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.

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.

ADAMTS13 TSP1 Repeat Domains Promote HUVEC Angiogenesis Via Phosphorylation of VEGFR2.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2182-2182
Author(s):  
Manfai Lee ◽  
Juan Xiao ◽  
X. Long Zheng ◽  
Jonathan Baza ◽  
Courtney Hoyt ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tube Formation ◽  
Negative Control ◽  
Endothelial Cell Migration ◽  
Endothelial Cell Proliferation ◽  
Primary Role ◽  
Polycarbonate Membrane

Abstract Abstract 2182 The primary role of ADAMTS13 (A Disintegrin And Metalloproteinase with ThromboSpondin type 1 motifs, 13) is to cleave unusually large von Willebrand factor (ULVWF) multimers under shear stress. Recently, we reported that ADAMTS13 may be a potent mitogen and chemoattractant, and it modulates angiogenesis in vitro (Microvas Res. 2012, 84, 109–115). However, the structural components and mechanism of ADAMTS13 modulating angiogenesis are not understood. Herein, we report the effect of ADAMTS13 variants on cell proliferation, migration, and, tube formation of human umbilical vein endothelial cells (HUVEC). In addition, we determined the signaling pathways by which ADAMTS13 promotes angiogenesis. ADAMTS13 fragments containing TSP1 repeat (i.e. MDT, MDTCS, TSP1 2–8, TSP1 5–8 plus CUB, and TSP1 2–8 plus CUB) were used in this study. In the proliferation model, TSP1 2–8 at the concentration of 34.6 ng/mL (651 pM) increased endothelial cell proliferation by 267 %. In the chemotaxis assay, TSP1 2–8 at the concentration of 27.7 ng/mL (521 pM) increased endothelial cell migration across a gelatinized polycarbonate membrane by 71 %. Similarly, TSP1 2–8 at the concentration of 55.4 ng/mL (1.0 nM) induced endothelial cell tube formation in Matrigel by 45 %. In all three models, the TSP1 2–8 induced angiogenic responses with similar efficacy to full-length ADAMTS13. MDT and MDTCS fragments did not affect proliferation, migration, or tube formation significantly, as compared to the negative control. To determine the mechanism by which ADAMTS13 induces angiogenesis, we incubated endothelial cells with ADAMTS13 at the concentration of 147 ng/mL (1.0 nM). We showed that ADAMTS13 increased the phosphorylation of VEGFR2, Akt, and, P44/42 MAPK, which may trigger downstream activation to promote cell proliferation and migration. Addition of anti-VEGF antibody in the culture system significantly blocked the ADAMTS13-induced effect, indicating that ADAMTS13 plays a role in promoting angiogenesis by inducing VEGF secretion from endothelial cells (Fig 1). The biological role of ADAMTS13 in angiogenesis was further demonstrated in a chick embryo model. Collagen onplants supplemented with EBM-2 (as negative control), 40 ng/mL VEGF165 (2.1 nM) (as positive control), and 306 ng/mL ADAMTS13 (2.2 nM) were placed on the chorioallantoic membrane of day 8 fertilized white leghorn chicken embryos. Localized and sustained release of VEGF and ADAMTS13 over a course of 72 hours resulted in 8-fold increase in capillary migration into the collagen onplants. Together, our findings suggest that the TSP1 repeats of ADAMTS13 metalloprotease promote angiogenesis by inducing VEGF secretion and VEGFR2 phosphorylation. Disclosures: No relevant conflicts of interest to declare.

Chronic hypoxia attenuates VEGF signaling and angiogenic responses by downregulation of KDR in human endothelial cells

2009 ◽  
Vol 296 (5) ◽  
pp. C1162-C1170 ◽  
Author(s):  
Barbara Olszewska-Pazdrak ◽  
Travis W. Hein ◽  
Paulina Olszewska ◽  
Darrell H. Carney
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Coronary Artery ◽  
Chronic Hypoxia ◽  
Tube Formation ◽  
Endothelial Cell Migration ◽  
No Production ◽  
Vegf Receptor ◽  
Human Coronary Artery ◽  
Vegf Signaling

Coronary artery disease results in progressive vascular stenosis associated with chronic myocardial ischemia. Vascular endothelial growth factor (VEGF) stimulates endothelial cell angiogenic responses to revascularize ischemic tissues; however, the effect of chronic hypoxia on the responsiveness of endothelial cells to VEGF remains unclear. We, therefore, investigated whether hypoxia alters VEGF-stimulated signaling and angiogenic responses in primary human coronary artery endothelial (HCAE) cells. Exposure of HCAE cells to hypoxia (1% O2) for 24 h decreased VEGF-stimulated endothelial cell migration (∼82%), proliferation (∼30%), and tube formation. Hypoxia attenuated VEGF-stimulated activation of endothelial nitric oxide (NO) synthase (eNOS) (∼72%) and reduced NO production in VEGF-stimulated cells from 237 ± 38.8 to 61.3 ± 28.4 nmol/l. Moreover, hypoxia also decreased the ratio of phosphorylated eNOS to total eNOS in VEGF-stimulated cells by ∼50%. This effect was not observed in thrombin-stimulated cells, suggesting that hypoxia specifically inhibited VEGF signaling upstream of eNOS phosphorylation. VEGF-induced activation of Akt, ERK1/2, p38, p70S6 kinases, and S6 ribosomal protein was also attenuated in hypoxic cells. Moreover, VEGF-stimulated phosphorylation of VEGF receptor-2 (KDR) at Y996 and Y1175 was decreased by hypoxia. This decrease correlated with a 70 ± 12% decrease in KDR protein expression. Analysis of mRNA from these cells showed that hypoxia reduced steady-state levels of KDR mRNA by 52 ± 16% and decreased mRNA stability relative to normoxic cells. Our findings demonstrate that chronic hypoxia attenuates VEGF-stimulated signaling in HCAE cells by specific downregulation of KDR expression. These data provide a novel explanation for the impaired angiogenic responses to VEGF in endothelial cells exposed to chronic hypoxia.

scholarly journals Cell Contact–dependent Activation of α3β1 Integrin Modulates Endothelial Cell Responses to Thrombospondin-1

2000 ◽  
Vol 11 (9) ◽  
pp. 2885-2900 ◽  
Author(s):  
Lakshmi Chandrasekaran ◽  
Chao-Zhen He ◽  
Hebah Al-Barazi ◽  
Henry C. Krutzsch ◽  
M. Luisa Iruela-Arispe ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Endothelial Cell Migration ◽  
Endothelial Cell Proliferation ◽  
Cell Contact ◽  
Binding Peptide ◽  
Α3β1 Integrin ◽  
Thrombospondin 1 ◽  
Cell Cell

Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified α3β1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an α3β1 integrin–binding peptide from TSP1 by normal endothelial cells is induced after loss of cell–cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, α3β1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell–cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the α3β1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the α3β1 integrin–binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by α3β1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors.

Prolylcarboxypeptidase Promotes Endothelial Cell Proliferation and Vascular Repair

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1142-1142 ◽  
Author(s):  
Gregory N Adams ◽  
Gretchen LaRusch ◽  
Alvin H. Schmaier
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Wound Repair ◽  
Endothelial Cell Migration ◽  
Endothelial Cell Proliferation ◽  
Vascular Health ◽  
Transfected Cells ◽  
Vessel Growth

Abstract Abstract 1142 Background. The S28 serine protease, prolylcarboxypeptidase (PRCP) degrades bradykinin, angiotensin II, alpha melanocyte stimulating hormone and actives plasma prekallikrein. Additionally our studies indicate that PRCP depletions in vivo and in cultured cells are associated with increased reactive oxygen species (ROS) and loss of constitutive anticoagulant function of endothelium (Blood 2011; 117:3929). PRCP-depleted mice are prothrombotic and hypertensive. We observed that PRCP-depleted cells in culture have reduced growth. We posited that PRCP promotes vascular health by influencing cell proliferation, angiogenesis, and wound repair. Methods and Results. Initial investigations determined that PRCP influences vascular endothelial cell proliferation. Bovine aortic endothelial cells (BAEC) were depleted of PRCP by siRNA knockdown resulting in 5% residual mRNA. After transfecting equal numbers of BAEC, at 24 h, the PRCP siRNA transfected cells have reduced proliferation, −18±3 change in cells/high power field (HPF) (mean±SEM), compared to the sham transfected cells, +23±8 cells/HPF, p<0.05. Additionally, PRCP siRNA-treated BAEC demonstrate less proliferation as measured by the MTS assay (Promega) (0.23±0.01 OD490 nm in PRCP-depleted cells vs 0.31±0.01 OD490 nm in sham transfected cells, p<0.02). Alternatively, when BAEC are transfected with full-length PRCP cDNA, at 24 h there is increased proliferation, +58±9 cells/HPF, vs +31±2 of sham-transfected cells, p<0.05. On a BAEC scratch assay, the degree of endothelial cell migration at 5 h in PRCP siRNA-knocked down cells is only 69% of that seen with sham-transfected cells (38±4% scratch coverage in PRCP knockdown BAEC vs 55±5% in sham knockdowns). These combined studies indicate that the content of PRCP in endothelial cells directly correlates with the degree of cell migration and proliferation. Studies next determined the influence of PRCP on angiogenesis. PRCP-depleted mice (PRCPgt/gt) have reduced new vessel growth into sub-cutaneous matrigel plugs containing FGF and VEGF. Matrigel plugs from the PRCP gt/gt mice show 3.5±0.5 Hgb mg/dL/mg-matrigel vs 6.7±1.2 Hgb mg/dL/mg matrigel in plugs in littermate wild type (WT) mice (p<0.03). When sections from the matrigel plugs are stained for the vascular marker CD31, the percent area of new vessels in the PRCPgt/gt (5.5±0.9%), as determined by ImageJ analysis, is significantly less (p<0.04) than that seen (11.9±2.1%) in WT plugs. These data indicate that host PRCP levels influence induced angiogenesis in the whole animal. Additional studies examined if PRCPgt/gt have reduced wound repair angiogenesis. Punch biopsies (5 mm) were performed on PRCPgt/gt. At day 7, no wound healed in PRCPgt/gt but 5/10 wounds healed in WT. The mean size of the PRCPgt/gt wounds is 5.5±1.1 mm2 vs 1.4±0.7 mm2 for WT, p<0.05. Also, at day 7, the wounds of PRCPgt/gt have 11.6±1.0 % area of CD31 stained vessels vs 15.0±1.0 % area of CD31 stained vessels in control wounds, p<0.03. Since there is no difference in the number of vessels in unwounded skin biopsies in PRCPgt/gt vs WT, the reduced vessel growth and delayed wound closure indicates that PRCPgt/gt mice have reduced repair angiogenesis. Conclusions. These combined studies indicate that PRCP levels in endothelial cells influence cell proliferation and growth. In the whole animal this cell biology observation translates into less induced and wound repair angiogenesis. Since PRCP-depleted endothelial cells and vessels from PRCPgt/gt have increased ROS with loss of anticoagulant properties and PRCPgt/gt have higher thrombosis risk, the finding that PRCP also influences endothelial cell growth and angiogenesis suggests that PRCP promotes vascular health and injury repair. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lukas S. Tombor ◽  
David John ◽  
Simone F. Glaser ◽  
Guillermo Luxán ◽  
Elvira Forte ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Endothelial Cell Migration ◽  
Metabolic Adaptation ◽  
Endothelial Cell Proliferation ◽  
Mesenchymal Transition ◽  
Single Cell Rna Sequencing

AbstractEndothelial cells play a critical role in the adaptation of tissues to injury. Tissue ischemia induced by infarction leads to profound changes in endothelial cell functions and can induce transition to a mesenchymal state. Here we explore the kinetics and individual cellular responses of endothelial cells after myocardial infarction by using single cell RNA sequencing. This study demonstrates a time dependent switch in endothelial cell proliferation and inflammation associated with transient changes in metabolic gene signatures. Trajectory analysis reveals that the majority of endothelial cells 3 to 7 days after myocardial infarction acquire a transient state, characterized by mesenchymal gene expression, which returns to baseline 14 days after injury. Lineage tracing, using the Cdh5-CreERT2;mT/mG mice followed by single cell RNA sequencing, confirms the transient mesenchymal transition and reveals additional hypoxic and inflammatory signatures of endothelial cells during early and late states after injury. These data suggest that endothelial cells undergo a transient mes-enchymal activation concomitant with a metabolic adaptation within the first days after myocardial infarction but do not acquire a long-term mesenchymal fate. This mesenchymal activation may facilitate endothelial cell migration and clonal expansion to regenerate the vascular network.

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.

Vascular endothelial growth factor (VEGF) in cartilage neovascularization and chondrocyte differentiation: auto-paracrine role during endochondral bone formation

2000 ◽  
Vol 113 (1) ◽  
pp. 59-69 ◽  
Author(s):  
M.F. Carlevaro ◽  
S. Cermelli ◽  
R. Cancedda ◽  
F. Descalzi Cancedda
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Hypertrophic Chondrocytes ◽  
Endothelial Cell Migration ◽  
Vegf Receptor ◽  
Hypertrophic Cartilage ◽  
Endothelial Growth Factor

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induces endothelial cell migration and proliferation in culture and is strongly angiogenic in vivo. VEGF synthesis has been shown to occur in both normal and transformed cells. The receptors for the factor have been shown to be localized mainly in endothelial cells, however, the presence of VEGF synthesis and the VEGF receptor in cells other than endothelial cells has been demonstrated. Neoangiogenesis in cartilage growth plate plays a fundamental role in endochondral ossification. We have shown that, in an avian in vitro system for chondrocyte differentiation, VEGF was produced and localized in cell clusters totally resembling in vivo cartilage. The factor was synthesized by hypertrophic chondrocytes and was released into their conditioned medium, which is highly chemotactic for endothelial cells. Antibodies against VEGF inhibited endothelial cell migration induced by chondrocyte conditioned media. Similarly, endothelial cell migration was inhibited also by antibodies directed against the VEGF receptor 2/Flk1 (VEGFR2). In avian and mammalian embryo long bones, immediately before vascular invasion, VEGF was distinctly localized in growth plate hypertrophic chondrocytes. In contrast, VEGF was not observed in quiescent and proliferating chondrocytes earlier in development. VEGF receptor 2 colocalized with the factor both in hypertrophic cartilage in vivo and hypertrophic cartilage engineered in vitro, suggesting an autocrine loop in chondrocytes at the time of their maturation to hypertrophic cells and of cartilage erosion. Regardless of cell exposure to exogenous VEGF, VEGFR-2 phosphorylation was recognized in cultured hypertrophic chondrocytes, supporting the idea of an autocrine functional activation of signal transduction in this non-endothelial cell type as a consequence of the endogenous VEGF production. In summary we propose that VEGF is actively responsible for hypertrophic cartilage neovascularization through a paracrine release by chondrocytes, with invading endothelial cells as a target. Furthermore, VEGF receptor localization and signal transduction in chondrocytes strongly support the hypothesis of a VEGF autocrine activity also in morphogenesis and differentiation of a mesoderm derived cell.

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.

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