinase A inhibitors RpcAMP and KT5720 or by substituting analogues without reported glucocorticoid activity for HC. The addition of MMP inhibitors human recombinant TIMP-1 or 1,10-phenanthroline to cultures under basal conditions induced matrix deposition in a dose-dependent manner, which was not observed with the serine protease inhibitor epsilon-amino-n-caproic acid (ACA). The deposited basement membrane-type of matrix reproducibly suppressed HDMEC proliferation and increased HDMEC adhesion to the substratum. These processes of matrix deposition and downregulation of endothelial cell proliferation, hallmarks of differentiating new capillaries in the end of angiogenesis, were recapitulated in our cell culture system by decreasing the matrix-degrading activity. These data suggest that our cell culture assay provides a simple and feasible model system for the study of capillary endothelial cell differentiation and vessel maturation in vitro.

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

2017 ◽  
Vol 37 (12) ◽  
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.

Angiopoietin-1 promotes endothelial cell proliferation and migration through AP-1–dependent autocrine production of interleukin-8

Blood ◽  
2008 ◽  
Vol 111 (8) ◽  
pp. 4145-4154 ◽  
Author(s):  
Nelly A. Abdel-Malak ◽  
Coimbatore B. Srikant ◽  
Arnold S. Kristof ◽  
Sheldon A. Magder ◽  
John A. Di Battista ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Interleukin 8 ◽  
Endothelial Cell Migration ◽  
Endothelial Cell Proliferation ◽  
Cell Proliferation And Migration ◽  
And Migration ◽  
Proliferation And Migration ◽  
Angiopoietin 1

Abstract Angiopoietin-1 (Ang-1), ligand for the endothelial cell–specific Tie-2 receptors, promotes migration and proliferation of endothelial cells, however, whether these effects are promoted through the release of a secondary mediator remains unclear. In this study, we assessed whether Ang-1 promotes endothelial cell migration and proliferation through the release of interleukin-8 (IL-8). Ang-1 elicited in human umbilical vein endothelial cells (HUVECs) a dose- and time-dependent increase in IL-8 production as a result of induction of mRNA and enhanced mRNA stability of IL-8 transcripts. IL-8 production is also elevated in HUVECs transduced with retroviruses expressing Ang-1. Neutralization of IL-8 in these cells with a specific antibody significantly attenuated proliferation and migration and induced caspase-3 activation. Exposure to Ang-1 triggered a significant increase in DNA binding of activator protein-1 (AP-1) to a relatively short fragment of IL-8 promoter. Upstream from the AP-1 complex, up-regulation of IL-8 transcription by Ang-1 was mediated through the Erk1/2, SAPK/JNK, and PI-3 kinase pathways, which triggered c-Jun phosphorylation on Ser63 and Ser73. These results suggest that promotion of endothelial migration and proliferation by Ang-1 is mediated, in part, through the production of IL-8, which acts in an autocrine fashion to suppress apoptosis and facilitate cell proliferation and migration.

INHIBITION OF ENDOTHELIAL CELL PROLIFERATION BY NORMAL HUMAN MONOCYTES

1987 ◽  
Author(s):  
F Liote ◽  
M P Wautier ◽  
E Savariau ◽  
H Setiadi ◽  
J L Wautier
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Umbilical Vein ◽  
Endothelial Cell Proliferation ◽  
Thymidine Uptake ◽  
Blood Monocytes ◽  
Fibronectin Receptor

Human peripheral blood monocytes and macrophages possess factors which are capable of inhibiting or stimulating endothelial cell proliferation. We have further explored if such activity is due to cytotoxic effects of monocytes. Normal mononuclear cells were isolated first by density gradient. Monocytes were then purified by three different techniques: 1) counter centrifugation elutriation (CCE) (Beckman) 2) selective adhesion to gelatin-plasma (GPI) 3) selective adhesion to fibronectin (Fn). Cytotoxicity was estimated by counting the release of 51cr used to label the human umbilical vein endothelial cells (HUVE) prior to the addition of monocytes. Whilst [3H] thymidine incorporation by HUVE permitted us to measure the effect of monocytes on the growth of the endothelial cells. Monocytes were incubated with HUVE (12×103) for 24 to 36h at various concentrations '(1.5-12×103). No cytotoxic effect could be demonstrated but an inhibition of [3h] thymidine uptake was observed and was dependent upon monocytes concentration. Monocytes isolated on GP1 exhibited a significantly higher inhibitory effect (p<0.05) compared to those purified on Fn or by CCE.(GP1: 85±6%, Fn:58±6%, CCE:67±5%). These results indicated t*hat normal monocytes can inhibit endothelial cell proliferation. This activity appeared to be higher when monocytes were isolated on GP1 which suggest that the adhesion on this surface could stimulate monocytes not only by its fibronectin receptor. This inhibitory activity of monocyte on endothelial cells proliferation could be different in patients with vascular disorders.

scholarly journals Monocyte activation state regulates monocyte-induced endothelial proliferation through Met signaling

Blood ◽  
2010 ◽  
Vol 115 (16) ◽  
pp. 3407-3412 ◽  
Author(s):  
Shai Y. Schubert ◽  
Alejandro Benarroch ◽  
Juan Monter-Solans ◽  
Elazer R. Edelman
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Direct Interaction ◽  
Endothelial Cell Proliferation ◽  
Specific Cell ◽  
Mrna Levels ◽  
Hepatocyte Growth

Abstract Direct interaction of unactivated primary monocytes with endothelial cells induces a mitogenic effect in subconfluent, injured endothelial monolayers through activation of endothelial Met. We now report that monocytes' contact-dependent mitogenicity is controlled by activation-mediated regulation of hepatocyte growth factor. Direct interaction of unactivated monocytes with subconfluent endothelial cells for 12 hours resulted in 9- and 120-fold increase in monocyte tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) mRNA levels and bitemporal spike in hepatocyte growth factor that closely correlates with endothelial Met and extracellular signal-related kinase (ERK) phosphorylation. Once activated, monocytes cannot induce a second wave of endothelial cell proliferation and endothelial Met phosphorylation and soluble hepatocyte growth factor levels fall off. Monocyte-induced proliferation is dose dependent and limited to the induction of a single cell cycle. Monocytes retain their ability to activate other endothelial cells for up to 8 hours after initial interaction, after which they are committed to the specific cell. There is therefore a profoundly sophisticated mode of vascular repair. Confluent endothelial cells ensure vascular quiescence, whereas subconfluence promotes vessel activation. Simultaneously, circulating monocytes stimulate endothelial cell proliferation, but lose this potential once activated. Such a system provides for the fine balance that can restore vascular and endothelial homeostasis with minimal overcompensation.

Lung endothelial cell proliferation in normal and pulmonary hypertensive neonatal calves

1998 ◽  
Vol 275 (3) ◽  
pp. L593-L600 ◽  
Author(s):  
Leopold Stiebellehner ◽  
James K. Belknap ◽  
Beverly Ensley ◽  
Alan Tucker ◽  
E. Christopher Orton ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Pulmonary Artery ◽  
Main Pulmonary Artery ◽  
Endothelial Cell Proliferation ◽  
Hemodynamic Changes ◽  
Vascular Segment ◽  
Neonatal Calves

Tremendous changes in pressure and flow occur in the pulmonary and systemic circulations after birth, and these hemodynamic changes should markedly affect endothelial cell replication. However, in vivo endothelial replication rates in the neonatal period have not been reported. To label replicating endothelial cells, we administered the thymidine analog bromodeoxyuridine to calves ∼1, 4, 7, 10, and 14 days old before they were killed. Because we expected the ratio of replicating to nonreplicating cells to vary with vascular segment, we examined the main pulmonary artery, a large elastic artery, three sizes of intrapulmonary arteries, the aorta, and the carotid artery. In normoxia for arteries < 1,500 μm, ∼27% of the endothelial cells were labeled on day 1 but only ∼2% on day 14. In the main pulmonary artery, only ∼4% of the endothelial cells were labeled on day 1 and ∼2% on day 14. In contrast, in the aorta, ∼12% of the endothelial cells were labeled on day 1 and ∼2% on day 14. In chronically hypoxic animals, only ∼14% of the endothelial cells were labeled on day 1 in small lung arteries and ∼8% were still labeled on day 14. We conclude that the postnatal circulatory adaptation to extrauterine life includes significant changes in endothelial cell proliferation that vary dramatically with time and vascular location and that these changes are altered in chronic hypoxia.

scholarly journals Regulation of lymph node vascular growth by dendritic cells

2006 ◽  
Vol 203 (8) ◽  
pp. 1903-1913 ◽  
Author(s):  
Brian Webster ◽  
Eric H. Ekland ◽  
Lucila M. Agle ◽  
Susan Chyou ◽  
Regina Ruggieri ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Proliferation ◽  
Endothelial Cells ◽  
Dendritic Cells ◽  
Lymph Node ◽  
Endothelial Cell ◽  
Endothelial Cell Proliferation ◽  
Cell Trafficking ◽  
Vascular Growth ◽  
Draining Lymph Node

Lymph nodes grow rapidly and robustly at the initiation of an immune response, and this growth is accompanied by growth of the blood vessels. Although the vessels are critical for supplying nutrients and for controlling cell trafficking, the regulation of lymph node vascular growth is not well understood. We show that lymph node endothelial cells begin to proliferate within 2 d of immunization and undergo a corresponding expansion in cell numbers. Endothelial cell proliferation is dependent on CD11c+ dendritic cells (DCs), and the subcutaneous injection of DCs is sufficient to trigger endothelial cell proliferation and growth. Lymph node endothelial cell proliferation is dependent on vascular endothelial growth factor (VEGF), and DCs are associated with increased lymph node VEGF levels. DC-induced endothelial cell proliferation and increased VEGF levels are mediated by DC-induced recruitment of blood-borne cells. Vascular growth in the draining lymph node includes the growth of high endothelial venule endothelial cells and is functionally associated with increased cell entry into the lymph node. Collectively, our results suggest a scenario whereby endothelial cell expansion in the draining lymph node is induced by DCs as part of a program that optimizes the microenvironment for the ensuing immune response.

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