Cerebral microvascular endothelial cell tube formation: role of astrocytic epoxyeicosatrienoic acid release

2000 ◽  
Vol 278 (4) ◽  
pp. H1163-H1167 ◽  
Author(s):  
Diane H. Munzenmaier ◽  
David R. Harder
Keyword(s):  
Thymidine Incorporation ◽  
Tube Formation ◽  
Microvascular Endothelial Cell ◽  
Epoxyeicosatrienoic Acids ◽  
Epoxyeicosatrienoic Acid ◽  
Acid Release ◽  
Microvascular Endothelial ◽  
Cytochrome P 450 ◽  
The Brain

Cerebral microvascular endothelial cells (CMVEC) form tubes when cocultured with astrocytes (AS). Therefore, it appears that AS may be important in mediating angiogenesis in the brain. We hypothesized that AS modulate CMVEC tube formation by releasing a soluble factor. Thymidine incorporation in cultured CMVEC increased 305% when incubated with 50% conditioned AS medium for 24 h [control: 52,755 ± 4,838 counts per minute (cpm) per well, conditioned 161,082 ± 12,099 cpm/well, n = 8]. Because our laboratory has previously shown that AS can produce epoxyeicosatrienoic acids (EETs), which are known mitogens, we investigated whether release of EETs by AS is responsible for tube formation in the CMVEC-AS coculture. AS were seeded on Lab-Tek slides, CMVEC were seeded on the AS the next day, and cultures were allowed to progress for another 5 days with and without cytochrome P-450 epoxygenase blockade by 17-octadecynoic acid (17-ODYA). Tube formation in cocultures receiving 17-ODYA was significantly inhibited compared with control (93.8%). These data suggest that tube formation requires the release of EETs by AS.

Role of PGI2 and epoxyeicosatrienoic acids in relaxation of bovine coronary arteries to arachidonic acid

1993 ◽  
Vol 264 (2) ◽  
pp. H327-H335 ◽  
Author(s):  
M. Rosolowsky ◽  
W. B. Campbell
Keyword(s):  
Arachidonic Acid ◽  
Coronary Arteries ◽  
Vascular Tone ◽  
Epoxyeicosatrienoic Acids ◽  
Lipoxygenase Inhibitor ◽  
Physiological Processes ◽  
Coronary Vascular Tone ◽  
Cytochrome P 450 ◽  
Endothelium Dependent Relaxation

Metabolites of arachidonic acid regulate several physiological processes, including vascular tone. The purpose of this study was to determine which metabolites of arachidonic acid are produced by bovine coronary arteries and which may regulate coronary vascular tone. Arachidonic acid induced a concentration-related, endothelium-dependent relaxation [one-half maximum effective concentration (EC50) of 2 x 10(-7) M and a maximal relaxation of 91 +/- 2% at 10(-5) M] of bovine coronary arteries that were contracted with U-46619, a thromboxane mimetic. The concentration of 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), a metabolite of prostaglandin I2 (PGI2), increased from 82 +/- 6 to 328 +/- 24 pg/ml with arachidonic acid (10(-5) M). Treatment with the cyclooxygenase inhibitor indomethacin attenuated arachidonic acid-induced relaxations by approximately 50% and blocked the synthesis of 6-keto-PGF1 alpha. PGI2 caused a concentration-related relaxation (EC50 of 10(-8) M and a maximal relaxation of 125 +/- 11% at 10(-7) M). BW755C, a cyclooxygenase and lipoxygenase inhibitor, inhibited arachidonic acid-induced relaxation to the same extent as indomethacin. When vessels were treated with both indomethacin and BW755C, the inhibition of relaxation was the same as either inhibitor alone. SKF 525a, a cytochrome P-450 inhibitor, reduced arachidonic acid-induced relaxation by approximately 50%. When SKF 525a was given in combination with indomethacin, the relaxation by arachidonic acid was almost completely inhibited. SKF 525a inhibited the synthesis of epoxyeicosatrienoic acids (EETs).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Exosomes Derived from CXCR4-Overexpressing BMSC Promoted Activation of Microvascular Endothelial Cells in Cerebral Ischemia/Reperfusion Injury

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Xutong Li ◽  
Ye Zhang ◽  
Yong Wang ◽  
Dan Zhao ◽  
Chengcheng Sun ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Reperfusion Injury ◽  
Vascular Function ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Tube Formation ◽  
Microvascular Endothelial Cells ◽  
Significant Difference ◽  
Microvascular Endothelial

Background. Ischemic stroke is a severe acute cerebrovascular disease which can be improved with neuroprotective therapies at an early stage. However, due to the lack of effective neuroprotective drugs, most stroke patients have varying degrees of long-term disability. In the present study, we investigated the role of exosomes derived from CXCR4-overexpressing BMSCs in restoring vascular function and neural repair after ischemic cerebral infarction. Methods. BMSCs were transfected with lentivirus encoded by CXCR4 (BMSCCXCR4). Exosomes derived from BMSCCXCR4 (ExoCXCR4) were isolated and characterized by transmission electron microscopy and dynamic light scattering. Western blot and qPCR were used to analyze the expression of CXCR4 in BMSCs and exosomes. The acute middle cerebral artery occlusion (MCAO) model was prepared, ExoCXCR4 were injected into the rats, and behavioral changes were analyzed. The role of ExoCXCR4 in promoting the proliferation and tube formation for angiogenesis and protecting brain endothelial cells was determined in vitro. Results. Compared with the control groups, the ExoCXCR4 group showed a significantly lower mNSS score at 7 d, 14 d, and 21 d after ischemia/reperfusion ( P < 0.05 ). The bEnd.3 cells in the ExoCXCR4 group have stronger proliferation ability than other groups ( P < 0.05 ), while the CXCR4 inhibitor can reduce this effect. Exosomes control (ExoCon) can significantly promote the migration of bEnd.3 cells ( P < 0.05 ), while there was no significant difference between the ExoCXCR4 and ExoCon groups ( P > 0.05 ). ExoCXCR4 can further promote the proliferation and tube formation for the angiogenesis of the endothelium compared with ExoCon group ( P < 0.05 ). In addition, cobalt chloride (COCl2) can increase the expression of β-catenin and Wnt-3, while ExoCon can reduce the expression of these proteins ( P < 0.05 ). ExoCXCR4 can further attenuate the activation of Wnt-3a/β-catenin pathway ( P < 0.05 ). Conclusions. In ischemia/reperfusion injury, ExoCXCR4 promoted the proliferation and tube formation of microvascular endothelial cells and play an antiapoptotic role via the Wnt-3a/β-catenin pathway.

scholarly journals Contribution of epoxyeicosatrienoic acids to flow-induced dilation in arteries of male ERα knockout mice: role of aromatase

2007 ◽  
Vol 293 (3) ◽  
pp. R1239-R1246 ◽  
Author(s):  
Dong Sun ◽  
Changdong Yan ◽  
Azita Jacobson ◽  
Houli Jiang ◽  
Mairead A. Carroll ◽  
...  
Keyword(s):  
Gas Chromatography Mass Spectrometry ◽  
Epoxyeicosatrienoic Acids ◽  
Epoxyeicosatrienoic Acid ◽  
Wild Type ◽  
Electrophysiological Technique ◽  
Isolated Arteries ◽  
Oxide Synthase ◽  
Interfering Rna ◽  
Endothelial Nitric Oxide

We studied the roles of estrogen receptors (ER) and aromatase in the mediation of flow-induced dilation (FID) in isolated arteries of male ERα-knockout (ERα-KO) and wild-type (WT) mice. FID was comparable between gracilis arteries of WT and ERα-KO mice. In WT arteries, inhibition of NO and prostaglandins eliminated FID. In ERα-KO arteries, Nω-nitro-l-arginine methyl ester (l-NAME) inhibited FID by ∼26%, whereas indomethacin inhibited dilations by ∼50%. The remaining portion of the dilation was abolished by additional administration of 6-(2-proparglyoxyphenyl)hexanoic acid (PPOH) or iberiotoxin, inhibitors of epoxyeicosatrienoic acid (EET) synthesis and large-conductance potassium channels, respectively. By using an electrophysiological technique, we found that, in the presence of 10 dyne/cm2 shear stress, perfusate passing through donor vessels isolated from gracilis muscle of ERα-KO mice subjected to l-NAME and indomethacin elicited smooth muscle hyperpolarization and a dilator response of endothelium-denuded detector vessels. These responses were prevented by the presence of iberiotoxin in detector or PPOH in donor vessels. Gas chromatography-mass spectrometry (GC-MS) analysis indicated a significant increase in arterial production of EETs in ERα-KO compared with WT mice. Western blot analysis showed a significantly reduced endothelial nitric oxide synthase expression but enhanced expressions of aromatase and ERβ in ERα-KO arteries. Treatment of ERα-KO arteries with specific aromatase short-interfering RNA for 72 h, knocked down the aromatase mRNA and protein associated with elimination of EET-mediation of FID. Thus, FID in male ERα-KO arteries is maintained via an endothelium-derived hyperpolarizing factor/EET-mediated mechanism compensating for reduced NO mediation due, at least in part, to estrogen aromatized from testosterone.

scholarly journals MicroRNA-126-3p Inhibits Angiogenic Function of Human Lung Microvascular Endothelial Cells via LAT1 (L-Type Amino Acid Transporter 1)-Mediated mTOR (Mammalian Target of Rapamycin) Signaling

2020 ◽  
Vol 40 (5) ◽  
pp. 1195-1206 ◽  
Author(s):  
Danting Cao ◽  
Andrew M. Mikosz ◽  
Alexandra J. Ringsby ◽  
Kelsey C. Anderson ◽  
Erica L. Beatman ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Amino Acid ◽  
Cell Apoptosis ◽  
Human Lung ◽  
Mammalian Target Of Rapamycin ◽  
Tube Formation ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelial

Objective: MicroRNA-126-3p (miR-126) is required for angiogenesis during organismal development or the repair of injured arterial vasculature. The role of miR-126 in lung microvascular endothelial cells, which are essential for gas exchange and for lung injury repair and regeneration, remains poorly understood. Considering the significant heterogeneity of endothelial cells from different vascular beds, we aimed to determine the role of miR-126 in regulating lung microvascular endothelial cell function and to elucidate its downstream signaling pathways. Approach and Results: Overexpression and knockdown of miR-126 in primary human lung microvascular endothelial cells (HLMVEC) were achieved via transfections of miR-126 mimics and antisense inhibitors. Increasing miR-126 levels in HLMVEC reduced cell proliferation, weakened tube formation, and increased cell apoptosis, whereas decreased miR-126 levels stimulated cell proliferation and tube formation. Whole-genome RNA sequencing revealed that miR-126 was associated with an antiangiogenic and proapoptotic transcriptomic profile. Using validation assays and knockdown approaches, we identified that the effect of miR-126 on HLMVEC angiogenesis was mediated by the LAT1 (L-type amino acid transporter 1), via regulation of mTOR (mammalian target of rapamycin) signaling. Furthermore, downregulation of miR-126 in HLMVEC inhibited cell apoptosis and improved endothelial tube formation during exposure to environmental insults such as cigarette smoke. Conclusions: miR-126 inhibits HLMVEC angiogenic function by targeting the LAT1-mTOR signaling axis, suggesting that miR-126 inhibition may be useful for conditions associated with microvascular loss, whereas miR-126 augmentation may help control unwanted microvascular angiogenesis.

scholarly journals MiR-375-3p regulates rat pulmonary microvascular endothelial cell activity by targeting Notch1 during hypoxia

2020 ◽  
Vol 48 (7) ◽  
pp. 030006052092685
Author(s):  
Yuan An ◽  
Ziquan Liu ◽  
Hui Ding ◽  
Qi Lv ◽  
Haojun Fan ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Cell Activity ◽  
Tube Formation ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Microvascular Endothelial Cell ◽  
Adaptation To Hypoxia ◽  
Pulmonary Microvascular Endothelial Cell ◽  
Reporter Assays ◽  
Microvascular Endothelial

Objective Pulmonary microvascular endothelial cells (PMECs) exhibit specific responses in adaptation to hypoxia. However, the mechanisms regulating PMEC activities during hypoxia remain unclear. This study investigated the potential involvement of a microRNA, miR-375-3p, in the regulation of PMEC activities. Methods Primary PMECs were isolated from rats. The expression levels of miR-375-3p and Notch1 in the PMECs were detected by quantitative PCR and western blotting. Luciferase reporter assays were performed to explore the transcriptional regulation of Notch1 by miR-375-3p. The proliferation and chemotaxis of the PMECs were measured with the Cell Counting Kit-8 and Transwell invasion assays, respectively. Additionally, the capacity of hypoxia-treated PMECs for angiogenesis and inflammatory response was determined with tube formation assays and ELISA, respectively. Results The expression of miR-375-3p and Notch1 in the PMECs was significantly down-regulated and up-regulated during hypoxia, respectively. The results demonstrated that miR-375-3p directly targets Notch1 in PMECs, thereby suppressing the transcriptional expression of Notch1. It was further revealed that miR-375-3p regulates the proliferation, chemotaxis, angiogenesis, and inflammatory response of PMECs. Conclusions Our findings revealed the important role of miR-375-3p in the regulation of PMEC function and suggest the potential involvement of miR-375-3p in the development of lung diseases.

scholarly journals Differential effects of cyclic and static stretch on coronary microvascular endothelial cell receptors and vasculogenic/angiogenic responses

2008 ◽  
Vol 295 (2) ◽  
pp. H794-H800 ◽  
Author(s):  
Wei Zheng ◽  
Lance P. Christensen ◽  
Robert J. Tomanek
Keyword(s):  
Growth Factor ◽  
Growth Factor Receptor ◽  
Tube Formation ◽  
Receptor Expression ◽  
Cyclic Stretch ◽  
Tube Length ◽  
Microvascular Endothelial Cell ◽  
Vegf Receptor ◽  
Static Stretch ◽  
Microvascular Endothelial

Mechanical stretch, an important growth stimulus, results not only from pulsatile blood flow and diastolic stretch of the ventricles [cyclic stretch (CS)] but also from tissue expansion during growth [constant static stretch (SS)]. We compared growth factor receptor expression and vasculogenic/angiogenic responses of rat coronary microvascular endothelial cells (ECs) by exposing cells to CS (10% elongation at 30 cycles/min) and SS (constant 10% elongation). Both CS and SS increased VEGF receptor (VEGF-R)2 protein levels and the extent of tube formation and branching. Moreover, both CS and SS enhanced VEGF-induced cell proliferation and tube formation, indicating that both types of stretch increase the sensitivity of ECs to VEGF. Blockade of VEGF-R2 prevented the increases in EC proliferation and aggregate tube length. However, CS but not SS enhanced EC Tie-2 protein and migration. CS affected a greater increase in tube length and branch formation than did SS. A unique finding was that SS but not CS increased VEGFR-1 in ECs. Our study is the first to distinguish between the effects of CS and SS on growth factor receptor expression and rat coronary microvascular EC proliferation, migration, and tube formation. In conclusion, EC angiogenic responses to these two types of stretch display both differences and similarities, but both CS and SS are dependent on VEGF-R2 signaling for their vasculogenic/angiogenic effects.

Role of CPI-17 in the regulation of endothelial cytoskeleton

2004 ◽  
Vol 287 (5) ◽  
pp. L970-L980 ◽  
Author(s):  
Irina A. Kolosova ◽  
Shwu-Fan Ma ◽  
Djanybek M. Adyshev ◽  
Peyi Wang ◽  
Motoi Ohba ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Molecular Mechanisms ◽  
Rho Kinase ◽  
Focal Adhesions ◽  
Endothelial Permeability ◽  
Dominant Negative ◽  
Microvascular Endothelial Cell ◽  
Microvascular Endothelial ◽  
Lung Microvascular Endothelial Cell

We have previously shown that myosin light chain (MLC) phosphatase (MLCP) is critically involved in the regulation of agonist-mediated endothelial permeability and cytoskeletal organization (Verin AD, Patterson CE, Day MA, and Garcia JG. Am J Physiol Lung Cell Mol Physiol 269: L99–L108, 1995). The molecular mechanisms of endothelial MLCP regulation, however, are not completely understood. In this study we found that, similar to smooth muscle, lung microvascular endothelial cells expressed specific endogenous inhibitor of MLCP, CPI-17. To elucidate the role of CPI-17 in the regulation of endothelial cytoskeleton, full-length CPI-17 plasmid was transiently transfected into pulmonary artery endothelial cells, where the background of endogenous protein is low. CPI-17 had no effect on cytoskeleton under nonstimulating conditions. However, stimulation of transfected cells with direct PKC activator PMA caused a dramatic increase in F-actin stress fibers, focal adhesions, and MLC phosphorylation compared with untransfected cells. Inflammatory agonist histamine and, to a much lesser extent, thrombin were capable of activating CPI-17. Histamine caused stronger CPI-17 phosphorylation than thrombin. Inhibitory analysis revealed that PKC more significantly contributes to agonist-induced CPI-17 phosphorylation than Rho-kinase. Dominant-negative PKC-α abolished the effect of CPI-17 on actin cytoskeleton, suggesting that the PKC-α isoform is most likely responsible for CPI-17 activation in the endothelium. Depletion of endogenous CPI-17 in lung microvascular endothelial cell significantly attenuated histamine-induced increase in endothelial permeability. Together these data suggest the potential importance of PKC/CPI-17-mediated pathway in histamine-triggered cytoskeletal rearrangements leading to lung microvascular barrier compromise.

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