scholarly journals TNF‐alpha signaling collaborates with Src family kinases (SFK) to promote actin rearrangement and loss of barrier function in endothelial cells

2011 ◽  
Vol 25 (S1) ◽  
Author(s):  
Alejandro Pablo Adam ◽  
Peter Vincent ◽  
Lowery Anthony
Keyword(s):  
Endothelial Cells ◽  
Barrier Function ◽  
Src Family Kinases ◽  
Tnf Alpha ◽  
Actin Rearrangement

Proproliferative phenotype of pulmonary microvascular endothelial cells

2007 ◽  
Vol 292 (3) ◽  
pp. L671-L677 ◽  
Author(s):  
Victor Solodushko ◽  
Brian Fouty
Keyword(s):  
Endothelial Cells ◽  
Barrier Function ◽  
Pulmonary Arteries ◽  
D1 Protein ◽  
Coagulation Cascade ◽  
Microvascular Endothelial Cells ◽  
Significant Heterogeneity ◽  
Pulmonary Microvascular Endothelial Cells ◽  
Microvascular Endothelial ◽  
Pulmonary Microcirculation

Endothelial cells perform a number of important functions including release of vasodilators, control of the coagulation cascade, and restriction of solutes and fluid from the extravascular space. Regulation of fluid balance is of particular importance in the microcirculation of the lung where the loss of endothelial barrier function can lead to alveolar flooding and life-threatening hypoxemia. Significant heterogeneity exists between endothelial cells lining the microcirculation and cells from larger pulmonary arteries, however, and these differences may be relevant in restoring barrier function following vascular injury. Using well-defined populations of rat endothelial cells harvested from the pulmonary microcirculation [pulmonary microvascular endothelial cells (PMVEC)] and from larger pulmonary arteries [pulmonary artery endothelial cells (PAEC)], we compared their growth characteristics in low serum conditions. Withdrawal of serum inhibited proliferation and induced G0/G1 arrest in PAEC, whereas PMVEC failed to undergo G0/G1 arrest and continued to proliferate. Consistent with this observation, PMVEC had an increased cdk4 and cdk2 kinase activity with hyperphosphorylated (inactive) retinoblastoma (Rb) relative to PAEC as well as a threefold increase in cyclin D1 protein levels; overexpression of the cdk inhibitors p21Cip1/Waf1 and p27Kip1 induced G0/G1 arrest. While serum withdrawal failed to induce G0/G1 arrest in nonconfluent PMVEC, confluence was associated with hypophosphorylated Rb and growth arrest; loss of confluence led to resumption of growth. These data suggest that nonconfluent PMVEC continue to proliferate independently of growth factors. This proliferative characteristic may be important in restoring confluence (and barrier function) in the pulmonary microcirculation following endothelial injury.

Induction of ICAM-1 by TNF-alpha, IL-1 beta, and LPS in human endothelial cells after downregulation of PKC

1992 ◽  
Vol 263 (4) ◽  
pp. C767-C772 ◽  
Author(s):  
C. L. Myers ◽  
S. J. Wertheimer ◽  
J. Schembri-King ◽  
T. Parks ◽  
R. W. Wallace
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Umbilical Vein ◽  
Interleukin 1 ◽  
Protein Kinase Inhibitors ◽  
Intercellular Adhesion Molecule ◽  
Tnf Alpha ◽  
Necrosis Factor Alpha ◽  
Western Blots ◽  
Mrna Induction

The intercellular adhesion molecule 1 (ICAM-1) is induced on endothelial cells by tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and lipopolysaccharide (LPS). We have reported the sensitivity of cytokine-induced ICAM-1 expression to protein kinase inhibitors, including inhibitors of protein kinase C (PKC) [C. L. Myers, S. N. Desai, J. Schembri-King, G. L. Letts, and R. W. Wallace. Am. J. Physiol. 262 (Cell Physiol. 31): C365-C373, 1992]. To directly investigate the role of PKC in ICAM-1 induction, we downregulated PKC by pretreatment of human umbilical vein endothelial cells with phorbol 12-myristate 13-acetate (PMA) and assessed ICAM-1 protein and mRNA induction elicited by subsequent exposure to inflammatory stimuli. PMA treatment results in ICAM-1 protein induction that declines to basal levels by 3 days. Western blots of endothelial cell lysates reveal a nearly complete loss of immunologically reactive PKC. Subsequent activation with cytokine or LPS leads to reinduction of ICAM-1 protein and mRNA; however, the cells no longer produced substantial amounts of ICAM-1 protein or mRNA in response to PMA stimulation. Cross desensitization is observed with phorbol dibutyrate, while 4 alpha-phorbol has no desensitizing effect. The data indicate that PKC activation, while capable of inducing ICAM-1 expression, is not essential for ICAM-1 induction by the inflammatory mediators TNF-alpha, IL-1 beta, or LPS.

Control of cAMP in lung endothelial cell phenotypes. Implications for control of barrier function

1999 ◽  
Vol 277 (1) ◽  
pp. L119-L126 ◽  
Author(s):  
Troy Stevens ◽  
Judy Creighton ◽  
W. Joseph Thompson
Keyword(s):  
Endothelial Cells ◽  
Adenylyl Cyclase ◽  
Barrier Function ◽  
Enzyme Regulation ◽  
Calcium Sensitivity ◽  
Cytosolic Calcium ◽  
Calcium Entry ◽  
Camp Accumulation ◽  
Camp Phosphodiesterase ◽  
Conversion Rates

Pulmonary microvascular endothelial cells (PMVECs) form a more restrictive barrier to macromolecular flux than pulmonary arterial endothelial cells (PAECs); however, the mechanisms responsible for this intrinsic feature of PMVECs are unknown. Because cAMP improves endothelial barrier function, we hypothesized that differences in enzyme regulation of cAMP synthesis and/or degradation uniquely establish an elevated content in PMVECs. PMVECs possessed 20% higher basal cAMP concentrations than did PAECs; however, increased content was accompanied by 93% lower ATP-to-cAMP conversion rates. In PMVECs, responsiveness to β-adrenergic agonist (isoproterenol) or direct adenylyl cyclase (forskolin) activation was attenuated and responsiveness to phosphodiesterase inhibition (rolipram) was increased compared with those in PAECs. Although both types of endothelial cells express calcium-inhibited adenylyl cyclase, constitutive PMVEC cAMP accumulation was not inhibited by physiological rises in cytosolic calcium, whereas PAEC cAMP accumulation was inhibited 30% by calcium. Increasing either PMVEC calcium entry by maximal activation of store-operated calcium entry or ATP-to-cAMP conversion with rolipram unmasked calcium inhibition of adenylyl cyclase. These data indicate that suppressed calcium entry and low ATP-to-cAMP conversion intrinsically influence calcium sensitivity. Adenylyl cyclase-to-cAMP phosphodiesterase ratios regulate cAMP at elevated levels compared with PAECs, which likely contribute to enhanced microvascular barrier function.

scholarly journals Activation of AMP-Activated Protein Kinase by Adenine Alleviates TNF-Alpha-Induced Inflammation in Human Umbilical Vein Endothelial Cells

PLoS ONE ◽  
2015 ◽  
Vol 10 (11) ◽  
pp. e0142283 ◽  
Author(s):  
Yi-Fang Cheng ◽  
Guang-Huar Young ◽  
Jiun-Tsai Lin ◽  
Hyun-Hwa Jang ◽  
Chin-Chen Chen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Umbilical Vein ◽  
Tnf Alpha ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells ◽  
Amp Activated Protein Kinase

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

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