scholarly journals A nonapoptotic endothelial barrier-protective role for caspase-3

2019 ◽  
Vol 316 (6) ◽  
pp. L1118-L1126 ◽  
Author(s):  
Karthik Suresh ◽  
Kathleen Carino ◽  
Laura Johnston ◽  
Laura Servinsky ◽  
Carolyn E. Machamer ◽  
...  
Keyword(s):  
Barrier Function ◽  
Caspase 3 ◽  
Specific Inhibitor ◽  
Cell Types ◽  
Protective Role ◽  
Endothelial Barrier ◽  
Cell Stiffness ◽  
Gap Formation ◽  
Endothelial Barrier Function ◽  
Barrier Integrity

Noncanonical roles for caspase-3 are emerging in the fields of cancer and developmental biology. However, little is known of nonapoptotic functions of caspase-3 in most cell types. We have recently demonstrated a disassociation between caspase-3 activation and execution of apoptosis with accompanying cytoplasmic caspase-3 sequestration and preserved endothelial barrier function. Therefore, we tested the hypothesis that nonapoptotic caspase-3 activation promotes endothelial barrier integrity. Human lung microvascular endothelial cells were exposed to thrombin, a nonapoptotic stimulus, and endothelial barrier function was assessed using electric cell-substrate impedance sensing. Actin cytoskeletal rearrangement and paracellular gap formation were assessed using phalloidin staining. Cell stiffness was evaluated using magnetic twisting cytometry. In addition, cell lysates were harvested for protein analyses. Caspase-3 was inhibited pharmacologically with pan-caspase and a caspase-3-specific inhibitor. Molecular inhibition of caspase-3 was achieved using RNA interference. Cells exposed to thrombin exhibited a cytoplasmic activation of caspase-3 with transient and nonapoptotic decrease in endothelial barrier function as measured by a drop in electrical resistance followed by a rapid recovery. Inhibition of caspases led to a more pronounced and rapid drop in thrombin-induced endothelial barrier function, accompanied by increased endothelial cell stiffness and paracellular gaps. Caspase-3-specific inhibition and caspase-3 knockdown both resulted in more pronounced thrombin-induced endothelial barrier disruption. Taken together, our results suggest cytoplasmic caspase-3 has nonapoptotic functions in human endothelium and can promote endothelial barrier integrity.

ID: 114: AN ANTAGONIST OF LYSOPHOSPHATIDIC ACID RECEPTOR1, AM966, INCREASES PULMONARY ENDOTHELIAL PERMEABILITY THROUGH ACTIVATION OF VE-CADHERIN

2016 ◽  
Vol 64 (4) ◽  
pp. 965.3-966
Author(s):  
J Cai ◽  
J Wei ◽  
AM Jacko ◽  
J Zhao
Keyword(s):  
Lysophosphatidic Acid ◽  
Barrier Function ◽  
Endothelial Permeability ◽  
Heart Association ◽  
Endothelial Barrier ◽  
Dependent Manner ◽  
Gap Formation ◽  
Endothelial Barrier Function ◽  
Barrier Dysfunction ◽  
Barrier Integrity

BackgroundMaintenance of pulmonary endothelial barrier integrity is important for reducing severity of lung injury. VE-cadherin is a major component of cell–cell adherens junctions in endothelium. In response to inflammatory stimuli, VE-cadherin is tyrosine phosphorylated, resulting in dissociation with catenins, which links to f-actin. Lysophosphatidic acid (LPA) is a bioactive lysophospholipid, which regulates cell motility. LPA has been shown to increase lung epithelial barrier integrity, while it reduces endothelial barrier function. AM966 is an antagonist exhibiting an anti-fibrotic property. However, the effect of AM966 on pulmonary endothelial barrier integrity has not been well studied.Methods and ResultsTo investigate endothelial barrier integrity, electric cell-substrate sensing (ECIS) system was used to measure permeability in human lung microvascular endothelial cells (HLMVECs). Similar to the effect of LPA, AM966 increases permeability immediately in a dose dependent manner. To investigate the molecular mechanism by which regulates AM966-mediated reduction of endothelial barrier function, HLMVECs were treated with AM966, and then phosphorylation of myosin light chain (MLC) and VE-cadherin were determined by immunoblotting. AM966 increased phosphorylation of MLC and VE-cadherin. VE-cadherin and f-actin double immunostaining revealed that AM966 induces gap formation and f-actin stress fibers as well as dissociation between VE-cadherin and f-actin.ConclusionThis study reveals that AM966 induces lung endothelial barrier dysfunction, which is regulated by phosphorylation of VE-cadherin.This work was supported by the National Institutes of Health (R01GM115389 to J.Z.), American Heart Association 12SDG9050005 (J.Z.), American Lung Association Biomedical Research Grant RG350146 (J.Z.).

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

Vascular endothelial cells express isoforms of protein kinase A inhibitor

2002 ◽  
Vol 282 (1) ◽  
pp. C59-C66 ◽  
Author(s):  
Hazel Lum ◽  
Zengping Hao ◽  
Dave Gayle ◽  
Priyadarsini Kumar ◽  
Carolyn E. Patterson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Barrier Function ◽  
Vascular Endothelial Cells ◽  
Cell Types ◽  
Endothelial Barrier ◽  
Rabbit Muscle ◽  
Endothelial Barrier Function ◽  
Rnase Protection ◽  
Brain Microvessel

First published September 5, 2001; 10.1152/ ajpcell.00256.2001.—The expression and function of the endogenous inhibitor of cAMP-dependent protein kinase (PKI) in endothelial cells are unknown. In this study, overexpression of rabbit muscle PKI gene into endothelial cells inhibited the cAMP-mediated increase and exacerbated thrombin-induced decrease in endothelial barrier function. We investigated PKI expression in human pulmonary artery (HPAECs), foreskin microvessel (HMECs), and brain microvessel endothelial cells (HBMECs). RT-PCR using specific primers for human PKIα, human PKIγ, and mouse PKIβ sequences detected PKIα and PKIγ mRNA in all three cell types. Sequencing and BLAST analysis indicated that forward and reverse DNA strands for PKIα and PKIγ were of >96% identity with database sequences. RNase protection assays showed protection of the 542 nucleotides in HBMEC and HPAEC PKIα mRNA and 240 nucleotides in HBMEC, HPAEC, and HMEC PKIγ mRNA. Western blot analysis indicated that PKIγ protein was detected in all three cell types, whereas PKIα was found in HBMECs. In summary, endothelial cells from three different vascular beds express PKIα and PKIγ, which may be physiologically important in endothelial barrier function.

TNF-alpha induces endothelial cell F-actin depolymerization, new actin synthesis, and barrier dysfunction

1993 ◽  
Vol 264 (4) ◽  
pp. C894-C905 ◽  
Author(s):  
S. E. Goldblum ◽  
X. Ding ◽  
J. Campbell-Washington
Keyword(s):  
Endothelial Cell ◽  
Barrier Function ◽  
Endothelial Barrier ◽  
Tnf Alpha ◽  
Vascular Endothelial ◽  
Gap Formation ◽  
Endothelial Barrier Function ◽  
Barrier Dysfunction ◽  
Necrosis Factor Alpha ◽  
Actin Depolymerization

Tumor necrosis factor-alpha (TNF-alpha) influences pulmonary vascular endothelial barrier function in vitro. We studied whether recombinant TNF-alpha (rTNF-alpha) regulates endothelial barrier function through actin reorganization. Postconfluent bovine pulmonary artery endothelial cell monolayers were exposed to human rTNF-alpha (1,000 U/ml) and evaluated for 1) transendothelial [14C]albumin flux, 2) F-actin organization with fluorescence microscopy, 3) F-actin quantitation by spectrofluorometry, and 4) monomeric G-actin levels by the deoxyribonuclease I inhibition assay. rTNF-alpha induced increments in [14C]albumin flux (P < 0.04) and intercellular gap formation at > or = 2-6 h. During this same time, the endothelial F-actin pool decreased (P = 0.0064), with reciprocal increases in the G-actin pool (P < 0.0001). Prior F-actin stabilization with phallicidin protected against the rTNF-alpha-induced increments in G-actin (P < 0.002) as well as changes in barrier function (P < 0.01). Prior protein synthesis inhibition enhanced the rTNF-alpha-induced decrement in F-actin (P < 0.0001), blunted the G-actin increment (P < 0.002), and increased rTNF-alpha-induced changes in endothelial barrier function (P < 0.003). Therefore, rTNF-alpha induces pulmonary vascular endothelial F-actin depolymerization, intercellular gap formation, and barrier dysfunction. rTNF-alpha also increased total actin (P < 0.02) and new actin synthesis (P < 0.002), which may be a compensatory endothelial cell response to rTNF-alpha-induced F-actin depolymerization.

scholarly journals Downregulation of S1P Lyase Improves Barrier Function in Human Cerebral Microvascular Endothelial Cells Following an Inflammatory Challenge

2020 ◽  
Vol 21 (4) ◽  
pp. 1240 ◽  
Author(s):  
Bisera Stepanovska ◽  
Antonia I. Lange ◽  
Stephanie Schwalm ◽  
Josef Pfeilschifter ◽  
Sina M. Coldewey ◽  
...  
Keyword(s):  
Protein Expression ◽  
Barrier Function ◽  
Endothelial Barrier ◽  
Receptor Subtypes ◽  
Endothelial Barrier Function ◽  
Barrier Integrity ◽  
Junction Molecules ◽  
Microvascular Endothelial ◽  
Factor C ◽  
S1p Lyase

Sphingosine 1-phosphate (S1P) is a key bioactive lipid that regulates a myriad of physiological and pathophysiological processes, including endothelial barrier function, vascular tone, vascular inflammation, and angiogenesis. Various S1P receptor subtypes have been suggested to be involved in the regulation of these processes, whereas the contribution of intracellular S1P (iS1P) through intracellular targets is little explored. In this study, we used the human cerebral microvascular endothelial cell line HCMEC/D3 to stably downregulate the S1P lyase (SPL-kd) and evaluate the consequences on endothelial barrier function and on the molecular factors that regulate barrier tightness under normal and inflammatory conditions. The results show that in SPL-kd cells, transendothelial electrical resistance, as a measure of barrier integrity, was regulated in a dual manner. SPL-kd cells had a delayed barrier build up, a shorter interval of a stable barrier, and, thereafter, a continuous breakdown. Contrariwise, a protection was seen from the rapid proinflammatory cytokine-mediated barrier breakdown. On the molecular level, SPL-kd caused an increased basal protein expression of the adherens junction molecules PECAM-1, VE-cadherin, and β-catenin, increased activity of the signaling kinases protein kinase C, AMP-dependent kinase, and p38-MAPK, but reduced protein expression of the transcription factor c-Jun. However, the only factors that were significantly reduced in TNFα/SPL-kd compared to TNFα/control cells, which could explain the observed protection, were VCAM-1, IL-6, MCP-1, and c-Jun. Furthermore, lipid profiling revealed that dihydro-S1P and S1P were strongly enhanced in TNFα-treated SPL-kd cells. In summary, our data suggest that SPL inhibition is a valid approach to dampenan inflammatory response and augmente barrier integrity during an inflammatory challenge.

ID: 131: ACTIN RELATED PROTEIN 2/3 COMPLEX REGULATES ACTIN MEMBRANE STRUCTURES TO DETERMINE ENDOTHELIAL BARRIER FUNCTION

2016 ◽  
Vol 64 (4) ◽  
pp. 971.2-972
Author(s):  
P Belvitch ◽  
S Dudek ◽  
ME Brown ◽  
JG Garcia
Keyword(s):  
Barrier Function ◽  
Actin Polymerization ◽  
Membrane Dynamics ◽  
Endothelial Barrier ◽  
Actin Dynamics ◽  
Related Protein ◽  
Endothelial Barrier Function ◽  
Membrane Structures ◽  
Barrier Integrity ◽  
Time Points

RationaleDisruption of the pulmonary endothelial barrier is a hallmark feature of sepsis and acute lung injury/ARDS. Cytoskeletal structures such as the peripheral protrusive structures lamellipodia and filopodia are hypothesized to be important determinants of endothelial barrier function. The actin related protein 2/3 complex (Arp 2/3) is a key regulator of branched actin polymerization and may play a role in the determination and recovery of endothelial cell (EC) barrier integrity. In the current study, we make detailed observations of actin structures and membrane formations in the presence of a specific Arp 2/3 inhibitor. In addition, we study the subcellular co-localization of Arp 2/3 and cortactin, another known protein regulator of peripheral actin dynamics.MethodsCultured human lung microvascular endothelial cells (HLMVEC) were subjected to pre-treatment with the specific Arp 2/3 inhibitor (CK-666 50 µM) or vehicle (DMSO) x 1 hour. Cells were then treated with barrier enhancing sphingosine-1-phosphate (S1P 1 µM) or barrier disruptive thrombin (1 U/ml) and fixed at various time points (2–90 min) for subsequent imaging. Cells were permeabilized and treated with far-red phalloidin to stain actin, an anti-cortactin-GFP mAb as well as DAPI and imaged by confocal microscopy. Peripheral actin formations and associated membrane lamellipodia and filopodia were then measured and characterized. Additionally, the co-localization of Arp 2/3 and cortactin was determined.ResultsArp 2/3 inhibition markedly reduced lamellipodia formation in response to S1P (1 µM) over a range of time points (2–30 min). Lamellipodia depth was decreased in Arp 2/3 inhibited cells compared to control both at baseline (1.825 +/− 0.407 µM) vs. (2.545 +/− 0.459 µM) and following 30 min treatment with 1 µM S1P (1.534 +/− 0.365 µM) vs. (2.090 +/− 0.356 µM). Similarly, filopodia were shorter following Arp 2/3 inhibition (2.392 +/− 0.393 µM) vs. control (2.753 +/− 0.274 µM). Robust colocalization of Arp 2/3 and cortactin was observed very early (2–5 min) following S1P (1 µM) treatment in vehicle treated cells but was attenuated in the presence of the Arp 2/3 inhibitor. Following thrombin treatment (1 U/ml), peripheral lamellipodia were observed during the barrier recovery phase (30–60 min) but were markedly reduced following Arp 2/3 inhibition along with the persistence of intercellular gaps.ConclusionThese results further demonstrate the importance of the Arp 2/3 complex in pulmonary endothelial barrier integrity and recovery. These experiments also serve to relate the concept of altered peripheral actin and membrane dynamics leading to changes in EC barrier function.

scholarly journals Novel regulators of endothelial barrier function

2014 ◽  
Vol 307 (12) ◽  
pp. L924-L935 ◽  
Author(s):  
Dolly Mehta ◽  
Krishnan Ravindran ◽  
Wolfgang M. Kuebler
Keyword(s):  
Small Molecules ◽  
Barrier Function ◽  
Intracellular Signaling ◽  
Distress Syndrome ◽  
Endothelial Permeability ◽  
Signaling Cascades ◽  
Endothelial Barrier ◽  
Endothelial Glycocalyx ◽  
Endothelial Barrier Function ◽  
Barrier Integrity

Endothelial barrier function is an essential and tightly regulated process that ensures proper compartmentalization of the vascular and interstitial space, while allowing for the diffusive exchange of small molecules and the controlled trafficking of macromolecules and immune cells. Failure to control endothelial barrier integrity results in excessive leakage of fluid and proteins from the vasculature that can rapidly become fatal in scenarios such as sepsis or the acute respiratory distress syndrome. Here, we highlight recent advances in our understanding on the regulation of endothelial permeability, with a specific focus on the endothelial glycocalyx and endothelial scaffolds, regulatory intracellular signaling cascades, as well as triggers and mediators that either disrupt or enhance endothelial barrier integrity, and provide our perspective as to areas of seeming controversy and knowledge gaps, respectively.

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