Insulin inhibition of platelet-endothelial interaction is mediated by insulin effects on endothelial cells without direct effects on platelets: reply to a rebuttal

Hydrogen sulfide (H 2 S), produced by the enzyme cystathionine-γ lyase (CSE), dilates arteries by hyperpolarizing and relaxing vascular smooth muscle cells (VSMC) and CSE knock-out causes hypertension and endothelial dysfunction showing the importance of this system. However, it is not clear if H 2 S-induced VSMC depolarization and relaxation is mediated by direct effects on VSMC or indirectly through actions on endothelial cells (EC). We reported previously that disrupting EC prevents H 2 S-induced vasodilation suggesting H 2 S might act directly on EC. Because inhibiting large-conductance Ca 2+ -activated K + (BK Ca ) channels also inhibits H 2 S-induced dilation, we hypothesized that H 2 S activates EC BK Ca channels to hyperpolarize EC and increase EC Ca 2+ which stimulates release of a secondary hyperpolarizing factor. Small mesenteric arteries from male Sprague-Dawley rats were used for all experiments. We found that EC disruption prevented H 2 S-induced VSMC membrane potential ( E m ) hyperpolarization. Blocking EC BK Ca channels with luminal application of the BK Ca inhibitor, iberiotoxin (IbTx, 100 nM), also prevented NaHS-induced dilation and VSMC hyperpolarization but did not affect resting VSMC E m showing EC specific actions. Sharp electrode recordings in arteries cut open to expose EC demonstrated H 2 S-induced hyperpolarization of EC while Ca 2+ imaging studies in fluor-4 loaded EC showed that H 2 S increases EC Ca 2+ event frequency. Thus H 2 S can act directly on EC. Inhibiting the EC enzyme cytochrome P 450 2C (Cyp2C) with sulfaphenazole also prevented VSMC depolarization and vasodilation. Finally, inhibiting TRPV4 channels to block the target of the Cyp2C product 11,12-EET inhibited NaHS-induced dilation. Combined with our previous report that CSE inhibition decreases BK Ca currents in EC, these results suggest that H 2 S stimulates EC BK Ca channels and activates Cyp2C upstream of VSMC hyperpolarization and vasodilation.

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Poloxamer 407-induced atherosclerosis in mice appears to be due to lipid derangements and not due to its direct effects on endothelial cells and macrophages

Mediators of Inflammation ◽

10.1080/0962935031000134860 ◽

2003 ◽

Vol 12 (3) ◽

pp. 147-155 ◽

Cited By ~ 19

Author(s):

Thomas P. Johnston ◽

Yuai Li ◽

Ahmed S. Jamal ◽

Daniel J. Stechschulte ◽

Kottarappat N. Dileepan

Keyword(s):

Endothelial Cells ◽

Mouse Model ◽

Umbilical Vein ◽

Density Lipoprotein ◽

Poloxamer 407 ◽

Direct Effects ◽

Direct Stimulation

Coronary heart disease secondary to atherosclerosis is still the leading cause of death in the US. Animal models used for elucidating the pathogenesis of this disease primarily involve rabbits and pigs. Previous studies from this laboratory have demonstrated intraperitoneal injections of poloxamer 407 (P-407) in both male and female mice will lead to hyperlipidemia and atherosclerosis, suggesting the use of this polymer to develop a mouse model of atherosclerosis. In order to understand the mechanism of P-407-induced hyperlipidemia and vascular lesion formation, we evaluated the direct effects of P-407 on endothelial cell and macrophage functionsin vitro, and itsin vivoeffects on the oxidation of circulating lipids following long-term (4 month) administration. Our results demonstrated that incubation of P-407 with human umbilical vein endothelial cells in culture did not influence either cell proliferation or interleukin-6 and interleukin-8 production over a concentration range of 0-40 μM. In addition, nitric oxide production by macrophages was not affected by P-407 over a concentration range of 0-20 μM. Finally, we demonstrated that while P-407 could not induce the oxidation of LDL-Cin vitro, long-term (4 month) administration of P-407 in mice resulted in elevated levels of oxidized lipids in the plasma. Thus, it is suggested that the formation of atherosclerotic lesions in this mouse model of atherosclerosis does not result from either direct stimulation of endothelial cells or macrophage activation by P-407. Instead, these data would support the premise that oxidation of lipids (perhaps low-density lipoprotein cholesterol) by an indirect mechanism following injection of P-407 may represent one of the mechanisms responsible for atheroma formation.

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Interleukin-19 increases angiogenesis in ischemic hind limbs by direct effects on both endothelial cells and macrophage polarization

Journal of Molecular and Cellular Cardiology ◽

10.1016/j.yjmcc.2014.11.002 ◽

2015 ◽

Vol 79 ◽

pp. 21-31 ◽

Cited By ~ 24

Author(s):

James Richards ◽

Khatuna Gabunia ◽

Sheri E. Kelemen ◽

Farah Kako ◽

Eric T. Choi ◽

...

Keyword(s):

Endothelial Cells ◽

Macrophage Polarization ◽

Direct Effects ◽

Hind Limbs

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Pro-atherothrombotic effects of leptin in human coronary endothelial cells

Thrombosis and Haemostasis ◽

10.1160/th09-06-0392 ◽

2010 ◽

Vol 103 (05) ◽

pp. 1065-1075 ◽

Cited By ~ 28

Author(s):

Valeria Angri ◽

Salvatore De Rosa ◽

Gaetano Calì ◽

Gianluca Petrillo ◽

Fabio Maresca ◽

...

Keyword(s):

Cardiovascular Disease ◽

Endothelial Cells ◽

Cardiovascular Risk ◽

Acute Coronary Syndromes ◽

Leukocyte Adhesion ◽

Cellular Adhesion ◽

Direct Effects ◽

Coronary Syndromes ◽

Coronary Endothelial Cells

SummaryAdipocytes are nowadays recognised as cells able to produce and secrete a large variety of active substances termed adipokines, which exert direct effects on vascular cells. Among these adipokines, leptin has been proposed to play a role in the pathophysiology of acute coronary syndromes, as well as in increasing cardiovascular risk. At the moment, however, the mechanisms linking leptin to cardiovascular disease are not completely understood. This study investigates the effects of leptin, in a concentration range usually observed in the plasma of patients with increased cardiovascular risk or measurable in patients with acute coronary syndromes, on tissue factor (TF) and cellular adhesion molecules (CAMs) expression in human coronary endothelial cells (HCAECs). We demonstrate that leptin induces transcription of mRNA for TF and CAMs by real-time PCR. In addition, we show that this adipokine promotes surface expression of TF and CAMs that are functionally active since we observed increased procoagulant activity and leukocyte adhesion on cell surface. Leptin effects appear modulated by eNOS-production of oxygen free radicals through the activation of the transcription factor, nuclear factor(NF)-κB, since L-NAME, Superoxide Dismutase and NF-κB inhibitors suppressed CAMs and TF expression. Data of the present study, although in vitro, indicate that leptin may exert direct effects on human coronary endothelial cells by promoting CAMs and TF expression and support the hypothesis that this adipokines, besides being involved in the pathophysiology of obesity, might play a relevant role as an active mediator linking obesity to cardiovascular disease.

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Diazoxide preserves hypercapnia-induced arteriolar vasodilation after global cerebral ischemia in piglets

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00887.2004 ◽

2005 ◽

Vol 289 (1) ◽

pp. H368-H373 ◽

Cited By ~ 14

Author(s):

Ferenc Domoki ◽

Béla Kis ◽

Krisztina Nagy ◽

Eszter Farkas ◽

David W. Busija ◽

...

Keyword(s):

Endothelial Cells ◽

Mitochondrial Membrane ◽

Ischemia Reperfusion ◽

Global Cerebral Ischemia ◽

Direct Effects ◽

Pial Arterioles ◽

Newborn Pigs ◽

Cerebrovascular Endothelial Cells ◽

Arteriolar Vasodilation ◽

Dose Dependent

Diazoxide (Diaz), an activator of mitochondrial ATP-sensitive K+ (mitoKATP) channels, is neuroprotective, but the mechanism of action is unclear. We tested whether Diaz preserves endothelium-dependent (hypercapnia) or -independent [iloprost (Ilo)] cerebrovascular dilator responses after ischemia-reperfusion (I/R) in newborn pigs and whether the effect of Diaz is sensitive to 5-hydroxydecanoate (5-HD), an inhibitor of mitoKATP channels. Anesthetized, ventilated piglets ( n = 48) were equipped with closed cranial windows. Changes in diameter of pial arterioles were determined with intravital microscopy in response to graded hypercapnia (5–10% CO2-21% O2-balance N2, n = 25) or Ilo (0.1–1 μg/ml, n = 18) before and 1 h after 10 min of global I/R. Experimental groups were pretreated with vehicle, NS-398 (a selective cyclooxygenase-2 inhibitor, 1 mg/kg), Diaz (3 mg/kg), or 5-HD (20 mg/kg) + Diaz. Potential direct effects of Diaz and 5-HD on hypercapnic vasodilation were also tested in the absence of I/R ( n = 5). To confirm the direct effect of Diaz on mitochondria, mitochondrial membrane potential in cultured piglet cerebrovascular endothelial cells was monitored using Mito Tracker Red. Hypercapnia resulted in dose-dependent pial arteriolar vasodilation, which was attenuated by ∼70% after I/R in vehicle- and NS-398-treated animals. Diaz and 5-HD did not affect the CO2 response. Diaz significantly preserved the postischemic vasodilation response to hypercapnia, but not to Ilo. Diaz depolarized mitochondria in cultured piglet cerebrovascular endothelial cells, and 5-HD completely abolished the protective effect of Diaz, both findings indicate a role for mitoKATP channels. In summary, preservation of arteriolar dilator responsiveness by Diaz may contribute to neuroprotection.

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Inorganic Phosphate (Pi) Signaling in Endothelial Cells: A Molecular Basis for Generation of Endothelial Microvesicles in Uraemic Cardiovascular Disease

International Journal of Molecular Sciences ◽

10.3390/ijms21196993 ◽

2020 ◽

Vol 21 (19) ◽

pp. 6993 ◽

Cited By ~ 1

Author(s):

Nima Abbasian ◽

Alan Bevington ◽

James O. Burton ◽

Karl E. Herbert ◽

Alison H. Goodall ◽

...

Keyword(s):

Endothelial Cells ◽

Mammalian Cells ◽

Inorganic Phosphate ◽

Feedback Loop ◽

Direct Effects ◽

Direct Inhibition ◽

Phosphoprotein Phosphatase ◽

Activity Inhibition ◽

Actin Stress Fibre ◽

Cytoskeleton Disruption

Hyperphosphataemia increases cardiovascular mortality in patients with kidney disease. Direct effects of high inorganic phosphate (Pi) concentrations have previously been demonstrated on endothelial cells (ECs), including generation of procoagulant endothelial microvesicles (MVs). However, no mechanism directly sensing elevated intracellular Pi has ever been described in mammalian cells. Here, we investigated the hypothesis that direct inhibition by Pi of the phosphoprotein phosphatase PP2A fulfils this sensing role in ECs, culminating in cytoskeleton disruption and MV generation. ECs were treated with control (1 mM [Pi]) vs. high (2.5 mM [Pi]), a condition that drives actin stress fibre depletion and MV generation demonstrated by confocal microscopy of F-actin and NanoSight Nanoparticle tracking, respectively. Immuno-blotting demonstrated that high Pi increased p-Src, p-PP2A-C and p-DAPK-1 and decreased p-TPM-3. Pi at 100 μM directly inhibited PP2A catalytic activity. Inhibition of PP2A enhanced inhibitory phosphorylation of DAPK-1, leading to hypophosphorylation of Tropomyosin-3 at S284 and MV generation. p-Src is known to perform inhibitory phosphorylation on DAPK-1 but also on PP2A-C. However, PP2A-C can itself dephosphorylate (and therefore inhibit) p-Src. The direct inhibition of PP2A-C by Pi is, therefore, amplified by the feedback loop between PP2A-C and p-Src, resulting in further PP2A-C inhibition. These data demonstrated that PP2A/Src acts as a potent sensor and amplifier of Pi signals which can further signal through DAPK-1/Tropomyosin-3 to generate cytoskeleton disruption and generation of potentially pathological MVs.

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