CCM2 and PAK4 act downstream of atrial natriuretic peptide signaling to promote cell spreading

2017 ◽  
Vol 474 (11) ◽  
pp. 1897-1918 ◽  
Author(s):  
Koichi Miura ◽  
Takashi Nojiri ◽  
Yoshiharu Akitake ◽  
Koji Ando ◽  
Shigetomo Fukuhara ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Barrier Function ◽  
Molecular Mechanisms ◽  
Cell Spreading ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Cavernous Malformations ◽  
Atrial Natriuretic

Atrial natriuretic peptide (ANP) is a cardiac hormone released by the atrium in response to stretching forces. Via its receptor, guanylyl cyclase-A (GC-A), ANP maintains cardiovascular homeostasis by exerting diuretic, natriuretic, and hypotensive effects mediated, in part, by endothelial cells. Both in vivo and in vitro, ANP enhances endothelial barrier function by reducing RhoA activity and reorganizing the actin cytoskeleton. We established mouse endothelial cells that stably express GC-A and used them to analyze the molecular mechanisms responsible for actin reorganization. Stimulation by ANP resulted in phosphorylation of myosin light chain (MLC) and promotion of cell spreading. p21-activated kinase 4 (PAK4) and cerebral cavernous malformations 2 (CCM2), a scaffold protein involved in a cerebrovascular disease, were required for the phosphorylation of MLC and promotion of cell spreading by ANP. Finally, in addition to the GC domain, the kinase homology domain of GC-A was also required for ANP/GC-A signaling. Our results indicate that CCM2 and PAK4 are important downstream mediators of ANP/GC-A signaling involved in cell spreading, an important initial step in the enhancement of endothelial barrier function.

scholarly journals Atrial Natriuretic Peptide Protects against Histamine-Induced Endothelial Barrier Dysfunction in Vivo

2008 ◽  
Vol 74 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Robert Fürst ◽  
Martin F. Bubik ◽  
Peter Bihari ◽  
Bettina A. Mayer ◽  
Alexander G. Khandoga ◽  
...  
Keyword(s):  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Endothelial Barrier ◽  
Barrier Dysfunction ◽  
Endothelial Barrier Dysfunction ◽  
Atrial Natriuretic

scholarly journals Effect of inflammation-mediated endothelial metabolic shift on endothelial barrier function

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
M Aslam ◽  
H Idrees ◽  
C W Hamm ◽  
Y Ladilov
Keyword(s):  
Endothelial Cells ◽  
Glucose Uptake ◽  
Barrier Function ◽  
Atp Synthesis ◽  
Fold Increase ◽  
Endothelial Barrier ◽  
Synthesis Rate ◽  
Endothelial Barrier Function ◽  
Metabolic Switch ◽  
Barrier Integrity

Abstract Background The integrity of the endothelial cell barrier of the microvasculature is compromised by inflammation. The increased vascular permeability leads to tissue injury and organ dysfunction. In recent years, considerable advances have been made in the understanding of signalling mechanisms regulating the endothelial barrier integrity. The role of endothelial metabolism as a modulator of endothelial barrier integrity is not yet well-studied. The aim of the present study was to investigate the effect of inflammation on endothelial metabolism and its role in the maintenance of endothelial barrier integrity. Methods The study was carried out on cultured human umbilical vein endothelial cells and rat coronary microvascular endothelial cells. Inflammatory condition was simulated by treating cells with low concentrations (1 ng/mL) of TNFα for 24h. Endothelial barrier function was analysed by measuring the flux of albumen through endothelial monolayers cultured on filter membranes. Gene expression was analysed by qPCR-based assays. The capacity of endothelial cells for maximal ATP synthesis rate was investigated by the real-time live-cell imaging using FRET-based ATP-biosensor (live cell FRET). Total cellular ATP concentration was measured using luminescence-based commercial kit (ATPLite, PerkinElmer). Mitochondrial mass was analysed by the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA). The cellular glucose uptake was measured by fluorescent microscopy using a fluorescent analogue of glucose (2-NBDG). Results Treatment of human endothelial cells with TNFα resulted in significant suppression of mitochondrial and upregulation of glycolytic ATP synthesis rate, suggesting a metabolic switch. This was accompanied by a reduction in mitochondrial content (mtDNA/nDNA), reduction in total cellular ATP levels, an enhanced expression of glycolytic enzymes 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and phosphofructokinase 1 (PFK1), and enhanced glucose uptake by endothelial cells (n=5; p<0.05 for all parameters tested). Moreover, TNFα caused a 3-fold increase in endothelial permeability. Pharmacological inhibition of glycolysis either by partial replacement of glucose with 2-deoxy glucose (2DG) or an inhibition of PFKFB3 resulted in further worsening (a 5-fold increase in permeability) of TNFα-induced endothelial barrier failure. On the other hand pharmacological activation of AMPK, a potent inducer of mitochondrial biogenesis, could attenuate TNFα-induced but not 2DG-induced endothelial hyperpermeability. Conclusion The study demonstrates that TNFα induces metabolic switch towards glycolysis in endothelial cells. Moreover, the data suggest that upregulation of glycolysis may serve as an endogenous metabolic adaptation to the TNFα-induced suppression of mitochondrial ATP synthesis, which protects endothelial barrier integrity. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Justus-Liebig University GiessenDZHK (German Centre for Cardiovascular Research), partner site Rhein-Main, Bad Nauheim, Germany

scholarly journals FBXW7 regulates endothelial barrier function by suppression of the cholesterol synthesis pathway and prenylation of RhoB

2019 ◽  
Vol 30 (5) ◽  
pp. 607-621 ◽  
Author(s):  
Manon C. A. Pronk ◽  
Jisca Majolée ◽  
Anke Loregger ◽  
Jan S. M. van Bezu ◽  
Noam Zelcer ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Barrier Function ◽  
Rho Gtpases ◽  
Umbilical Vein ◽  
Cholesterol Biosynthesis ◽  
Endothelial Barrier ◽  
Biosynthesis Pathway ◽  
Endothelial Barrier Function ◽  
Cholesterol Biosynthesis Pathway

Rho GTPases control both the actin cytoskeleton and adherens junction stability and are recognized as essential regulators of endothelial barrier function. They act as molecular switches and are primarily regulated by the exchange of GDP and GTP. However, posttranslational modifications such as phosphorylation, prenylation, and ubiquitination can additionally alter their localization, stability, and activity. F-box proteins are involved in the recognition of substrate proteins predestined for ubiquitination and subsequent degradation. Given the importance of ubiquitination, we studied the effect of the loss of 62 members of the F-box protein family on endothelial barrier function in human umbilical vein endothelial cells. Endothelial barrier function was quantified by electrical cell impedance sensing and macromolecule passage assay. Our RNA interference–based screen identified FBXW7 as a key regulator of endothelial barrier function. Mechanistically, loss of FBXW7 induced the accumulation of the RhoB GTPase in endothelial cells, resulting in their increased contractility and permeability. FBXW7 knockdown induced activation of the cholesterol biosynthesis pathway and changed the prenylation of RhoB. This effect was reversed by farnesyl transferase inhibitors and by the addition of geranylgeranyl pyrophosphate. In summary, this study identifies FBXW7 as a novel regulator of endothelial barrier function in vitro. Loss of FBXW7 indirectly modulates RhoB activity via alteration of the cholesterol biosynthesis pathway and, consequently, of the prenylation status and activity of RhoB, resulting in increased contractility and disruption of the endothelial barrier.

scholarly journals Effects of Ebola Virus Glycoproteins on Endothelial Cell Activation and Barrier Function

2005 ◽  
Vol 79 (16) ◽  
pp. 10442-10450 ◽  
Author(s):  
Victoria M. Wahl-Jensen ◽  
Tatiana A. Afanasieva ◽  
Jochen Seebach ◽  
Ute Ströher ◽  
Heinz Feldmann ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Barrier Function ◽  
Cell Activation ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Endothelial Cell Activation ◽  
Tnf Α

ABSTRACT Ebola virus causes severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. Vascular instability and dysregulation are disease-decisive symptoms during severe infection. While the transmembrane glycoprotein GP1,2 has been shown to cause endothelial cell destruction, the role of the soluble glycoproteins in pathogenesis is largely unknown; however, they are hypothesized to be of biological relevance in terms of target cell activation and/or increase of endothelial permeability. Here we show that virus-like particles (VLPs) consisting of the Ebola virus matrix protein VP40 and GP1,2 were able to activate endothelial cells and induce a decrease in barrier function as determined by impedance spectroscopy and hydraulic conductivity measurements. In contrast, the soluble glycoproteins sGP and Δ-peptide did not activate endothelial cells or change the endothelial barrier function. The VLP-induced decrease in barrier function was further enhanced by the cytokine tumor necrosis factor alpha (TNF-α), which is known to induce a long-lasting decrease in endothelial cell barrier function and is hypothesized to play a key role in Ebola virus pathogenesis. Surprisingly, sGP, but not Δ-peptide, induced a recovery of endothelial barrier function following treatment with TNF-α. Our results demonstrate that Ebola virus GP1,2 in its particle-associated form mediates endothelial cell activation and a decrease in endothelial cell barrier function. Furthermore, sGP, the major soluble glycoprotein of Ebola virus, seems to possess an anti-inflammatory role by protecting the endothelial cell barrier function.

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