Abstract 547: TGFß Response Coupled to Stress Signaling by TGRL Lipolysis in Vascular Endothelial Cells

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Hnin Aung ◽  
Larissa Eiselein ◽  
Michael W Lamé ◽  
Kit Ng ◽  
Dennis W Wilson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Endothelial Cell ◽  
Map Kinases ◽  
Gene Array ◽  
Receptor Activation ◽  
Type I ◽  
Tgfβ Signaling ◽  
Tgfβ Receptor ◽  
Activating Transcription Factor

Elevation of circulating triglyceride-rich lipoproteins (TGRL) in the postprandial state is associated with increased endothelial cell inflammation and dysfunction potentially contributing to atherosclerosis. Previous studies from our laboratory showed that lipolysis products of TGRL induced endothelial cell barrier permeability, up-regulation of cytokines and adhesion molecules, and apoptosis. Gene array studies implicated the Stress Associated Protein Kinase and c-Jun N-terminal kinase (SAPK/JNK) pathway and identified the transcription factor activating transcription factor 3 (ATF3) as highly induced in this process. Additional studies demonstrated activation of second messengers for transforming growth factor beta (TGFβ) signaling (SMAD2/SMAD4) in response to lipolysis product treatment. In the present study, we asked whether TGFβ signaling was implicated in the induction of apoptosis by lipolysis products and whether there was a linkage between TGFβ receptor activation and pro-inflammatory responses. siRNA knockdown of ATF3 transcription abrogated both IL-8 and E-selectin up-regulation in lipolysis product-treated human aortic endothelial cells (HAEC). In addition, ATF3 knockdown prevented activation of caspase 3/7 and induction of p53, markers of apoptosis. Inhibition of the TGFβ Type I activin receptor-like kinase (ALK) receptors (ALKs 4, 5 and 7) by SB 431542 (10 μM) also inhibited inflammatory up-regulation, ATF3 induction and apoptosis. Both ATF3 knockdown and TGFβ receptor blockade prevented c-Jun phosphorylation, a co-activating transcription factor with ATF3 of AP-1 transcription sites. Our findings implicate TGFβ signaling in the inflammatory and apoptotic responses to TGRL lipolysis and suggest a complex interaction between TGFβ receptors and the SAPK subset of MAP Kinases.

scholarly journals TGFβ receptor internalization into EEA1-enriched early endosomes

2002 ◽  
Vol 158 (7) ◽  
pp. 1239-1249 ◽  
Author(s):  
Susan Hayes ◽  
Anil Chawla ◽  
Silvia Corvera
Keyword(s):  
Transforming Growth Factor ◽  
Nuclear Translocation ◽  
Receptor Activation ◽  
Receptor Internalization ◽  
Type I ◽  
Tgfβ Signaling ◽  
Serine Kinase ◽  
Tgfβ Receptors ◽  
Early Endosomes ◽  
Tgfβ Receptor

Transforming growth factor (TGF)β is an important physiological regulator of cellular growth and differentiation. It activates a receptor threonine/serine kinase that phosphorylates the transcription factor Smad2, which then translocates into the nucleus to trigger specific transcriptional events. Here we show that activated type I and II TGFβ receptors internalize into endosomes containing the early endosomal protein EEA1. The extent of TGFβ-stimulated Smad2 phosphorylation, Smad2 nuclear translocation, and TGFβ-stimulated transcription correlated closely with the extent of internalization of the receptor. TGFβ signaling also requires SARA (Smad anchor for receptor activation), a 135-kD polypeptide that contains a FYVE Zn++ finger motif. Here we show that SARA localizes to endosomes containing EEA1, and that disruption of this localization inhibits TGFβ-induced Smad2 nuclear translocation. These results indicate that traffic of the TGFβ receptor into the endosome enables TGFβ signaling, revealing a novel function for the endosome as a compartment specialized for the amplification of certain extracellular signals.

scholarly journals An autoantibody directed against human thrombin anion-binding exosite in a patient with arterial thrombosis: effects on platelets, endothelial cells, and protein C activation

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1843-1850 ◽  
Author(s):  
E Arnaud ◽  
M Lafay ◽  
P Gaussem ◽  
V Picard ◽  
M Jandrot-Perrus ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Arterial Thrombosis ◽  
Protein C ◽  
Receptor Activation ◽  
Anion Binding ◽  
Thrombin Receptor ◽  
Cell Functions ◽  
Human Thrombin

Abstract An autoantibody, developed by a patient with severe and recurrent arterial thrombosis, was characterized to be directed against the anion- binding exosite of thrombin, and inhibited all thrombin interactions requiring this secondary binding site without interfering with the catalytic site. The effect of the antibody was studied on thrombin interactions with platelets and endothelial cells from human umbilical veins (HUVEC). The autoantibody specifically and concentration- dependently inhibited alpha-thrombin-induced platelet activation and prostacyclin (PGI2) synthesis from HUVEC. It had no effect when gamma- thrombin or the thrombin receptor activation peptide SFLLR were the inducers. The effect of the antibody on protein C activation has been studied. The antibody blocked the thrombin-thrombomodulin activation of protein C. The inhibition of the activation was maximal with a low concentration of thrombomodulin. The fact that the autoantibody inhibited concentration-dependent alpha-thrombin-induced platelet and endothelial cell functions emphasizes the crucial role of the anion- binding exosite of thrombin to activate its receptor. In regard to the pathology, the antibody inhibited two vascular processes implicated in thrombin-antithrombotic functions, PGI2 secretion, and protein C activation, which could be implicated in this arterial thrombotic disease.

scholarly journals Role of Activating Transcription Factor 4 in Murine Choroidal Neovascularization Model

2021 ◽  
Vol 22 (16) ◽  
pp. 8890
Author(s):  
Hiroto Yasuda ◽  
Miruto Tanaka ◽  
Anri Nishinaka ◽  
Shinsuke Nakamura ◽  
Masamitsu Shimazawa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Er Stress ◽  
Choroidal Neovascularization ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Activating Transcription Factor 4 ◽  
Age Related ◽  
Activating Transcription Factor ◽  
Transcription Factor 4

Neovascular age-related macular degeneration (nAMD) featuring choroidal neovascularization (CNV) is the principal cause of irreversible blindness in elderly people in the world. Integrated stress response (ISR) is one of the intracellular signals to be adapted to various stress conditions including endoplasmic reticulum (ER) stress. ISR signaling results in the upregulation of activating transcription factor 4 (ATF4), which is a mediator of ISR. Although recent studies have suggested ISR contributes to the progression of some age-related disorders, the effects of ATF4 on the development of CNV remain unclear. Here, we performed a murine model of laser-induced CNV and found that ATF4 was highly expressed in endothelial cells of the blood vessels of the CNV lesion site. Exposure to integrated stress inhibitor (ISRIB) reduced CNV formation, vascular leakage, and the upregulation of vascular endothelial growth factor (VEGF) in retinal pigment epithelium (RPE)-choroid-sclera complex. In human retinal microvascular endothelial cells (HRMECs), ISRIB reduced the level of ATF4 and VEGF induced by an ER stress inducer, thapsigargin, and recombinant human VEGF. Moreover, ISRIB decreased the VEGF-induced cell proliferation and migration of HRMECs. Collectively, our findings showed that pro-angiogenic effects of ATF4 in endothelial cells may be a potentially therapeutic target for patients with nAMD.

Abstract 16: BcrKinase is a Novel Akt Kinase That Modulates Scavenger Receptor BI- and PDZK1-dependent Actions of HDL in Endothelium

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Anastasia Sacharidou ◽  
Wan-Ru Lee ◽  
Philip E Shaul ◽  
Chieko Mineo
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Kinase Activity ◽  
Scavenger Receptor ◽  
Adaptor Protein ◽  
Myelogenous Leukemia ◽  
Endothelial Cell Migration ◽  
Kinase Assay ◽  
Type I ◽  
Akt Kinase

High density lipoprotein cholesterol (HDL) has direct atheroprotective actions on endothelium. These are mediated by scavenger receptor class B, type I (SR-BI) and its adaptor protein PDZK1, and they entail the activation of Akt kinase, which phosphorylates and thereby stimulates endothelial nitric oxide synthase (eNOS). In the present work we sought to determine how PDZK1 couples HDL/SR-BI to Akt and eNOS to modulate endothelial function. Using tandem affinity purification (TAP) following the infection of the human endothelial cell line EAhy926 with adenovirus expressing TAP-tagged PDZK1, we identified Breakpoint Cluster Region (Bcr) kinase as a PDZK1 interacting protein in endothelium. Whereas Bcr is well-known as a component of the Bcr-Abl fusion protein that results from translocation of the Philadelphia chromosome in chronic myelogenous leukemia, little is known of its function in endothelial cells. Bcr contains several distinctive domains including a C-terminal PDZ binding motif and a serine/threonine protein kinase domain. In primary human aortic endothelial cells (HAEC), we determined that endogenous Bcr interacts with PDZK1 in an HDL-dependent manner, and that Bcr is required for HDL-induced activation of eNOS and HDL stimulation of endothelial cell migration, which underlies the ability of the lipoprotein to promote endothelial monolayer integrity. Studies of mutant forms of Bcr with disruption of PDZK1 binding or kinase activity introduced into endothelial cells further revealed that Bcr-PDZK1 interaction and its kinase function are required for HDL activation of Akt and eNOS. Using a novel kinase assay that we recently developed that employs time-resolved Forster resonance energy transfer, we found that via SR-BI and PDZK1, HDL stimulates Bcr kinase activity in endothelial cells more than 20-fold. In addition, using Akt-based peptides in studies of the two known kinases for Akt, mTOR and PDK1, we determined that HDL activates Bcr kinase to directly phosphorylate Akt-Ser473 in an mTOR independent manner, and that Akt-Thr308 is a direct substrate of PDK1. These collective findings have identified Bcr to be a novel kinase for Akt, and they have revealed that Bcr is critically involved in HDL modulation of endothelial cell phenotype.

Abstract P217: Mitochondrial N-Formyl Peptides Elicit Changes in Endothelial Cell Cytoskeleton via Formyl Peptide Receptor Activation

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Camilla F Wenceslau ◽  
Cameron G McCarthy ◽  
R.Clinton Webb
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Vascular Dysfunction ◽  
Receptor Activation ◽  
Vascular Leakage ◽  
Formyl Peptide Receptor ◽  
Experimental Hypertension ◽  
Peptide Receptor ◽  
Formyl Peptide ◽  
Formyl Peptides

One major pathophysiological characteristic of cardiovascular disease, including hypertension, is vascular dysfunction. Recently, we demonstrated that mitochondrial damage-associated molecular patterns are elevated in the circulation of SHR. Mitochondria carry hallmarks of their bacterial ancestry and one of these hallmarks is that this organelle still uses an N-formyl-methionyl-tRNA as an initiator of protein synthesis. We observed that mitochondrial N-formyl peptides (F-MIT) infusion into rats induces inflammation and vascular dysfunction, including vascular leakage, via formyl peptide receptor (FPR) activation. However, neutrophil depletion did not change this response. Therefore, we hypothesize that F-MIT via FPR activation elicits changes directly to cytoskeleton-regulating proteins in vascular cells, which may lead to increased vascular permeability. To test this hypothesis we used vascular smooth muscle cells (VSMC) and endothelial cells harvested from aortas of Sprague-Dawley rats (n=5) and human donors (n=5), respectively. Cells were divided into three groups for Western blot analysis of cytoskeleton-regulating proteins. The cells were incubated for 20 minutes in medium with either vehicle (non-formylated peptide), F-MIT (10 μM), or F-MIT after a 5-minute pre-incubation with FPR1 and 2 antagonists (Cyclosporine H, CsH, 1 μM and WRW4, 10 μM). In endothelial cells, the treatment with F-MIT increased the protein expression of RhoA/ROCK (Rho: 1.8 fold vs. Veh; ROCK: 1.4 fold vs. Veh, p<0.05), cell division control protein 42 (CDC42) (2.0 fold vs. Veh, p<0.05) and phospho-myosin light chain (MLC) Thr/Ser19 (1.5 fold vs. Veh, p<0.05). These changes were all abolished in the presence of FPR antagonists. On the other hand, F-MIT decreased expression of phospho-MLC (0.6 fold vs. Veh, p<0.05) and CDC42 (0.5 fold vs. Veh, p<0.05) and did not change RhoA/ROCK expression in VSMC. In conclusion, F-MIT, via FPR activation, elicits direct changes in endothelial cell and VSMC cytoskeleton-regulating proteins. This interaction can lead to endothelial contraction, increased vascular leakage and attenuated barrier function as observed in clinical and experimental hypertension.

scholarly journals Endothelial to Mesenchymal Transition: Role in Physiology and in the Pathogenesis of Human Diseases

2019 ◽  
Vol 99 (2) ◽  
pp. 1281-1324 ◽  
Author(s):  
Sonsoles Piera-Velazquez ◽  
Sergio A. Jimenez
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Transforming Growth Factor ◽  
Mesenchymal Cell ◽  
Human Diseases ◽  
Type I ◽  
Mesenchymal Transition ◽  
Regulatory Pathways ◽  
Complex Biological Process ◽  
Endothelial To Mesenchymal Transition

Numerous studies have demonstrated that endothelial cells are capable of undergoing endothelial to mesenchymal transition (EndMT), a newly recognized type of cellular transdifferentiation. EndMT is a complex biological process in which endothelial cells adopt a mesenchymal phenotype displaying typical mesenchymal cell morphology and functions, including the acquisition of cellular motility and contractile properties. Endothelial cells undergoing EndMT lose the expression of endothelial cell-specific proteins such as CD31/platelet-endothelial cell adhesion molecule, von Willebrand factor, and vascular-endothelial cadherin and initiate the expression of mesenchymal cell-specific genes and the production of their encoded proteins including α-smooth muscle actin, extra domain A fibronectin, N-cadherin, vimentin, fibroblast specific protein-1, also known as S100A4 protein, and fibrillar type I and type III collagens. Transforming growth factor-β1 is considered the main EndMT inducer. However, EndMT involves numerous molecular and signaling pathways that are triggered and modulated by multiple and often redundant mechanisms depending on the specific cellular context and on the physiological or pathological status of the cells. EndMT participates in highly important embryonic development processes, as well as in the pathogenesis of numerous genetically determined and acquired human diseases including malignant, vascular, inflammatory, and fibrotic disorders. Despite intensive investigation, many aspects of EndMT remain to be elucidated. The identification of molecules and regulatory pathways involved in EndMT and the discovery of specific EndMT inhibitors should provide novel therapeutic approaches for various human disorders mediated by EndMT.

Shear stress enhances human endothelial cell wound closure in vitro

2000 ◽  
Vol 279 (1) ◽  
pp. H293-H302 ◽  
Author(s):  
Maria Luiza C. Albuquerque ◽  
Christopher M. Waters ◽  
Ushma Savla ◽  
H. William Schnaper ◽  
Annette S. Flozak
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Wound Closure ◽  
Type I Collagen ◽  
Umbilical Vein ◽  
Type I ◽  
Laminar Shear Stress ◽  
Human Coronary Artery ◽  
Closure Rate

Repair of the endothelium occurs in the presence of continued blood flow, yet the mechanisms by which shear forces affect endothelial wound closure remain elusive. Therefore, we tested the hypothesis that shear stress enhances endothelial cell wound closure. Human umbilical vein endothelial cells (HUVEC) or human coronary artery endothelial cells (HCAEC) were cultured on type I collagen-coated coverslips. Cell monolayers were sheared for 18 h in a parallel-plate flow chamber at 12 dyn/cm2 to attain cellular alignment and then wounded by scraping with a metal spatula. Subsequently, the monolayers were exposed to a laminar shear stress of 3, 12, or 20 dyn/cm2 under shear-wound-shear (S-W-sH) or shear-wound-static (S-W-sT) conditions for 6 h. Wound closure was measured as a percentage of original wound width. Cell area, centroid-to-centroid distance, and cell velocity were also measured. HUVEC wounds in the S-W-sH group exposed to 3, 12, or 20 dyn/cm2 closed to 21, 39, or 50%, respectively, compared with only 59% in the S-W-sT cells. Similarly, HCAEC wounds closed to 29, 49, or 33% (S-W-sH) compared with 58% in the S-W-sT cells. Cell spreading and migration, but not proliferation, were the major mechanisms accounting for the increases in wound closure rate. These results suggest that physiological levels of shear stress enhance endothelial repair.

Natural surfactant and hyperoxic lung injury in primates. II. Morphometric analyses

1994 ◽  
Vol 76 (3) ◽  
pp. 1002-1010 ◽  
Author(s):  
P. J. Fracica ◽  
S. P. Caminiti ◽  
C. A. Piantadosi ◽  
F. G. Duhaylongsod ◽  
J. D. Crapo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Epithelial Cells ◽  
Lung Injury ◽  
Type I ◽  
Fibroblast Proliferation ◽  
Surfactant Treatment ◽  
Natural Surfactant ◽  
Hyperoxic Lung Injury ◽  
Diffuse Lung

Diffuse lung injury is accompanied by low compliance and hypoxemia with histological evidence of endothelial and alveolar epithelial cell disruption. The histological effects of treatment of an acute diffuse lung injury with a natural surfactant product were evaluated in a primate model because surfactant function and content have been shown to be abnormal in diffuse lung injury in both animals and humans. Ten baboons were ventilated with 100% O2 for 96 h, and 5 were given an aerosol of natural porcine surfactant. Physiological and biochemical measurements of the effects of hyperoxia and surfactant treatment are presented in a companion paper. After O2 exposure, lungs were fixed and processed for quantitative electron microscopy. The responses to O2 included epithelial and endothelial cell injuries, interstitial edema, and inflammation. The hyperoxic animals treated with surfactant were compared with the untreated animals; the treatments altered neutrophil distribution, fibroblast proliferation, and changes in the volumes of type I epithelial cells and endothelial cells. Surfactant-treated animals also had decreased lamellar body volume density in type II epithelial cells and preservation of endothelial cell integrity. These changes suggest complex effects of natural surfactant on the pulmonary response to hyperoxia, including protection against epithelial and endothelial cell destruction as well as significant interstitial inflammation and fibroblast proliferation. We conclude that natural surfactant treatment of hyperoxic lung injury in primates resulted in partial protection of epithelial and endothelial cells but also increased the accumulation of fibroblasts in the lung.

Transcriptional regulation of endothelial-to-mesenchymal transition in cardiac fibrosis: role of myocardin-related transcription factor A and activating transcription factor 3

2017 ◽  
Vol 95 (10) ◽  
pp. 1263-1270 ◽  
Author(s):  
Vibhuti Sharma ◽  
Nilambra Dogra ◽  
Uma Nahar Saikia ◽  
Madhu Khullar
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Cardiac Fibrosis ◽  
Activating Transcription Factor 3 ◽  
Mesenchymal Transition ◽  
Related Transcription Factor ◽  
Endothelial To Mesenchymal Transition ◽  
Activating Transcription Factor ◽  
Transcription Factor 3

The etiology of cardiac fibrogenesis is quite diverse, but a common feature is the presence of activated fibroblasts. Experimental evidence suggests that a subset of cardiac fibroblasts is derived via transition of vascular endothelial cells into fibroblasts by endothelial-to-mesenchymal transition (EndMT). During EndMT, endothelial cells lose their endothelial characteristics and acquire a mesenchymal phenotype. Molecular mechanisms and the transcriptional mediators controlling EndMT in heart during development or disease remain relatively undefined. Myocardin-related transcription factor A facilitates the transcription of cytoskeletal genes by serum response factor during fibrosis; therefore, its specific role in cardiac EndMT might be of importance. Activation of activating transcription factor 3 (ATF-3) during cardiac EndMT is speculative, since ATF-3 responds to a transforming growth factor β (TGF-β) stimulus and controls the expression of the primary epithelial-to-mesenchymal transition markers Snail, Slug, and Twist. Although the role of TGF-β in EndMT-mediated cardiac fibrosis has been established, targeting of the TGF-β ligand has not proven to be a viable anti-fibrotic strategy owing to the broad functional importance of this ligand. Thus, targeting of downstream transcriptional mediators may be a useful therapeutic approach in attenuating cardiac fibrosis. Here, we discuss some of the transcription factors that may regulate EndMT-mediated cardiac fibrosis and their involvement in type 2 diabetes.

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