Simvastatin-induced endothelial cell detachment and microparticle release are prenylation dependent

Statins reduce cardiovascular disease risk and affect endothelial function by cholesterol-dependent and independent mechanisms. Recently, circulating (detached) endothelial cells and endothelial microparticles (EMP) have been associated with endothelial functioning in vitro and in vivo. We investigated whether simvastatin affects endothelial detachment and release of EMP. Human umbilical vein endothelial cells (HUVECs) were incubated with clinically relevant concentrations of simvastatin (1.0 and 5.0 microM), with or without mevalonic acid (100 microM) or geranylgeranylpyrophosphate (GGPP; 20 microM) for 24 hours, and analyzed by flowcytometry and Western blot. Simvastatin at 1.0 and 5.0 microM increased cell detachment from 12.5+/-4.1% to 26.0+/-7.6% (p=0.013) and 28.9 +/- 2.2% (p=0.002) as well as EMP release (p=0.098 and p=0.041, respectively). The majority of detached cells was apoptotic, although the fraction of detached cells that showed signs of apoptosis (>70%) was unaffected by simvastatin. Detached cells and EMP contained caspase 3 and caspase 8, but not caspase 9. Restoring either cholesterol biosynthesis and prenylation (mevalonate) or prenylation alone (GGPP) reversed all simvastatin-induced effects on cell detachment and EMP release. Adherent cells showed no signs of simvastatin-induced apoptosis. Simvastatin promotes detachment and EMP release by inhibiting prenylation, presumably via a caspase 8-dependent mechanism. We hypothesize that by facilitating detachment and EMP release, statins improve the overall condition of the remaining vascular endothelium.     

Inhibition of hydrogen peroxide-induced apoptosis but not arachidonic acid release in GH3 cell by EGF

Reactive oxygen species (ROS) and arachidonic acid (AA) can both function as extra- and intra-cellular messengers to regulate various cell functions including cell death. The effect of ROS on phospholipase A₂ (PLA₂) activity and/or AA release has not been extensively studied in neuronal cells. In this study, we investigated the effects of H₂O₂ on AA release and apoptosis in GH3 cells, a clonal strain from rat anterior pituitary. Incubation with H₂O₂ for 1 h stimulated [³H]AA release in a concentration-dependent manner from prelabeled GH3 cells. [³H]AA release was inhibited by arachidonyl trifluoromethyl ketone, a specific inhibitor of cytosolic PLA₂, and cytosolic PLA₂ protein with a molecular mass of 100 kDa was detected by immunoblotting. Culture with 0.2 mM H₂O₂ and 30 μM AA for 24 h induced lactate dehydrogenase (LDH) leakage, DNA laddering and DNA fragmentation in GH3 cells. In GH3 cells pretreated with EGF (50 ng/ml) for 24 h, LDH leakage and DNA fragmentation by H₂O₂ and AA were inhibited, although H₂O₂-induced [³H]AA release was not modified. Mastoparan, a wasp venom peptide, induced [³H]AA release and cell death in GH3 cells. Neither effect of mastoparan was inhibited by EGF treatment. These findings suggest that (1) H₂O₂ stimulates AA release via activation of cytosolic PLA₂, (2) H₂O₂ and AA induce apoptotic death of GH3 cells and (3) treatment with EGF protects H₂O₂- and AA-, but not mastoparan-, induced GH3 cell death.     

Deficient release of plasminogen activator inhibitor-1 from astrocytes triggers apoptosis in neuronal cells.

Plasminogen activator inhibitor-1 (PAI-1) plays an important role in the processes of peripheral tissue remodeling and fibrinolysis through the regulation of plasminogen activation. We found that cultured human astrocytes efficiently released PAI-1, and that both mRNA expression and protein release of PAI-1 were suppressed by pretreatment of the cells with daunorubicin. To examine the role of PAI-1 in the nervous system, neuronally differentiated PC-12 cells (PC-12 neurons) were maintained in a PAI-1-deficient culture medium derived from daunorubicin-pretreated astrocytes. The deficiency of PAI-1 in the medium caused a significant reduction in Bcl-2 and Bcl-XL mRNAs and an increase in Bcl-XS and Bax mRNAs in PC-12 neurons at 3 h. The changes in balance between mRNA expressions of the anti- and pro-apoptotic Bcl-2 family proteins caused caspase-3 activation following the release of cytochrome c from mitochondria. Apoptotic morphological change and DNA fragmentation were also observed in the neuronal cells at 24 h. Addition of exogenous PAI-1 protein to the inhibitor-deficient medium blocked the apoptotic changes in PC-12 neurons. However, addition of PAI-1 antibodies to control medium caused similar apoptotic changes in PC-12 neurons. During the apoptotic processes, plasminogen activator (PA) activity in the PAI-1-deficient medium was as low as the control level. The present data suggest that PAI-1 has physiological functions other than its role as PA inhibitor for the survival of neurons.     

Inhibition of the endothelial cell activation by antithrombin in vitro

We examined whether antithrombin (AT) inhibits tumor necrosis factor (TNF)-alpha-induced endothelial cell activation to elucidate molecular mechanism(s) of the anti-inflammatory activity of AT. AT inhibited the increase in E-selectin expression in cultured human umbilical vein endothelial cells (HUVECs) stimulated with TNF-alpha. In contrast, chemically modified AT that lacks affinity for heparin did not. AT inhibited the TNF-alpha-induced interaction of NF-kappaB p65 with p300, a homologue of cAMP-responsive element binding protein (CREB)-binding protein (CBP). AT increased both intracellular levels of cAMP and binding of phosphorylated-CREB to DNA in HUVECs. Forskolin showed the inhibitory effect similar to that of AT and pretreatment of HUVECs with KT-5720, an inhibitor of protein kinase A, reversed the inhibitory effect of AT. These observations suggested that AT inhibited the TNF-alpha-induced increase in E-selectin expression in HUVECs by inhibiting the interaction of NF-kappaB with CBP/p300 through cAMP-dependent protein kinase A-induced CREB activation. This inhibitory activity of AT might depend on its binding to heparin-like substances on the endothelial cell. Such an inhibitory effect of AT on TNF-alpha-induced endothelial cell activation might at least partly contribute to its anti-inflammatory activity.     

Inhibition of endothelial cell adhesion molecule expression improves colonic hyperalgaesia

Abstract  Leucocyte–endothelial cell interactions are prerequisite to leucocyte infiltration and intestinal inflammation. GI270384X is a novel inhibitor of ICAM-1 and E-selectin expression and inhibits leucocyte adhesion and improves experimental colitis. We hypothesized that GI270384X maybe effective in treatment of visceral hyperalgaesia. Visceromotor behavioural responses to colorectal distension (CRD) were obtained in naïve rats or rats treated with zymosan (3 h) or 2,4,6-trinitrobenzene sulphonic acid (TNBS) (4 and 30 days) or rats exposed to acute restraint stress. Studies were also performed in a high-anxiety genetic model of colonic hyperalgaesia using Wistar–Kyoto (WKY) rats. Rats were treated orally with GI270384X or vehicle either prior to or after the administration of sensitizing stimulus. The visceromotor response to CRD was significantly enhanced in all models. GI270384X attenuated the enhanced responses to distension induced by inflammatory stimuli (TNBS and zymosan) and in the high-anxiety WKY rats; however, the drug did not inhibit the hypersensitivity induced by acute restraint stress. GI270384X was most potent in the models of acute inflammatory hyperalgaesia with a minimum efficacious dose (MED) of 0.3 and 1 mg kg⁻¹ observed in the TNBS and zymosan models respectively. The compound was less potent in the chronic and postinflammatory models with an MED of 10 and 30 mg kg⁻¹ observed in the WKY and 30-day TNBS models respectively. These findings show for the first time that inhibition of leucocyte–endothelial cell interactions can have a beneficial effect on visceral hyperalgaesia associated with inflammatory and chronic anxiety states, but is less effective against stress-associated visceral hyperalgaesia.     

Expressed isolated integrin beta1 subunit cytodomain induces endothelial cell death secondary to detachment

Expression of isolated beta integrin cytoplasmic domains in cultured endothelial cells was reported to induce cell detachment and death. To test whether cell death was the cause or the consequence of cell detachment, we expressed isolated integrin beta1 cytoplasmic and transmembrane domains (CH1) in cultured human umbilical vein endothelial cells (HUVEC), and monitored detachment, viability, caspase activation and signaling. CH1 expression induced dose-dependent cell detachment. At 24 h over 90% of CH1-expressing HUVEC were detached but largely viable (>85%). No evidence of pro-caspase-8,-3, and PARP cleavage or suppression of phosphorylation of ERK, PKB and Ikappa-B was observed. The caspase inhibitor z-VAD did not prevent cell detachment. At 48 h, however, CH1-expressing cells were over 50% dead. As a comparison trypsin-mediated detachment resulted in a time-dependent cell death, paralleled by caspase-3 activation and suppression of ERK, PKB and Ikappa-B phosphoyrylation at 24 h or later after detachment. HUVEC stimulation with agents that strengthen integrin-mediated adhesion (i.e. PMA, the Src inhibitor PP2 and COMP-Ang1) did not prevent CH1-induced detachment. Expression of CH1 in rat carotid artery endothelial cells in vivo caused endothelial cell detachment and increased nuclear DNA fragmentation among detached cells. A construct lacking the integrin cytoplasmic domain (CH2) had no effect on adhesion and cell viability in vitro and in vivo. These results demonstrate that isolated beta1 cytoplasmic domain expression induces caspase-independent detachment of viable endothelial cells and that death is secondary to detachment (i.e. anoikis). They also reveal an essential role for integrins in the adhesion and survival of quiescent endothelial cells in vivo.     

Elevated endothelial microparticle-monocyte complexes induced by multiple sclerosis plasma and the inhibitory effects of interferon-beta 1b on release of endothelial microparticles, formation and transendothelial migration of monocyte-endothelial microparticle complexes

Monocyte migration through the disrupted cerebral endothelial cell (EC) junctions plays an essential role in formation of multiple sclerosis (MS) demyelinating lesions. During pathogenesis of MS, activated ECs release endothelial microparticles (EMP), which possibly facilitate transendothelial migration (TEMIG) of monocytes. To assess functional roles of EMP in MS, specifically, their (i) interaction with monocytes, (ii) effect on monocyte TEMIG in an in vitro model of the brain microvascular endothelial cells (BMVEC), (iii) phenotypic profiles of EMP elicited by MS plasma and (iv) the effects of IFN-beta 1b on release of EMP and on TEMIG of monocytes (mono) and monocytes:EMP complexes (mono:EMP) through the BMVEC. The effect of IFN-beta 1b on the release of EMP and the TEMIG of mono and mono:EMP was assessed by preincubating BMVEC cultures of IFN-beta 1b prior to addition of plasma. Three EMP phenotypes, CD54, CD62E and CD31 were assayed. Plasma specimens from 20 patients with relapsing remitting MS (11 in exacerbation, MS-E, and 9 in remission, ME-R) and 10 healthy controls were studied. Incubation of BMVEC with MS-E plasma yielded elevated levels of EMPCD54, EMP62E and EMPCD31 relative to MS-R and control plasmas. MS-E but not MS-R or control plasma also augmented TEMIG of monocytes, respectively. Mono:EMP complexes further augmented TEMIG relative to mono alone, but only in the presence of MS-E plasma; there was no significant effect with MS-R or control plasmas. The presence of IFN-beta 1b inhibited TEMIG of mono and mono:EMP by 20% and 30%, respectively. MS-E but not MS-R plasma elicited release of activation-derived EMP and enhanced TEMIG of mono and mono:EMP. IFN-beta 1b inhibited TEMIG and release of EMP, suggesting a role of EMP and a novel therapeutic mechanism for IFN-beta 1b in MS.     

Parallel decrease of tissue factor surface exposure and increase of tissue factor microparticle release by the n-3 fatty acid docosahexaenoate in endothelial cells

Tissue factor (TF) is expressed on the endothelium in response to inflammatory mediators, giving endothelial cells a pro-thrombotic phenotype. Since fish-derived n-3 fatty acids (FA) have been associated with reduced incidence of myocardial infarction, we investigated the endothelial effects of the most abundant n-3 FA, docosahexaenoate (DHA), on TF expression. Human umbilical vein endothelial cells were pre-incubated with DHA (or stearate and arachidonate as controls) for 48-72 hours, and then stimulated with bacterial lipopolysaccharide (LPS) or tumor necrosis factor-alpha. Pre-incubation of endothelial cells with DHA (but not stearate or arachidonate) concentration-dependently reduced surface protein exposure, independent of TF mRNA or total protein expression regulation. Conversely, DHA treatment in conjunction with activating stimuli, induced the release of endothelial TF-exposing microparticles from endothelial cells, quantitatively accounting for the decreased TF cell surface exposure. In conclusion, DHA treatment, with a time-course consistent with its incorporation in membrane phospholipids, increases the release of TF-exposing microparticles from endothelial cells, accounting for decreased endothelial cell TF surface exposure, thus potentially modifying the overall endothelial control of microparticle-related effects.     

Amyloid-beta peptide triggers Fas-independent apoptosis and differentiation of neural progenitor cells

Amyloid-beta peptide (A beta), derived from the amyloid-beta precursor protein (APP), plays a central role in the pathogenesis of Alzheimer's disease and induces neuronal apoptosis. Neural progenitor cells persist in the adult mammalian brain and continue to produce new neurons throughout the life. The aim of our study was to establish the effects of A beta on neural progenitor cells (NPC). We found that the neurotoxic peptide A beta 25-35 induced apoptosis of both neurons and NPC in wild-type (wt) primary cortical cultures derived from mouse embryos. Contrary to neurons, NPC were also subjected to apoptosis in response to A beta 25-35 in both fas-/- and Z-VAD/fmk (the broad-spectrum caspase inhibitor)-treated wt cortical cultures indicating that A beta triggers a Fas- and caspase-independent apoptotic pathway in NPC. Interestingly, we also show that A beta induces neurospheres adherence and NPC neuronal differentiation. Further studies are thus needed in order to understand the role of A beta effects on NPC in AD pathology. Understanding the mechanisms involved may also be essential for the development of new regenerative therapies in AD.     

Arsenic trioxide induces procoagulant activity through phosphatidylserine exposure and microparticle generation in endothelial cells

Background

Coagulopathy is a major cause of early death when arsenic trioxide (As₂O₃) therapy fails. In addition to the procoagulant properties of blast cells, the cytotoxic therapy may contribute to the coagulation disorders. The aim of the present study was to evaluate the possible impact of As₂O₃ on membrane alterations, including phosphatidylserine (PS) exposure and microparticle generation, and the consequent procoagulant properties of endothelial cells.

Methods

Procoagulant activity (PCA) of human umbilical vein endothelial cells (HUVECs) was assessed by measuring clotting time and through purified coagulation complex assays. PS exposure on HUVEC membrane was observed by confocal microscopy and quantified with flow cytometry. In addition, counts and PCA of endothelial microparticles were determined by flow cytometry and plasma coagulation assay.

Results

As₂O₃ increased the ability of HUVECs to accelerate coagulation process and promote formation of coagulation complexes. Procoagulant activity corresponded to PS exposed on HUVECs. In coincidence with the PS externalization, As₂O₃ increased the production of PS-bearing microparticles, which then accelerated fibrin strand formation significantly. By blocking PS, lactadherin was able to inhibit over 90% of the intrinsic tenase/prothrombinase activity of As₂O₃-treated HUVECs, and restored coagulation times of As₂O₃-treated cells and microparticles to control levels.

Conclusions

As₂O₃ increases PCA of HUVECs through PS exposure and PS-bearing microparticle generation, which might cause thrombosis and act as a contributing factor in As₂O₃ therapy-related coagulopathy.     

Inhibition of retinal ganglion cell apoptosis: regulation of mitochondrial function by PACAP

Pituitary adenylate cyclase-activating polypeptide(PACAP) is an endogenous peptide with neuroprotective effects on retinal neurons, but the precise mechanism underlying these effects remains unknown. Considering the abundance of mitochondria in retinal ganglion cells(RGCs), we postulate that the protective effect of PACAP is associated with the regulation of mitochondrial function. RGC-5 cells were subjected to serum deprivation for 48 hours to induce apoptosis in the presence or absence of 100 nM PACAP. As revealed with the Cell Counting Kit-8 assay, PACAP at different concentrations significantly increased the viability of RGC-5 cells. PACAP also inhibited the excessive generation of reactive oxygen species in RGC-5 cells subjected to serum deprivation. We also showed by flow cytometry that PACAP inhibited serum deprivation-induced apoptosis in RGC-5 cells. The proportions of apoptotic cells and cells with mitochondria depolarization were significantly decreased with PACAP treatment. Western blot assays demonstrated that PACAP increased the levels of Bcl-2 and inhibited the compensatory increase of PAC1. Together, these data indicate protective effects of PACAP against serum deprivation-induced apoptosis in RGCs, and that the mechanism of this action is associated with maintaining mitochondrial function.     

Cerebral endothelial cell apoptosis after ischemia-reperfusion: role of PARP activation and AIF translocation.

Cerebral ischemia-reperfusion leads to vascular dysfunction characterized by endothelial cell injury or death. In the present study, we used an in vitro model to elucidate mechanisms of human brain microvascular endothelial cell (HBMEC) injury after episodic ischemia-reperfusion. Near-confluent HBMEC cultures were exposed to intermittent hypoxia-reoxygenation (HX/RO) and, at different recovery time points, cell viability was assessed by the MTT assay, apoptotic death by fluorescence microscopy of terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL)-positive cells, and nuclear translocation of apoptosis-inducing factor (AIF) and cleavage of poly(ADP-ribose) polymerase-1 (PARP-1) by immunoblotting of subcellular fractions. Reductions in HBMEC viability were proportional to the number of HX/RO cycles, and not the total duration of hypoxia. Using four cycles of 1-h HX with 1 h of intervening normoxic RO, cell viability was reduced 30% to 40% between 12 and 48 h. Treatment with the PARP-1 inhibitors 3-aminobenzamide or 4-amino-1,8-naphthalimide during the insult improved HBMEC viability at 24 h after insult, and resulted in dose-dependent reductions in TUNEL-positivity at 16 h after insult, but not if these treatments were delayed by 4 h. HX/RO-induced increases in nuclear AIF translocation, as well as PARP-1 cleavage, were also reduced dose-dependently at 4 h after insult by the inhibitors. The caspase inhibitor z-VAD-fmk blocked PARP-1 cleavage, but did not affect AIF translocation and was only modestly cytoprotective. These findings indicate that PARP-1 activation and a PARP-1-dependent, caspase-independent, nuclear translocation of AIF contribute to apoptotic cerebral endothelial cell death after ischemia-reperfusion, underscoring the potential for ischemic microvascular protection by inhibiting PARP activation or preventing AIF translocation.     

抑制HSF-1增强PS-341诱导胶质瘤细胞凋亡

目的探索HSF1是否能诱导HSPs的高表达,以及抑制HSF1是否能够增强PS-341诱导胶质瘤细胞凋亡。方法 Western检测HSP70、HSF1的表达,以及JNK的磷酸化。转染siRNA敲除HSF1,胎盘蓝染色及sub-G1检测细胞凋亡。结果胶质瘤细胞中HSP70及HSF1的表达明显高于正常脑组织。敲除HSF1能通过抑制HSP70明显增强PS-341诱导的胶质瘤细凋亡,并能增强及延长JNK通路的活化。在HSF1+/+细胞中,PS-341能够强烈诱导HSP70的表达;而在HSF1–/–细胞中,PS-341诱导并延长了JNK通路的激活。热休克预处理对两种细胞活性都没明显影响,但能明显增强HSF1+/+细胞对抗PS-341诱导凋亡的能力。结论胶质瘤细胞中,HSF1的激活能促进HSPs的表达,进而对抗PS-341诱导的细胞凋亡。抑制HSF-1能增强PS-341诱导胶质瘤细胞凋亡,这有望成为一种新的胶质瘤治疗途径。     

Sympathetic activation triggers endogenous opioid release and analgesia within peripheral inflamed tissue

Stress induces analgesia by mechanisms within and outside the brain. Here we show that the sympathetic nervous system is an essential trigger of intrinsic opioid analgesia within peripheral injured tissue. Noradrenaline, injected directly into inflamed hind paws of male Wistar rats, produced dose-dependent antinociception, reversible by alpha(1)-, alpha(2)- and beta(2)-antagonists. alpha(1)-, alpha(2)- and beta(2)-adrenergic receptors were demonstrated on beta-endorphin-containing immune cells and noradrenaline induced adrenergic receptor-specific release of beta-endorphin from immune cell suspensions. This antinociceptive effect of noradrenaline was reversed by micro - and delta-opioid antagonists as well as by anti-beta-endorphin. Stress-induced peripheral analgesia was abolished by chemical sympathectomy and by adrenergic antagonists. These findings indicate that sympathetic neuron-derived noradrenaline stimulates adrenergic receptors on inflammatory cells to release beta-endorphin, which induces analgesia via activation of peripheral opioid receptors.     

Fipronil is a powerful uncoupler of oxidative phosphorylation that triggers apoptosis in human neuronal cell line SHSY5Y

Fipronil is a phenylpyrazole insecticide known to elicit neurotoxicity via an interaction with ionotropic receptors, namely GABA and glutamate receptors. Recently, we showed that fipronil and other phenylpyrazole compounds trigger cell death in Caco-2 cells. In this study, we investigated the mode of action and the type of cell death induced by fipronil in SH-SY5Y human neuroblastoma cells. Flow cytometric and western blot analyses demonstrated that fipronil induces cellular events belonging to the apoptosis process, such as mitochondrial potential collapse, cytochrome c release, caspase-3 activation, nuclear condensation and phosphatidylserine externalization. In addition, fipronil induces a rapid ATP depletion with concomitant activation of anaerobic glycolysis. This cellular response is characteristic of mitochondrial injury associated with a defect of the respiration process. Therefore, we also investigated the effect of fipronil on the oxygen consumption in isolated mitochondria. Interestingly, we show for the first time that fipronil is a strong uncoupler of oxidative phosphorylation at relative low concentrations. Thus in this study, we report a new mode of action by which the insecticide fipronil could triggers apoptosis.     

缓释BCNU对GL261胶质瘤细胞凋亡作用机制的研究

目的 研究BCNU-PLGA 对GL261 胶质瘤细胞凋亡的作用机制.方法 利用溶剂挥发萃取法制备BCNU-PLGA 缓释微球.将24 只C57BL/6 小鼠随机等分为治疗组与非治疗组,借助动物立体定向仪,将体外培养小鼠GL261 胶质瘤细胞接种于小鼠颅内,行MR 检查掌握颅内成瘤情况.治疗组于术后第2 周立体定位植入BCNU-PLGA 缓释片剂,观察小鼠生存期,MRI 了解瘤体变化.获取标本行HE 染色,并行GFAP、Bax、Bcl-2 免疫组化检查检测其表达情况,相关数据采用统计学方法 进行分析.结果 治疗组与对照组生存期有明显差异(P ＜0.05),治疗组与对照组凋亡相关基因Bcl-2 免疫组化检查提示明显差异性(P ＜ 0.05),Bax 基本无改变,Bax/Bcl-2 比值明显增加.GFAP 表达为阳性.结论 BCNU-PLGA 局部缓释制剂通过下调Bcl-2 蛋白表达,上调Bax/Bcl-2 的比值,从而使胶质瘤细胞发生凋亡.     

Disruption of focal adhesions mediates detachment during neuronal apoptosis.

Apoptosis participates in the development of the nervous system and in neurodegeneration. The aim of this study was to investigate the mechanisms of detachment of neuronal cells from the extracellular matrix (ECM) during apoptosis. Detachment of Ntera2 neuronal cells was accompanied by decreased surface expression of the beta1 integrin and redistribution of proteins from focal adhesions (FA). FA proteins were cleaved in a discrete sequence: p130cas, then paxillin, then talin. Caspase inhibition prevented detachment and cleavage of paxillin and p130cas, whilst calpain inhibition blocked talin cleavage. Neuronal cells therefore detach as a result of redistribution and caspase-dependent cleavage of focal adhesion proteins. Cleavage occurs sequentially such that critical ECM-integrin survival signalling cascades are severed before disruption of focal adhesion-cytoskeletal links.