Background: Apoptotic death of endothelial cells (EC) plays a crucial role for the development of... more Background: Apoptotic death of endothelial cells (EC) plays a crucial role for the development of ischemic injury. In the present study we investigated the impact of extracellular Adenosine-5 0-triphosphate (ATP), either released from cells or exogenously added, on ischemia-induced apoptosis of human EC. Methods and results: To simulate ischemic conditions, cultured human umbilical vein endothelial cells (HUVEC) were exposed to 2 h of hypoxia (Po 2 < 4 mm Hg) in serum-free medium. Ischemia led to a 1.7-fold (+/À0.4; P < 0.05) increase in EC apoptosis compared to normoxic controls as assessed by immu-noblotting and immunocytochemistry of cleaved caspase-3. Ischemia-induced apoptosis was accompanied by a 2.3-fold (+/À0.5; P < 0.05) increase of extracellular ATP detected by using a luciferin/ luciferase assay. Addition of the soluble ecto-ATPase apyrase, enhancing ATP degradation, increased ischemia-induced caspase-3 cleavage. Correspondingly, inhibition of ATP breakdown by addition of the selective ecto-ATPase inhibitor ARL67156 significantly reduced ischemia-induced apoptosis. Extracellu-lar ATP acts on membrane-bound P2Y-and P2X-receptors to induce intracellular signaling. Both, ATP and the P2Y-receptor agonist UTP significantly reduced ischemia-induced apoptosis in an equipotent manner, whereas the P2X-receptor agonist ab-me-ATP did not alter caspase-3 cleavage. The anti-apop-totic effects of ARL67156 and UTP were abrogated when P2-receptors were blocked by Suramin or PPADS. Furthermore, extracellular ATP led to an activation of MEK/ERK-and PI3K/Akt-signaling pathways. Accordingly, inhibition of MEK/ERK-signaling by UO126 or inhibition of PI3K/Akt-signaling by LY294002 abolished the anti-apoptotic effects of ATP. Conclusion: The data of the present study indicate that extracellular ATP counteracts ischemia-induced apoptosis of human EC by activating a P2Y-receptor-mediated signaling reducing caspase-3 cleavage.
Effects of oxygen depletion on cellular membranes are still poorly understood. Amphiphilic molecu... more Effects of oxygen depletion on cellular membranes are still poorly understood. Amphiphilic molecules are known to modulate the plasma membrane lipid bilayer's physical properties; in turn, mechanical properties of the lipid bilayer affect signal transduction through numerous mechanosensitive transmembrane proteins including ion channels, receptor tyrosine kinases, NADPH oxidases and G-protein coupled receptors. Thus, the concentration of oxygen in/at the lipid bilayer may modulate its mechanical properties. Here we propose that: (i) under hypoxia, the plasma membrane lipid bilayer would become oxygen depleted, (ii) depletion of oxygen molecules might induce mechanical stress in the lipid bilayer, and (iii) hypoxia-induced mechanical stress in the lipid bilayer activates mechanosensitive transmembrane proteins and downstream signalling pathways. We provide evidence – on the basis of published experimental data – that there can be links between oxygen depletion-induced mechanical stress in the membrane and activation of some mechanisms participating in oxygen sensing, including reactive oxygen species (ROS) produced by mitochondrial complex III, ROS generated at the plasma membrane by NADPH oxidases, ion channels of the transient receptor potential family and increase in intracellular Ca 2+ and stabilization of hypoxia-inducible factor 1 (HIF-1).
Objectives: Extracellular ATP stabilizes the endothelial barrier and inactivates the contractile ... more Objectives: Extracellular ATP stabilizes the endothelial barrier and inactivates the contractile machinery of endothelial cells. This inactivation relies on dephosphorylation of the regulatory myosin light chain (MLC) due to an activation of the MLC phosphatase (MLCP). To date, activation and function of MLCP in endothelial cells are only partially understood. Methods: Here, the mechanism of extracellular ATP-mediated activation of MLCP was analyzed in human endothelial cells from umbilical veins. Cells were transfected with the endogenous protein phosphatase 1 (PP1)-specific inhibitor-2 (I-2). Results: Overexpression of I-2 led to inhibition of PP1 activity and abrogation of the ATP-induced dephosphorylation of MLC. This indicates that the PP1 catalytic subunit is the principal phosphatase catalyzing the MLC dephosphorylation induced by extracellular ATP. As demonstrated by immunoprecipitation analysis, extracellular ATP recruits the PP1δ catalytic subunit and the myosin phosphatase targeting subunit (MYPT1) to form a complex. ATP stimulated dephosphorylation of MYPT1 at the inhibitory phosphorylation sites threonine 850 and 696. However, extracellular ATP failed to stimulate MYPT1 dephosphorylation in I-2-overexpressing cells. Conclusions: The present study shows for the first time that, in endothelial cells, extracellular ATP causes activation of MLCP through recruitment of PP1δ and MYPT1 into a MLCP holoenzyme complex and PP1-mediated reduction of the inhibitory phosphorylation of MYPT1.
Objective: Ischemia–reperfusion provokes barrier failure of the coronary microvasculature, leadin... more Objective: Ischemia–reperfusion provokes barrier failure of the coronary microvasculature, leading to myocardial edema development that jeopardizes functional recovery of the heart during reperfusion. Here, we tested whether adenosine 5′-triphosphate (ATP), either exogenously applied or spontaneously released during reperfusion, protects the endothelial barrier against an imminent reperfusion injury and whether interventions preventing ATP breakdown augment this protective ATP effect. Methods: Cultured microvascular coronary endothelial monolayers and isolated-perfused hearts of rat were used. Results: After ischemic conditions were induced, reperfusion of endothelial monolayers activated the endothelial contractile machinery and caused intercellular gap formation. It also led to the release of ATP. When its breakdown was inhibited by 6-N,N-diethyl-β,γ-dibromomethylene-D-ATP (ARL 67156; 100 μM), a selective ectonucleotidase inhibitor, contractile activation and gap formation were significantly reduced. Reperfusion in the presence of exogenously added ATP (10 μM) plus ARL caused an additional reduction of both aforementioned effects. In contrast, elevation of ATP degradation by apyrase (1 U/ml), a soluble ectonucleotidase, or addition of adenosine (10 μM) provoked an increase in gap formation during reperfusion that could be completely inhibited by 8-phenyltheophylline (8-PT; 10 μM), an adenosine receptor antagonist. In Langendorff-perfused rat hearts, the reperfusion-induced increase in water content was significantly reduced by ARL plus ATP. Under conditions favouring ATP degradation, an increase in myocardial edema was observed that could be blocked by 8-PT. Conclusion: ATP, either released from cells or exogenously applied, protects against reperfusion-induced failure of the coronary endothelial barrier. Inhibition of ATP degradation enhances the stabilizing effect of ATP on barrier function.
Objective—Insulin is a key regulator of metabolism, but it also confers protective effects on the... more Objective—Insulin is a key regulator of metabolism, but it also confers protective effects on the cardiovascular system. Here, we analyze the mechanism by which insulin stabilizes endothelial barrier function. Methods and Results—Insulin reduced basal and antagonized tumor necrosis factor-–induced macromolecule permeability of rat coronary microvascular endothelial monolayers. It also abolished reperfusion-induced vascular leakage in isolated-perfused rat hearts. Insulin induced dephosphorylation of the regulatory myosin light chains, as well as translocation of actin and vascular endothelial (VE)-cadherin to cell borders, indicating a reduction in contractile activation and stabilization of cell adhesion structures. These protective effects were blocked by genistein or Hydroxy-2-naphthalenylmethylphosphonic acid tris acetoxymethyl ester (HNMPA-[AM]3), a pan-tyrosine-kinase or specific insulin-receptor-kinase inhibitor, respectively. Insulin stimulated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and NO production, and it activated Rac1. Inhibition of PI3K/Akt abrogated Rac1 activation and insulin-induced barrier protection, whereas inhibition of the endothelial nitric oxide synthase/soluble guanylyl cyclase pathway partially inhibited them. Inhibition of Rac1 abrogated the assembly of actin at cell borders. Accordingly, it abolished the protective effect of insulin on barrier function of the cultured endothelial monolayer, as well as the intact coronary system of ischemic-reperfused hearts. Conclusion—Insulin stabilizes endothelial barrier via inactivation of the endothelial contractile machinery and enhancement of cell-cell adhesions. These effects are mediated via PI3K/Akt- and NO/cGMP-induced Rac1 activation.
MicroRNAs (miRs) are a class of small (~22 nucleotides), widely distributed, and highly conserved... more MicroRNAs (miRs) are a class of small (~22 nucleotides), widely distributed, and highly conserved non-coding RNA molecules and play an important post-transcriptional regulatory role by targeting mRNA. Embryonic and induced pluripotent stem cells (ESCs and iPSC, respectively) hold great promise for vascular regenerative therapies. However, several limitations currently prohibit their therapeutic use. The importance of miRs in controlling the gene expression profile of a particular cell type is emerging and a multitude of miRs have been identified that play key roles in vascular development and regeneration. A combination of pluripotency transcription factors and different miRs not only enhances the pluripotency of stem cells but also has been reported to enhance their endothelial differentiation. This review will summarize the findings that focus different miR clusters in the induction, maintenance, and directed endothelial differentiation of ESCs and iPSCs.
Compound C (comp. C) is a cell-permeable pyrrazolopyrimidine derivative and widely used as adenos... more Compound C (comp. C) is a cell-permeable pyrrazolopyrimidine derivative and widely used as adenosine monophosphate-activated protein kinase (AMPK) inhibitor to characterise the role of AMPK in various physiological processes. However, its AMPK-independent effects have also been reported. In the present study we investigated the effects of moderate dose (1–10 μM) comp. C on endothelial cell (EC) proliferation , in vitro angiogenesis, and endothelial barrier function. Comp. C was unable to inhibit AMPK phosphorylation (activation) induced by metformin and A-769662 in ECs even at concentration of 10 μM. At lower concentration (1 μM), comp. C inhibited and potentiated the inhibitory effects of metformin and A-769662 on EC proliferation, migration, tube formation, and sprouting without inducing apoptosis. However, at higher concentration (10 μM), it strongly induced apoptosis as measured by enhanced caspase 3/7 activity. Moreover, comp. C antagonised thrombin-induced EC hyperpermeability accompanied by activation of Rac1 and strengthening of adherens junctions (AJs). This EC barrier protective effect was not affected by the presence of AMPK activators. The data of the present study demonstrate that long-term treatment of ECs with low concentration comp. C inhibits EC proliferation and angio-genesis without induction of apoptosis. While short-term incubation antagonises thrombin-induced EC hyperpermeability presumably via Rac1-dependent strengthening of AJs. Furthermore, higher concentration of comp. C (10 μM or above) is toxic for ECs and warns that this agent should be used with caution to demonstrate the AMPK-mediated effects.
Effects of oxygen depletion on cellular membranes are still poorly understood. Amphiphilic molecu... more Effects of oxygen depletion on cellular membranes are still poorly understood. Amphiphilic molecules are known to modulate the plasma membrane lipid bilayer's physical properties; in turn, mechanical properties of the lipid bilayer affect signal transduction through numerous mechanosensitive transmembrane proteins including ion channels, receptor tyrosine kinases, NADPH oxidases and G-protein coupled receptors. Thus, the concentration of oxygen in/at the lipid bilayer may modulate its mechanical properties. Here we propose that: (i) under hypoxia, the plasma membrane lipid bilayer would become oxygen depleted, (ii) depletion of oxygen molecules might induce mechanical stress in the lipid bilayer, and (iii) hypoxia-induced mechanical stress in the lipid bilayer activates mechanosensitive transmembrane proteins and downstream signalling pathways. We provide evidence – on the basis of published experimental data – that there can be links between oxygen depletion-induced mechanical stress in the membrane and activation of some mechanisms participating in oxygen sensing, including reactive oxygen species (ROS) produced by mitochondrial complex III, ROS generated at the plasma membrane by NADPH oxidases, ion channels of the transient receptor potential family and increase in intracellular Ca 2+ and stabilization of hypoxia-inducible factor 1 (HIF-1).
ATP can differentially affect the micro-and macrovascular endothelial barrier. It has been shown ... more ATP can differentially affect the micro-and macrovascular endothelial barrier. It has been shown that it can both increase and/or decrease macromolecule permeability of microvascular endothelial cells and microves-sels, in vivo. We hypothesised that the barrier stabilising effect is mediated by ATP itself via P2 receptors, while barrier-disrupting effect is mediated by its metabolite adenosine via adenosine receptors. The effects of ATP, ADP, AMP and adenosine on barrier function were studied in cultured rat coronary microvascular en-dothelial monolayers (RCEC) in vitro, as well as in rat mesentery vessels, and in rat hearts in vivo. ATP and ADP showed a biphasic effect on permeability of RCEC monolayers with a reduction followed by a later increase in albumin permeability. The permeability decreasing effect of ATP was enhanced by ecto-nucleotidase inhibitor ARL67156 while permeability increasing effect was enhanced by apyrase, an extracel-lular ecto-nucleotidase. Moreover, the permeability increasing effect was abrogated by adenosine receptor antagonists, 8-phenyltheophylline (8-PT) and DMPX. Adenosine and adenosine receptor agonists 5′-(N-ethylcarboxamido)-adenosine (NECA), CGS21680, and R-PIA enhanced albumin permeability which was antagonised by 8-PT, A 1 , and A 2 but not by A 3 receptor antagonists. Likewise, immunofluorescence microsco-py of VE-cadherin and actin showed that NECA induces a disturbance of intercellular junctions. Pre-incubation of ATP antagonised the effects of NECA on permeability, actin cytoskeleton and intercellular junctions. Similar effects of the applied substances were observed in rat mesentery artery by determining the vascular leakage using intravital microscopy as well as in rat hearts by assessing myocardial water contents in vivo. In conclusion, the study demonstrates that in RCEC, ATP, ADP, and its metabolite adenosine play opposing roles on endothelial barrier function.
Background: Platelets express two ADP receptors namely P2Y 1 and P2Y 12 that regulate ADP and oth... more Background: Platelets express two ADP receptors namely P2Y 1 and P2Y 12 that regulate ADP and other agonists-induced platelet aggregation. P2Y 1 receptor activation causes platelet shape change while P2Y 12 receptor activation induces platelet aggregation. Previously, anti-aggregatory effects of ATP on ADP-induced and pro-aggregatory effects on epinephrine-induced platelet aggregation have been reported. However, the effects of other nucleoside triphosphates on platelet aggregation have never been described. The aim of the present study was to characterise the effects of nucleoside triphosphates (ATP, UTP, GTP, and CTP) on agonist-induced platelet aggregation. Methods: The experiments were performed on platelet rich plasma freshly isolated from blood donated by healthy human volunteers. Results: All the nucleoside triphosphates tested inhibited ADP-and collagen-induced platelet aggregation in a concentration-dependent manner with a rank order of potency, 2MeSATP N ATP ≥ α,β,methyleneATP N UTP NN CTP ≥ GTP. The IC 50 values against ADP (10 μM)-induced platelet aggregation were 0.039 ± 0.013, 18 ± 7, 25 ± 6, 32 ± 9, 360 ± 130, and 400 ± 160 μM, respectively. Low concentrations of ATP induced platelet shape change which was due to contaminating ADP. However, higher concentrations antagonised ADP and MRS2365-induced platelet shape change. The ATP analogue α,β,methyleneATP and CTP but not UTP and GTP also antagonised ADP-induced platelet shape change. Similarly, low ATP concentrations potentiated epinephrine-induced platelet aggregation that was abolished by P2Y 1 antagonist MRS2500 suggesting P2Y 1 receptor activation due to contaminating ADP. Higher ATP concentrations, α,β,methyleneATP, UTP, CTP, and GTP antagonised epinephrine-induced platelet aggregation. Conclusion: Thus, the data demonstrate nucleoside triphosphates in general act as P2Y 12 receptor antagonists and antagonise ADP-, collagen-, and epinephrine-induced platelet aggregation.
Inflammatory mediators like thrombin disrupt endothelial adherens junctions (AJs) and barrier int... more Inflammatory mediators like thrombin disrupt endothelial adherens junctions (AJs) and barrier integrity leading to oedema formation followed by resealing of AJs and a slow recovery of the barrier function. The molecular mechanisms of this process have not yet been fully delineated. The aim of the present study was to analyse the molecular mechanism of endothelial barrier recovery and thrombin was used as model inflammatory mediator. Thrombin caused a strong increase in endothelial permeability within 10 min accompanied by loss of Rac1 but not cdc42 activity, drop in cellular cAMP contents, and a strong activation of the endothelial contractile machinery mainly via RhoA/Rock signalling. Activation of RhoA/Rock signalling precedes and is dependent upon a rise in the cytosolic Ca 2+ concentration. Inhibition of cytosolic Ca 2+ rise but not MLCK or Rock enhances the recovery of endothelial barrier function. The cellular cAMP contents increased gradually during the barrier recovery phase (30–60 min after thrombin challenge) accompanied by an increase in Rac1 activity. Inhibition of Rac1 activity using a specific pharmacological inhibitor (NSC23766) abrogated the endothelial barrier recovery process, suggesting a Rac1-dependent phenomenon. Likewise, inhibition of either adenylyl cyclase or the cAMP-effectors PKA and Epac (with PKI and ESI-09, respectively) caused an abrogation of Rac1 activation, resealing of endothelial AJs and recovery of endothelial barrier function. The data demonstrate that endothelial barrier recovery after thrombin challenge is regulated by Rac1 GTPase activation. This Rac1 activation is due to increased levels of cellular cAMP and activation of downstream signalling during the barrier recovery phase.
Key points • Hypoxia–reoxygenation induces loss of endothelial barrier function and oedema format... more Key points • Hypoxia–reoxygenation induces loss of endothelial barrier function and oedema formation accompanied by a rise in intracellular Ca 2+ , an increase in myosin light chain (MLC) phosphorylation, and RhoA/Rho kinase (Rock) signalling and an inactivation of Rac1. • Neither inhibition of RhoA/Rock signalling nor antagonising Ca 2+ increase could protect against this hypoxia–reoxygenation-induced loss of barrier function. • Inhibition of MLC kinase (MLCK) abrogates hypoxia–reoxygenation-induced MLC phosphorylation and partially protects against hypoxia–reoxygenation-induced endothelial hyperpermeability. • Activation of Rac1 using a cAMP analogue, 8-CPT-O-Me-cAMP, which specifically activates Epac/Rap1 signalling abrogated reoxygenation-induced hyperpermeability. The data help us to better understand the role of Rho GTPases and contractile machinery in the regulation of endothelial barrier function during hypoxia–reoxygenation. Abstract Hypoxia–reoxygenation induces loss of endothelial barrier function and oedema formation, which presents a major impediment for recovery of the organ. The integrity of the endothelial barrier is highly dependent on its contractile machinery and actin dynamics, which are precisely regulated by Rho GTPases. Perturbed activities of these Rho-GTPases under hypoxia–reoxygenation lead to derangement of the actin cytoskeleton and therefore may affect the integrity of the endothelial barrier. The aim of the present study was to analyse the role of these GTPases in regulating endothelial barrier function during hypoxia–reoxygenation in cultured porcine aortic endothelial cells and isolated perfused rat hearts. Hypoxia–reoxygenation induced an increase in albumin permeability of endothelial monolayers accompanied by an activation of the endothelial contractile machinery, derangement of the actin cytoskeleton and loss of VE-cadherin from cellular junctions. Inhibition of contractile activation with ML-7 partially protected against hypoxia–reoxygenation-induced hyperpermeability. Likewise, reoxygenation caused an increase in RhoA and a reduction in Rac1 activity accompanied by enhanced stress fibre formation and loss of peripheral actin. Inhibition of RhoA/rho kinase (Rock) signalling with RhoA or Rock inhibitors led to a complete depolymerisation and derangement of the actin cytoskeleton and worsened hypoxia–reoxygenation-induced hyperpermeability. Activation of Rac1 using a cAMP analogue, 8-CPT-O-Me-cAMP, which specifically activates Epac/Rap1 signalling, restored peripheral localisation of actin and VE-cadherin at cellular junctions and abrogated reoxygenation-induced hyperpermeability. Similar results were reproduced in isolated C
Aims: Intermedin (IMD) is a novel member of calcitonin-gene-related-peptide family
which acts via... more Aims: Intermedin (IMD) is a novel member of calcitonin-gene-related-peptide family which acts via calcitonin-receptor-like-receptors (CLR) mediated activation of cAMP signalling. The main objective of the present study was to analyse the molecular mechanisms of differential effects of IMD on macromolecule permeability of endothelial cells of different vascular beds. Methods and Results: Here we demonstrate that IMD increases permeability of rat coronary microvascular endothelial cells (RCEC) and reduces permeability of human umbilical vein endothelial cells (HUVEC) and rat aortic endothelial cells via CLR and cAMP. IMD causes a derangement of actin cytoskeleton accompanied by loss of VEcadherin in RCEC, while it causes a rearrangement of actin cytoskeleton and VEcadherin at cell-cell junctions in HUVEC. IMD inactivates RhoA/Rock pathway in both cell types; however, it inactivates Rac1 in RCEC but not in HUVEC. Inhibition and rescue experiments demonstrate that both RhoA and Rac1 are required for the RCEC barrier stability while in HUVEC inhibition of RhoA/Rock signalling do not interfere with basal permeability. Conclusion: The opposite effect of IMD on RCEC and HUVEC permeability is due to differential regulation of actin cytoskeleton dynamics via RhoA and Rac1. Moreover, Rac1 activity is regulated by RhoA/Rock pathway in RCEC but not in HUVEC.
Aims Activation of cAMP signalling abrogates thrombin-induced hyperpermeability. One of the mecha... more Aims Activation of cAMP signalling abrogates thrombin-induced hyperpermeability. One of the mechanisms underlying this protective effect is the inactivation of endothelial contractile machinery, one of the major determinants of endothelial barrier function, mainly via the activation of myosin light chain phosphatase (MLCP). To date, the mechanisms of cAMP-mediated MLCP activation are only partially understood. Here the contribution of two cAMP effectors, PKA and Epac, in the regulation of endothelial contractile machinery and barrier function was studied. Methods and results Endothelial contractile machinery and barrier function were analysed in cultured human umbilical vein endothelial cells (HUVEC). The cAMP analogues 8-CPT-cAMP and 6-Bnz-cAMP were used to activate Epac and PKA, respectively, and forskolin (FSK) was used to activate adenylyl cyclase. The cells were challenged by thrombin to inhibit MLCP via the RhoA/Rock pathway. Activation of either PKA or Epac partially blocked thrombin-induced hyperpermeability. Simultaneous activation of PKA and Epac had additive effects that were comparable to that of FSK. Activation of PKA but not Epac inhibited thrombin-induced phosphorylation of MLC and the MLCP regulatory subunit MYPT1, partly via inhibition of the RhoA/Rock pathway. FSK activated the MLCP catalytic subunit PP1 via dephosphorylation and dissociation of the PP1 inhibitory protein CPI-17. FSK blunted thrombin-induced CPI-17 phosphorylation, CPI-17/PP1 complex formation, and PP1 inactivation. Down-regulation of CPI-17 attenuated thrombin-induced hyperpermeability and abolished the antagonistic effect of the PKA activator, whereas the Epac activator retained its antagonistic effect. Conclusion cAMP/PKA regulates the endothelial barrier via inhibition of the contractile machinery, mainly by the activation of MLCP via inhibition of CPI-17 and RhoA/Rock. The permeability-lowering effect of the cAMP/Epac pathway is independent of CPI-17.
Background: Intermedin/adrenomedullin2 (IMD) is a new member of calcitonin-generelated-
peptide (... more Background: Intermedin/adrenomedullin2 (IMD) is a new member of calcitonin-generelated- peptide (CGRP) family expressed in a variety of tissues including endothelial cells which acts via calcitonin-receptor-like-receptors (CLR). The main objective of the present study was to analyse the IMD receptors and its effect on endothelial barrier in human umbilical vein endothelial cells (HUVEC). Methods: We analysed the effect of IMD on albumin permeability, contractile machinery, actin cytoskeleton, and VE-cadherin on cultured HUVEC. Results: IMD, concentration-dependently, reduced endothelial albumin permeability under basal conditions and antagonised thrombin-induced hyperpermeability. The EC50 of IMD was 1.29±0.12 nM which was five times higher than adrenomedullin (AM; 0.24±0.07 nM) in reducing endothelial permeability. These IMD effects were sensitive to AM22-52 and higher conc. of αCGRP8-37. Furthermore, the pA2 values of αCGRP8-37 were 6.4 for both IMD and AM and PCR data shows that HUVEC expressed only CLR/RAMP2 receptor complex. IMD activated cAMP/PKA and cAMP/Epac signalling pathways and IMD effect on permeability was sensitive to PKA but not to eNOS inhibition. IMD antagonised thrombin-induced contractile activation, RhoA activation and stress fibre formation. It also induced Rac1 activation, enhanced cell-cell adhesion and antagonised thrombin-induced loss of cell-cell adhesion. Pharmacological inhibition of Rac1 with specific inhibitor abrogated IMD-mediated barrier stabilisation. Conclusion: IMD stabilises endothelial barrier of HUVEC monolayers via CLR/RAMP2 receptor complex. These IMD effects are mediated via cAMP-mediated contractile inactivation and strengthening of cell-cell adhesion. These findings identify IMD as barrier stabilising agent and point IMD as a potential interventional agent for the reduction of vascular leakage in inflammatory conditions.
Background: Apoptotic death of endothelial cells (EC) plays a crucial role for the development of... more Background: Apoptotic death of endothelial cells (EC) plays a crucial role for the development of ischemic injury. In the present study we investigated the impact of extracellular Adenosine-5 0-triphosphate (ATP), either released from cells or exogenously added, on ischemia-induced apoptosis of human EC. Methods and results: To simulate ischemic conditions, cultured human umbilical vein endothelial cells (HUVEC) were exposed to 2 h of hypoxia (Po 2 < 4 mm Hg) in serum-free medium. Ischemia led to a 1.7-fold (+/À0.4; P < 0.05) increase in EC apoptosis compared to normoxic controls as assessed by immu-noblotting and immunocytochemistry of cleaved caspase-3. Ischemia-induced apoptosis was accompanied by a 2.3-fold (+/À0.5; P < 0.05) increase of extracellular ATP detected by using a luciferin/ luciferase assay. Addition of the soluble ecto-ATPase apyrase, enhancing ATP degradation, increased ischemia-induced caspase-3 cleavage. Correspondingly, inhibition of ATP breakdown by addition of the selective ecto-ATPase inhibitor ARL67156 significantly reduced ischemia-induced apoptosis. Extracellu-lar ATP acts on membrane-bound P2Y-and P2X-receptors to induce intracellular signaling. Both, ATP and the P2Y-receptor agonist UTP significantly reduced ischemia-induced apoptosis in an equipotent manner, whereas the P2X-receptor agonist ab-me-ATP did not alter caspase-3 cleavage. The anti-apop-totic effects of ARL67156 and UTP were abrogated when P2-receptors were blocked by Suramin or PPADS. Furthermore, extracellular ATP led to an activation of MEK/ERK-and PI3K/Akt-signaling pathways. Accordingly, inhibition of MEK/ERK-signaling by UO126 or inhibition of PI3K/Akt-signaling by LY294002 abolished the anti-apoptotic effects of ATP. Conclusion: The data of the present study indicate that extracellular ATP counteracts ischemia-induced apoptosis of human EC by activating a P2Y-receptor-mediated signaling reducing caspase-3 cleavage.
Effects of oxygen depletion on cellular membranes are still poorly understood. Amphiphilic molecu... more Effects of oxygen depletion on cellular membranes are still poorly understood. Amphiphilic molecules are known to modulate the plasma membrane lipid bilayer's physical properties; in turn, mechanical properties of the lipid bilayer affect signal transduction through numerous mechanosensitive transmembrane proteins including ion channels, receptor tyrosine kinases, NADPH oxidases and G-protein coupled receptors. Thus, the concentration of oxygen in/at the lipid bilayer may modulate its mechanical properties. Here we propose that: (i) under hypoxia, the plasma membrane lipid bilayer would become oxygen depleted, (ii) depletion of oxygen molecules might induce mechanical stress in the lipid bilayer, and (iii) hypoxia-induced mechanical stress in the lipid bilayer activates mechanosensitive transmembrane proteins and downstream signalling pathways. We provide evidence – on the basis of published experimental data – that there can be links between oxygen depletion-induced mechanical stress in the membrane and activation of some mechanisms participating in oxygen sensing, including reactive oxygen species (ROS) produced by mitochondrial complex III, ROS generated at the plasma membrane by NADPH oxidases, ion channels of the transient receptor potential family and increase in intracellular Ca 2+ and stabilization of hypoxia-inducible factor 1 (HIF-1).
Objectives: Extracellular ATP stabilizes the endothelial barrier and inactivates the contractile ... more Objectives: Extracellular ATP stabilizes the endothelial barrier and inactivates the contractile machinery of endothelial cells. This inactivation relies on dephosphorylation of the regulatory myosin light chain (MLC) due to an activation of the MLC phosphatase (MLCP). To date, activation and function of MLCP in endothelial cells are only partially understood. Methods: Here, the mechanism of extracellular ATP-mediated activation of MLCP was analyzed in human endothelial cells from umbilical veins. Cells were transfected with the endogenous protein phosphatase 1 (PP1)-specific inhibitor-2 (I-2). Results: Overexpression of I-2 led to inhibition of PP1 activity and abrogation of the ATP-induced dephosphorylation of MLC. This indicates that the PP1 catalytic subunit is the principal phosphatase catalyzing the MLC dephosphorylation induced by extracellular ATP. As demonstrated by immunoprecipitation analysis, extracellular ATP recruits the PP1δ catalytic subunit and the myosin phosphatase targeting subunit (MYPT1) to form a complex. ATP stimulated dephosphorylation of MYPT1 at the inhibitory phosphorylation sites threonine 850 and 696. However, extracellular ATP failed to stimulate MYPT1 dephosphorylation in I-2-overexpressing cells. Conclusions: The present study shows for the first time that, in endothelial cells, extracellular ATP causes activation of MLCP through recruitment of PP1δ and MYPT1 into a MLCP holoenzyme complex and PP1-mediated reduction of the inhibitory phosphorylation of MYPT1.
Objective: Ischemia–reperfusion provokes barrier failure of the coronary microvasculature, leadin... more Objective: Ischemia–reperfusion provokes barrier failure of the coronary microvasculature, leading to myocardial edema development that jeopardizes functional recovery of the heart during reperfusion. Here, we tested whether adenosine 5′-triphosphate (ATP), either exogenously applied or spontaneously released during reperfusion, protects the endothelial barrier against an imminent reperfusion injury and whether interventions preventing ATP breakdown augment this protective ATP effect. Methods: Cultured microvascular coronary endothelial monolayers and isolated-perfused hearts of rat were used. Results: After ischemic conditions were induced, reperfusion of endothelial monolayers activated the endothelial contractile machinery and caused intercellular gap formation. It also led to the release of ATP. When its breakdown was inhibited by 6-N,N-diethyl-β,γ-dibromomethylene-D-ATP (ARL 67156; 100 μM), a selective ectonucleotidase inhibitor, contractile activation and gap formation were significantly reduced. Reperfusion in the presence of exogenously added ATP (10 μM) plus ARL caused an additional reduction of both aforementioned effects. In contrast, elevation of ATP degradation by apyrase (1 U/ml), a soluble ectonucleotidase, or addition of adenosine (10 μM) provoked an increase in gap formation during reperfusion that could be completely inhibited by 8-phenyltheophylline (8-PT; 10 μM), an adenosine receptor antagonist. In Langendorff-perfused rat hearts, the reperfusion-induced increase in water content was significantly reduced by ARL plus ATP. Under conditions favouring ATP degradation, an increase in myocardial edema was observed that could be blocked by 8-PT. Conclusion: ATP, either released from cells or exogenously applied, protects against reperfusion-induced failure of the coronary endothelial barrier. Inhibition of ATP degradation enhances the stabilizing effect of ATP on barrier function.
Objective—Insulin is a key regulator of metabolism, but it also confers protective effects on the... more Objective—Insulin is a key regulator of metabolism, but it also confers protective effects on the cardiovascular system. Here, we analyze the mechanism by which insulin stabilizes endothelial barrier function. Methods and Results—Insulin reduced basal and antagonized tumor necrosis factor-–induced macromolecule permeability of rat coronary microvascular endothelial monolayers. It also abolished reperfusion-induced vascular leakage in isolated-perfused rat hearts. Insulin induced dephosphorylation of the regulatory myosin light chains, as well as translocation of actin and vascular endothelial (VE)-cadherin to cell borders, indicating a reduction in contractile activation and stabilization of cell adhesion structures. These protective effects were blocked by genistein or Hydroxy-2-naphthalenylmethylphosphonic acid tris acetoxymethyl ester (HNMPA-[AM]3), a pan-tyrosine-kinase or specific insulin-receptor-kinase inhibitor, respectively. Insulin stimulated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and NO production, and it activated Rac1. Inhibition of PI3K/Akt abrogated Rac1 activation and insulin-induced barrier protection, whereas inhibition of the endothelial nitric oxide synthase/soluble guanylyl cyclase pathway partially inhibited them. Inhibition of Rac1 abrogated the assembly of actin at cell borders. Accordingly, it abolished the protective effect of insulin on barrier function of the cultured endothelial monolayer, as well as the intact coronary system of ischemic-reperfused hearts. Conclusion—Insulin stabilizes endothelial barrier via inactivation of the endothelial contractile machinery and enhancement of cell-cell adhesions. These effects are mediated via PI3K/Akt- and NO/cGMP-induced Rac1 activation.
MicroRNAs (miRs) are a class of small (~22 nucleotides), widely distributed, and highly conserved... more MicroRNAs (miRs) are a class of small (~22 nucleotides), widely distributed, and highly conserved non-coding RNA molecules and play an important post-transcriptional regulatory role by targeting mRNA. Embryonic and induced pluripotent stem cells (ESCs and iPSC, respectively) hold great promise for vascular regenerative therapies. However, several limitations currently prohibit their therapeutic use. The importance of miRs in controlling the gene expression profile of a particular cell type is emerging and a multitude of miRs have been identified that play key roles in vascular development and regeneration. A combination of pluripotency transcription factors and different miRs not only enhances the pluripotency of stem cells but also has been reported to enhance their endothelial differentiation. This review will summarize the findings that focus different miR clusters in the induction, maintenance, and directed endothelial differentiation of ESCs and iPSCs.
Compound C (comp. C) is a cell-permeable pyrrazolopyrimidine derivative and widely used as adenos... more Compound C (comp. C) is a cell-permeable pyrrazolopyrimidine derivative and widely used as adenosine monophosphate-activated protein kinase (AMPK) inhibitor to characterise the role of AMPK in various physiological processes. However, its AMPK-independent effects have also been reported. In the present study we investigated the effects of moderate dose (1–10 μM) comp. C on endothelial cell (EC) proliferation , in vitro angiogenesis, and endothelial barrier function. Comp. C was unable to inhibit AMPK phosphorylation (activation) induced by metformin and A-769662 in ECs even at concentration of 10 μM. At lower concentration (1 μM), comp. C inhibited and potentiated the inhibitory effects of metformin and A-769662 on EC proliferation, migration, tube formation, and sprouting without inducing apoptosis. However, at higher concentration (10 μM), it strongly induced apoptosis as measured by enhanced caspase 3/7 activity. Moreover, comp. C antagonised thrombin-induced EC hyperpermeability accompanied by activation of Rac1 and strengthening of adherens junctions (AJs). This EC barrier protective effect was not affected by the presence of AMPK activators. The data of the present study demonstrate that long-term treatment of ECs with low concentration comp. C inhibits EC proliferation and angio-genesis without induction of apoptosis. While short-term incubation antagonises thrombin-induced EC hyperpermeability presumably via Rac1-dependent strengthening of AJs. Furthermore, higher concentration of comp. C (10 μM or above) is toxic for ECs and warns that this agent should be used with caution to demonstrate the AMPK-mediated effects.
Effects of oxygen depletion on cellular membranes are still poorly understood. Amphiphilic molecu... more Effects of oxygen depletion on cellular membranes are still poorly understood. Amphiphilic molecules are known to modulate the plasma membrane lipid bilayer's physical properties; in turn, mechanical properties of the lipid bilayer affect signal transduction through numerous mechanosensitive transmembrane proteins including ion channels, receptor tyrosine kinases, NADPH oxidases and G-protein coupled receptors. Thus, the concentration of oxygen in/at the lipid bilayer may modulate its mechanical properties. Here we propose that: (i) under hypoxia, the plasma membrane lipid bilayer would become oxygen depleted, (ii) depletion of oxygen molecules might induce mechanical stress in the lipid bilayer, and (iii) hypoxia-induced mechanical stress in the lipid bilayer activates mechanosensitive transmembrane proteins and downstream signalling pathways. We provide evidence – on the basis of published experimental data – that there can be links between oxygen depletion-induced mechanical stress in the membrane and activation of some mechanisms participating in oxygen sensing, including reactive oxygen species (ROS) produced by mitochondrial complex III, ROS generated at the plasma membrane by NADPH oxidases, ion channels of the transient receptor potential family and increase in intracellular Ca 2+ and stabilization of hypoxia-inducible factor 1 (HIF-1).
ATP can differentially affect the micro-and macrovascular endothelial barrier. It has been shown ... more ATP can differentially affect the micro-and macrovascular endothelial barrier. It has been shown that it can both increase and/or decrease macromolecule permeability of microvascular endothelial cells and microves-sels, in vivo. We hypothesised that the barrier stabilising effect is mediated by ATP itself via P2 receptors, while barrier-disrupting effect is mediated by its metabolite adenosine via adenosine receptors. The effects of ATP, ADP, AMP and adenosine on barrier function were studied in cultured rat coronary microvascular en-dothelial monolayers (RCEC) in vitro, as well as in rat mesentery vessels, and in rat hearts in vivo. ATP and ADP showed a biphasic effect on permeability of RCEC monolayers with a reduction followed by a later increase in albumin permeability. The permeability decreasing effect of ATP was enhanced by ecto-nucleotidase inhibitor ARL67156 while permeability increasing effect was enhanced by apyrase, an extracel-lular ecto-nucleotidase. Moreover, the permeability increasing effect was abrogated by adenosine receptor antagonists, 8-phenyltheophylline (8-PT) and DMPX. Adenosine and adenosine receptor agonists 5′-(N-ethylcarboxamido)-adenosine (NECA), CGS21680, and R-PIA enhanced albumin permeability which was antagonised by 8-PT, A 1 , and A 2 but not by A 3 receptor antagonists. Likewise, immunofluorescence microsco-py of VE-cadherin and actin showed that NECA induces a disturbance of intercellular junctions. Pre-incubation of ATP antagonised the effects of NECA on permeability, actin cytoskeleton and intercellular junctions. Similar effects of the applied substances were observed in rat mesentery artery by determining the vascular leakage using intravital microscopy as well as in rat hearts by assessing myocardial water contents in vivo. In conclusion, the study demonstrates that in RCEC, ATP, ADP, and its metabolite adenosine play opposing roles on endothelial barrier function.
Background: Platelets express two ADP receptors namely P2Y 1 and P2Y 12 that regulate ADP and oth... more Background: Platelets express two ADP receptors namely P2Y 1 and P2Y 12 that regulate ADP and other agonists-induced platelet aggregation. P2Y 1 receptor activation causes platelet shape change while P2Y 12 receptor activation induces platelet aggregation. Previously, anti-aggregatory effects of ATP on ADP-induced and pro-aggregatory effects on epinephrine-induced platelet aggregation have been reported. However, the effects of other nucleoside triphosphates on platelet aggregation have never been described. The aim of the present study was to characterise the effects of nucleoside triphosphates (ATP, UTP, GTP, and CTP) on agonist-induced platelet aggregation. Methods: The experiments were performed on platelet rich plasma freshly isolated from blood donated by healthy human volunteers. Results: All the nucleoside triphosphates tested inhibited ADP-and collagen-induced platelet aggregation in a concentration-dependent manner with a rank order of potency, 2MeSATP N ATP ≥ α,β,methyleneATP N UTP NN CTP ≥ GTP. The IC 50 values against ADP (10 μM)-induced platelet aggregation were 0.039 ± 0.013, 18 ± 7, 25 ± 6, 32 ± 9, 360 ± 130, and 400 ± 160 μM, respectively. Low concentrations of ATP induced platelet shape change which was due to contaminating ADP. However, higher concentrations antagonised ADP and MRS2365-induced platelet shape change. The ATP analogue α,β,methyleneATP and CTP but not UTP and GTP also antagonised ADP-induced platelet shape change. Similarly, low ATP concentrations potentiated epinephrine-induced platelet aggregation that was abolished by P2Y 1 antagonist MRS2500 suggesting P2Y 1 receptor activation due to contaminating ADP. Higher ATP concentrations, α,β,methyleneATP, UTP, CTP, and GTP antagonised epinephrine-induced platelet aggregation. Conclusion: Thus, the data demonstrate nucleoside triphosphates in general act as P2Y 12 receptor antagonists and antagonise ADP-, collagen-, and epinephrine-induced platelet aggregation.
Inflammatory mediators like thrombin disrupt endothelial adherens junctions (AJs) and barrier int... more Inflammatory mediators like thrombin disrupt endothelial adherens junctions (AJs) and barrier integrity leading to oedema formation followed by resealing of AJs and a slow recovery of the barrier function. The molecular mechanisms of this process have not yet been fully delineated. The aim of the present study was to analyse the molecular mechanism of endothelial barrier recovery and thrombin was used as model inflammatory mediator. Thrombin caused a strong increase in endothelial permeability within 10 min accompanied by loss of Rac1 but not cdc42 activity, drop in cellular cAMP contents, and a strong activation of the endothelial contractile machinery mainly via RhoA/Rock signalling. Activation of RhoA/Rock signalling precedes and is dependent upon a rise in the cytosolic Ca 2+ concentration. Inhibition of cytosolic Ca 2+ rise but not MLCK or Rock enhances the recovery of endothelial barrier function. The cellular cAMP contents increased gradually during the barrier recovery phase (30–60 min after thrombin challenge) accompanied by an increase in Rac1 activity. Inhibition of Rac1 activity using a specific pharmacological inhibitor (NSC23766) abrogated the endothelial barrier recovery process, suggesting a Rac1-dependent phenomenon. Likewise, inhibition of either adenylyl cyclase or the cAMP-effectors PKA and Epac (with PKI and ESI-09, respectively) caused an abrogation of Rac1 activation, resealing of endothelial AJs and recovery of endothelial barrier function. The data demonstrate that endothelial barrier recovery after thrombin challenge is regulated by Rac1 GTPase activation. This Rac1 activation is due to increased levels of cellular cAMP and activation of downstream signalling during the barrier recovery phase.
Key points • Hypoxia–reoxygenation induces loss of endothelial barrier function and oedema format... more Key points • Hypoxia–reoxygenation induces loss of endothelial barrier function and oedema formation accompanied by a rise in intracellular Ca 2+ , an increase in myosin light chain (MLC) phosphorylation, and RhoA/Rho kinase (Rock) signalling and an inactivation of Rac1. • Neither inhibition of RhoA/Rock signalling nor antagonising Ca 2+ increase could protect against this hypoxia–reoxygenation-induced loss of barrier function. • Inhibition of MLC kinase (MLCK) abrogates hypoxia–reoxygenation-induced MLC phosphorylation and partially protects against hypoxia–reoxygenation-induced endothelial hyperpermeability. • Activation of Rac1 using a cAMP analogue, 8-CPT-O-Me-cAMP, which specifically activates Epac/Rap1 signalling abrogated reoxygenation-induced hyperpermeability. The data help us to better understand the role of Rho GTPases and contractile machinery in the regulation of endothelial barrier function during hypoxia–reoxygenation. Abstract Hypoxia–reoxygenation induces loss of endothelial barrier function and oedema formation, which presents a major impediment for recovery of the organ. The integrity of the endothelial barrier is highly dependent on its contractile machinery and actin dynamics, which are precisely regulated by Rho GTPases. Perturbed activities of these Rho-GTPases under hypoxia–reoxygenation lead to derangement of the actin cytoskeleton and therefore may affect the integrity of the endothelial barrier. The aim of the present study was to analyse the role of these GTPases in regulating endothelial barrier function during hypoxia–reoxygenation in cultured porcine aortic endothelial cells and isolated perfused rat hearts. Hypoxia–reoxygenation induced an increase in albumin permeability of endothelial monolayers accompanied by an activation of the endothelial contractile machinery, derangement of the actin cytoskeleton and loss of VE-cadherin from cellular junctions. Inhibition of contractile activation with ML-7 partially protected against hypoxia–reoxygenation-induced hyperpermeability. Likewise, reoxygenation caused an increase in RhoA and a reduction in Rac1 activity accompanied by enhanced stress fibre formation and loss of peripheral actin. Inhibition of RhoA/rho kinase (Rock) signalling with RhoA or Rock inhibitors led to a complete depolymerisation and derangement of the actin cytoskeleton and worsened hypoxia–reoxygenation-induced hyperpermeability. Activation of Rac1 using a cAMP analogue, 8-CPT-O-Me-cAMP, which specifically activates Epac/Rap1 signalling, restored peripheral localisation of actin and VE-cadherin at cellular junctions and abrogated reoxygenation-induced hyperpermeability. Similar results were reproduced in isolated C
Aims: Intermedin (IMD) is a novel member of calcitonin-gene-related-peptide family
which acts via... more Aims: Intermedin (IMD) is a novel member of calcitonin-gene-related-peptide family which acts via calcitonin-receptor-like-receptors (CLR) mediated activation of cAMP signalling. The main objective of the present study was to analyse the molecular mechanisms of differential effects of IMD on macromolecule permeability of endothelial cells of different vascular beds. Methods and Results: Here we demonstrate that IMD increases permeability of rat coronary microvascular endothelial cells (RCEC) and reduces permeability of human umbilical vein endothelial cells (HUVEC) and rat aortic endothelial cells via CLR and cAMP. IMD causes a derangement of actin cytoskeleton accompanied by loss of VEcadherin in RCEC, while it causes a rearrangement of actin cytoskeleton and VEcadherin at cell-cell junctions in HUVEC. IMD inactivates RhoA/Rock pathway in both cell types; however, it inactivates Rac1 in RCEC but not in HUVEC. Inhibition and rescue experiments demonstrate that both RhoA and Rac1 are required for the RCEC barrier stability while in HUVEC inhibition of RhoA/Rock signalling do not interfere with basal permeability. Conclusion: The opposite effect of IMD on RCEC and HUVEC permeability is due to differential regulation of actin cytoskeleton dynamics via RhoA and Rac1. Moreover, Rac1 activity is regulated by RhoA/Rock pathway in RCEC but not in HUVEC.
Aims Activation of cAMP signalling abrogates thrombin-induced hyperpermeability. One of the mecha... more Aims Activation of cAMP signalling abrogates thrombin-induced hyperpermeability. One of the mechanisms underlying this protective effect is the inactivation of endothelial contractile machinery, one of the major determinants of endothelial barrier function, mainly via the activation of myosin light chain phosphatase (MLCP). To date, the mechanisms of cAMP-mediated MLCP activation are only partially understood. Here the contribution of two cAMP effectors, PKA and Epac, in the regulation of endothelial contractile machinery and barrier function was studied. Methods and results Endothelial contractile machinery and barrier function were analysed in cultured human umbilical vein endothelial cells (HUVEC). The cAMP analogues 8-CPT-cAMP and 6-Bnz-cAMP were used to activate Epac and PKA, respectively, and forskolin (FSK) was used to activate adenylyl cyclase. The cells were challenged by thrombin to inhibit MLCP via the RhoA/Rock pathway. Activation of either PKA or Epac partially blocked thrombin-induced hyperpermeability. Simultaneous activation of PKA and Epac had additive effects that were comparable to that of FSK. Activation of PKA but not Epac inhibited thrombin-induced phosphorylation of MLC and the MLCP regulatory subunit MYPT1, partly via inhibition of the RhoA/Rock pathway. FSK activated the MLCP catalytic subunit PP1 via dephosphorylation and dissociation of the PP1 inhibitory protein CPI-17. FSK blunted thrombin-induced CPI-17 phosphorylation, CPI-17/PP1 complex formation, and PP1 inactivation. Down-regulation of CPI-17 attenuated thrombin-induced hyperpermeability and abolished the antagonistic effect of the PKA activator, whereas the Epac activator retained its antagonistic effect. Conclusion cAMP/PKA regulates the endothelial barrier via inhibition of the contractile machinery, mainly by the activation of MLCP via inhibition of CPI-17 and RhoA/Rock. The permeability-lowering effect of the cAMP/Epac pathway is independent of CPI-17.
Background: Intermedin/adrenomedullin2 (IMD) is a new member of calcitonin-generelated-
peptide (... more Background: Intermedin/adrenomedullin2 (IMD) is a new member of calcitonin-generelated- peptide (CGRP) family expressed in a variety of tissues including endothelial cells which acts via calcitonin-receptor-like-receptors (CLR). The main objective of the present study was to analyse the IMD receptors and its effect on endothelial barrier in human umbilical vein endothelial cells (HUVEC). Methods: We analysed the effect of IMD on albumin permeability, contractile machinery, actin cytoskeleton, and VE-cadherin on cultured HUVEC. Results: IMD, concentration-dependently, reduced endothelial albumin permeability under basal conditions and antagonised thrombin-induced hyperpermeability. The EC50 of IMD was 1.29±0.12 nM which was five times higher than adrenomedullin (AM; 0.24±0.07 nM) in reducing endothelial permeability. These IMD effects were sensitive to AM22-52 and higher conc. of αCGRP8-37. Furthermore, the pA2 values of αCGRP8-37 were 6.4 for both IMD and AM and PCR data shows that HUVEC expressed only CLR/RAMP2 receptor complex. IMD activated cAMP/PKA and cAMP/Epac signalling pathways and IMD effect on permeability was sensitive to PKA but not to eNOS inhibition. IMD antagonised thrombin-induced contractile activation, RhoA activation and stress fibre formation. It also induced Rac1 activation, enhanced cell-cell adhesion and antagonised thrombin-induced loss of cell-cell adhesion. Pharmacological inhibition of Rac1 with specific inhibitor abrogated IMD-mediated barrier stabilisation. Conclusion: IMD stabilises endothelial barrier of HUVEC monolayers via CLR/RAMP2 receptor complex. These IMD effects are mediated via cAMP-mediated contractile inactivation and strengthening of cell-cell adhesion. These findings identify IMD as barrier stabilising agent and point IMD as a potential interventional agent for the reduction of vascular leakage in inflammatory conditions.
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Papers by Muhammad Aslam
Here, we analyze the mechanism by which insulin stabilizes endothelial barrier function.
Methods and Results—Insulin reduced basal and antagonized tumor necrosis factor-–induced macromolecule permeability
of rat coronary microvascular endothelial monolayers. It also abolished reperfusion-induced vascular leakage in
isolated-perfused rat hearts. Insulin induced dephosphorylation of the regulatory myosin light chains, as well as
translocation of actin and vascular endothelial (VE)-cadherin to cell borders, indicating a reduction in contractile
activation and stabilization of cell adhesion structures. These protective effects were blocked by genistein or
Hydroxy-2-naphthalenylmethylphosphonic acid tris acetoxymethyl ester (HNMPA-[AM]3), a pan-tyrosine-kinase or
specific insulin-receptor-kinase inhibitor, respectively. Insulin stimulated the phosphatidylinositol 3-kinase (PI3K)/Akt
pathway and NO production, and it activated Rac1. Inhibition of PI3K/Akt abrogated Rac1 activation and
insulin-induced barrier protection, whereas inhibition of the endothelial nitric oxide synthase/soluble guanylyl cyclase
pathway partially inhibited them. Inhibition of Rac1 abrogated the assembly of actin at cell borders. Accordingly, it
abolished the protective effect of insulin on barrier function of the cultured endothelial monolayer, as well as the intact
coronary system of ischemic-reperfused hearts.
Conclusion—Insulin stabilizes endothelial barrier via inactivation of the endothelial contractile machinery and
enhancement of cell-cell adhesions. These effects are mediated via PI3K/Akt- and NO/cGMP-induced Rac1
activation.
which acts via calcitonin-receptor-like-receptors (CLR) mediated activation of cAMP
signalling. The main objective of the present study was to analyse the molecular
mechanisms of differential effects of IMD on macromolecule permeability of
endothelial cells of different vascular beds.
Methods and Results: Here we demonstrate that IMD increases permeability of rat
coronary microvascular endothelial cells (RCEC) and reduces permeability of human
umbilical vein endothelial cells (HUVEC) and rat aortic endothelial cells via CLR and
cAMP. IMD causes a derangement of actin cytoskeleton accompanied by loss of VEcadherin
in RCEC, while it causes a rearrangement of actin cytoskeleton and VEcadherin
at cell-cell junctions in HUVEC. IMD inactivates RhoA/Rock pathway in both
cell types; however, it inactivates Rac1 in RCEC but not in HUVEC. Inhibition and
rescue experiments demonstrate that both RhoA and Rac1 are required for the
RCEC barrier stability while in HUVEC inhibition of RhoA/Rock signalling do not
interfere with basal permeability.
Conclusion: The opposite effect of IMD on RCEC and HUVEC permeability is due to
differential regulation of actin cytoskeleton dynamics via RhoA and Rac1. Moreover,
Rac1 activity is regulated by RhoA/Rock pathway in RCEC but not in HUVEC.
protective effect is the inactivation of endothelial contractile machinery, one of the major determinants of endothelial
barrier function, mainly via the activation of myosin light chain phosphatase (MLCP). To date, the mechanisms of
cAMP-mediated MLCP activation are only partially understood. Here the contribution of two cAMP effectors,
PKA and Epac, in the regulation of endothelial contractile machinery and barrier function was studied.
Methods
and results
Endothelial contractile machinery and barrier function were analysed in cultured human umbilical vein endothelial cells
(HUVEC). The cAMP analogues 8-CPT-cAMP and 6-Bnz-cAMP were used to activate Epac and PKA, respectively, and
forskolin (FSK) was used to activate adenylyl cyclase. The cells were challenged by thrombin to inhibit MLCP via the
RhoA/Rock pathway. Activation of either PKA or Epac partially blocked thrombin-induced hyperpermeability. Simultaneous
activation of PKA and Epac had additive effects that were comparable to that of FSK. Activation of PKA but
not Epac inhibited thrombin-induced phosphorylation of MLC and the MLCP regulatory subunit MYPT1, partly via inhibition
of the RhoA/Rock pathway. FSK activated the MLCP catalytic subunit PP1 via dephosphorylation and dissociation
of the PP1 inhibitory protein CPI-17. FSK blunted thrombin-induced CPI-17 phosphorylation, CPI-17/PP1
complex formation, and PP1 inactivation. Down-regulation of CPI-17 attenuated thrombin-induced hyperpermeability
and abolished the antagonistic effect of the PKA activator, whereas the Epac activator retained its antagonistic effect.
Conclusion cAMP/PKA regulates the endothelial barrier via inhibition of the contractile machinery, mainly by the activation of
MLCP via inhibition of CPI-17 and RhoA/Rock. The permeability-lowering effect of the cAMP/Epac pathway is independent
of CPI-17.
peptide (CGRP) family expressed in a variety of tissues including endothelial
cells which acts via calcitonin-receptor-like-receptors (CLR). The main objective of the
present study was to analyse the IMD receptors and its effect on endothelial barrier in
human umbilical vein endothelial cells (HUVEC).
Methods: We analysed the effect of IMD on albumin permeability, contractile
machinery, actin cytoskeleton, and VE-cadherin on cultured HUVEC.
Results: IMD, concentration-dependently, reduced endothelial albumin permeability
under basal conditions and antagonised thrombin-induced hyperpermeability. The EC50
of IMD was 1.29±0.12 nM which was five times higher than adrenomedullin (AM;
0.24±0.07 nM) in reducing endothelial permeability. These IMD effects were sensitive to
AM22-52 and higher conc. of αCGRP8-37. Furthermore, the pA2 values of αCGRP8-37
were 6.4 for both IMD and AM and PCR data shows that HUVEC expressed only
CLR/RAMP2 receptor complex. IMD activated cAMP/PKA and cAMP/Epac signalling
pathways and IMD effect on permeability was sensitive to PKA but not to eNOS
inhibition. IMD antagonised thrombin-induced contractile activation, RhoA activation and
stress fibre formation. It also induced Rac1 activation, enhanced cell-cell adhesion and
antagonised thrombin-induced loss of cell-cell adhesion. Pharmacological inhibition of
Rac1 with specific inhibitor abrogated IMD-mediated barrier stabilisation.
Conclusion: IMD stabilises endothelial barrier of HUVEC monolayers via CLR/RAMP2
receptor complex. These IMD effects are mediated via cAMP-mediated contractile
inactivation and strengthening of cell-cell adhesion. These findings identify IMD as
barrier stabilising agent and point IMD as a potential interventional agent for the
reduction of vascular leakage in inflammatory conditions.
Here, we analyze the mechanism by which insulin stabilizes endothelial barrier function.
Methods and Results—Insulin reduced basal and antagonized tumor necrosis factor-–induced macromolecule permeability
of rat coronary microvascular endothelial monolayers. It also abolished reperfusion-induced vascular leakage in
isolated-perfused rat hearts. Insulin induced dephosphorylation of the regulatory myosin light chains, as well as
translocation of actin and vascular endothelial (VE)-cadherin to cell borders, indicating a reduction in contractile
activation and stabilization of cell adhesion structures. These protective effects were blocked by genistein or
Hydroxy-2-naphthalenylmethylphosphonic acid tris acetoxymethyl ester (HNMPA-[AM]3), a pan-tyrosine-kinase or
specific insulin-receptor-kinase inhibitor, respectively. Insulin stimulated the phosphatidylinositol 3-kinase (PI3K)/Akt
pathway and NO production, and it activated Rac1. Inhibition of PI3K/Akt abrogated Rac1 activation and
insulin-induced barrier protection, whereas inhibition of the endothelial nitric oxide synthase/soluble guanylyl cyclase
pathway partially inhibited them. Inhibition of Rac1 abrogated the assembly of actin at cell borders. Accordingly, it
abolished the protective effect of insulin on barrier function of the cultured endothelial monolayer, as well as the intact
coronary system of ischemic-reperfused hearts.
Conclusion—Insulin stabilizes endothelial barrier via inactivation of the endothelial contractile machinery and
enhancement of cell-cell adhesions. These effects are mediated via PI3K/Akt- and NO/cGMP-induced Rac1
activation.
which acts via calcitonin-receptor-like-receptors (CLR) mediated activation of cAMP
signalling. The main objective of the present study was to analyse the molecular
mechanisms of differential effects of IMD on macromolecule permeability of
endothelial cells of different vascular beds.
Methods and Results: Here we demonstrate that IMD increases permeability of rat
coronary microvascular endothelial cells (RCEC) and reduces permeability of human
umbilical vein endothelial cells (HUVEC) and rat aortic endothelial cells via CLR and
cAMP. IMD causes a derangement of actin cytoskeleton accompanied by loss of VEcadherin
in RCEC, while it causes a rearrangement of actin cytoskeleton and VEcadherin
at cell-cell junctions in HUVEC. IMD inactivates RhoA/Rock pathway in both
cell types; however, it inactivates Rac1 in RCEC but not in HUVEC. Inhibition and
rescue experiments demonstrate that both RhoA and Rac1 are required for the
RCEC barrier stability while in HUVEC inhibition of RhoA/Rock signalling do not
interfere with basal permeability.
Conclusion: The opposite effect of IMD on RCEC and HUVEC permeability is due to
differential regulation of actin cytoskeleton dynamics via RhoA and Rac1. Moreover,
Rac1 activity is regulated by RhoA/Rock pathway in RCEC but not in HUVEC.
protective effect is the inactivation of endothelial contractile machinery, one of the major determinants of endothelial
barrier function, mainly via the activation of myosin light chain phosphatase (MLCP). To date, the mechanisms of
cAMP-mediated MLCP activation are only partially understood. Here the contribution of two cAMP effectors,
PKA and Epac, in the regulation of endothelial contractile machinery and barrier function was studied.
Methods
and results
Endothelial contractile machinery and barrier function were analysed in cultured human umbilical vein endothelial cells
(HUVEC). The cAMP analogues 8-CPT-cAMP and 6-Bnz-cAMP were used to activate Epac and PKA, respectively, and
forskolin (FSK) was used to activate adenylyl cyclase. The cells were challenged by thrombin to inhibit MLCP via the
RhoA/Rock pathway. Activation of either PKA or Epac partially blocked thrombin-induced hyperpermeability. Simultaneous
activation of PKA and Epac had additive effects that were comparable to that of FSK. Activation of PKA but
not Epac inhibited thrombin-induced phosphorylation of MLC and the MLCP regulatory subunit MYPT1, partly via inhibition
of the RhoA/Rock pathway. FSK activated the MLCP catalytic subunit PP1 via dephosphorylation and dissociation
of the PP1 inhibitory protein CPI-17. FSK blunted thrombin-induced CPI-17 phosphorylation, CPI-17/PP1
complex formation, and PP1 inactivation. Down-regulation of CPI-17 attenuated thrombin-induced hyperpermeability
and abolished the antagonistic effect of the PKA activator, whereas the Epac activator retained its antagonistic effect.
Conclusion cAMP/PKA regulates the endothelial barrier via inhibition of the contractile machinery, mainly by the activation of
MLCP via inhibition of CPI-17 and RhoA/Rock. The permeability-lowering effect of the cAMP/Epac pathway is independent
of CPI-17.
peptide (CGRP) family expressed in a variety of tissues including endothelial
cells which acts via calcitonin-receptor-like-receptors (CLR). The main objective of the
present study was to analyse the IMD receptors and its effect on endothelial barrier in
human umbilical vein endothelial cells (HUVEC).
Methods: We analysed the effect of IMD on albumin permeability, contractile
machinery, actin cytoskeleton, and VE-cadherin on cultured HUVEC.
Results: IMD, concentration-dependently, reduced endothelial albumin permeability
under basal conditions and antagonised thrombin-induced hyperpermeability. The EC50
of IMD was 1.29±0.12 nM which was five times higher than adrenomedullin (AM;
0.24±0.07 nM) in reducing endothelial permeability. These IMD effects were sensitive to
AM22-52 and higher conc. of αCGRP8-37. Furthermore, the pA2 values of αCGRP8-37
were 6.4 for both IMD and AM and PCR data shows that HUVEC expressed only
CLR/RAMP2 receptor complex. IMD activated cAMP/PKA and cAMP/Epac signalling
pathways and IMD effect on permeability was sensitive to PKA but not to eNOS
inhibition. IMD antagonised thrombin-induced contractile activation, RhoA activation and
stress fibre formation. It also induced Rac1 activation, enhanced cell-cell adhesion and
antagonised thrombin-induced loss of cell-cell adhesion. Pharmacological inhibition of
Rac1 with specific inhibitor abrogated IMD-mediated barrier stabilisation.
Conclusion: IMD stabilises endothelial barrier of HUVEC monolayers via CLR/RAMP2
receptor complex. These IMD effects are mediated via cAMP-mediated contractile
inactivation and strengthening of cell-cell adhesion. These findings identify IMD as
barrier stabilising agent and point IMD as a potential interventional agent for the
reduction of vascular leakage in inflammatory conditions.