Apoptosis Of Cerebral Endothelial Cells Research Articles

In Vitro Induction of Endothelial Apoptosis of the Post-Hypoxic Blood-Brain Barrier by Isoflurane but Not by Sevoflurane and Midazolam.

BackgroundThe effects of anesthetics on the injured brain continue to be the subject of controversial discussion. Since isoflurane has recently been shown to induce apoptosis of cerebral endothelial cells, this study compared different anesthetic compounds regarding their potential to induce cerebro-vascular apoptosis.MethodsThe in vitro model of the blood-brain barrier used in this study consisted of astrocyte-conditioned human umbilical vein endothelial cells (AC-HUVEC) has been used. After 24 h of deep hypoxia and reoxygenation or control treatment, AC-HUVEC were exposed to 0, 0.5, 1.0, or 2.0 times the minimum alveolar concentration of isoflurane or sevoflurane, or 0, 75, 150, or 300 nM of midazolam for 2 h. After 24 h, AC-HUVEC were harvested, and the degree of apoptosis was assessed by means of Western blots for the Bax and Bcl-2 ratio and, for controls and the highest concentration groups, terminal deoxynucleotidyl-mediated dUTP-biotin nick end labeling (TUNEL).ResultsWithout hypoxic pretreatment, 2.0 MAC of isoflurane slightly increased TUNEL intensity compared to control and sevoflurane, but without any significant changes in the Bax and Bcl-2 ratio. After hypoxic pretreatment, exposure to isoflurane led to a multifold increase in the Bax and Bcl-2 ratio in a dose dependent manner, which was also significantly higher than the ratio observed in the 2 MAC sevoflurane group. TUNEL intensity in the post-hypoxic 2 MAC isoflurane group was increased by a factor of 11 vs. control and by 40 vs. sevoflurane. Sevoflurane and midazolam did not significantly alter these markers of apoptosis, when compared to the control group.ConclusionsIsoflurane administered after hypoxia elevates markers of apoptosis in endothelial cells transdifferentiated to the cerebro-vascular endothelium. Endothelial apoptosis may be a previously underestimated mechanism of anesthetic neurotoxicity. Administration of high concentrations of isoflurane in experimental settings may have negative effects on the blood-brain barrier.

15-Deoxy-∆12,14-PGJ2, by Activating Peroxisome Proliferator-Activated Receptor-Gamma, Suppresses p22phox Transcription to Protect Brain Endothelial Cells Against Hypoxia-Induced Apoptosis

15-Deoxy-∆(12,14)-PGJ(2) (15d-PGJ(2)) and thiazolidinedione attenuate reactive oxygen species (ROS) production via a peroxisome proliferator-activated receptor-gamma (PPAR-γ)-dependent pathway. Nonetheless, how PPAR-γ mediates ROS production to ameliorate ischemic brain injury is not clear. Recent studies indicated that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is the major source of ROS in the vascular system. In the present study, we used an in vitro oxygen-glucose deprivation and reoxygenation (hypoxia reoxygenation [HR]) paradigm to study whether PPAR-γ interacts with NADPH oxidase, thereby regulating ROS formation in cerebral endothelial cells (CECs). With pharmacological (PPAR-γ antagonist GW9662), loss-of-function (PPAR-γ siRNA), and gain-of-function (Ad-PPAR-γ) approaches, we first demonstrated that 15d-PGJ(2) protected HR-treated CECs against ROS-induced apoptosis in a PPAR-γ-dependent manner. Results of promoter and subcellular localization analyses further revealed that 15d-PGJ(2), by activating PPAR-γ, blocked HR-induced NF-κB nuclear translocation, which led to inhibited transcription of the NADPH oxidase subunit p22phox. In summary, we report a novel transrepression mechanism whereby PPAR-γ downregulates hypoxia-activated p22phox transcription and the subsequent NADPH oxidase activation, ROS formation, and CEC apoptosis.

A novel bioactivity of andrographolide from Andrographis paniculata on cerebral ischemia/reperfusion-induced brain injury through induction of cerebral endothelial cell apoptosis

Context: Andrographolide, extracted from the leaves of Andrographis paniculata (Burm. f.) Nees (Acanthaceae), is a labdane diterpene lactone. It is widely reported to possess anti-inflammatory and antitumorigenic activities. Cerebral endothelial cells (CECs) play a crucial role in supporting the integrity and the function of the blood–brain barrier (BBB). However, no data are available concerning the effects of andrographolide in CECs. The aim of this study was to examine the detailed mechanisms of andrographolide on CECs.Objective: This study investigated a novel bioactivity of andrographolide on cerebral ischemia/reperfusion-induced brain injury.Materials and methods: CECs were treated with andrographolide (20–100 µΜ) for the indicated times (0–24 h). After the reactions, cell survival rate and cytotoxicity were tested by the MTT assay and the lactate dehydrogenase (LDH) test, respectively. Western blotting was used to detect caspase-3 expression. In addition, analysis of cell cycle and apoptosis using PI staining and annexin V-FITC/PI labeling, respectively, was performed by flow cytometry. We also investigated the effect of andrographolide on middle cerebral artery occlusion (MCAO)/reperfusion-induced brain injury in a rat model.Results: In the present study, we found that andrographolide (50–100 µΜ) markedly inhibited CEC growth according to an MTT assay and caused CEC damage according to a LDH test. Our data also revealed that andrographolide (50 µM) induced CEC apoptosis and caspase-3 activation as respectively detected by PI/annexin-V double staining and western blotting. Moreover, andrographolide arrested the CEC cell cycle at the G0/G1 phase by PI staining. In addition, andrographolide (5 mg/kg) caused deterioration of MCAO/reperfusion-induced brain injury in a rat model.Conclusions: These data suggest that andrographolide may disrupt BBB integrity, thereby deteriorating MCAO/reperfusion-induced brain injury, which are, in part, associated with its capacity to arrest cell-cycle and induce CEC apoptosis.

Cerebroprotective effects of selective head cooling during epileptic seizures

Epileptic seizures in newborn pigs induce apoptosis in cerebral endothelial cells resulting in reduction of endothelial‐dependent cerebrovascular function. We hypothesized that selective head cooling during seizures reduces apoptosis and prevents postictal cerebral vascular endothelial dysfunction. Seizures were induced by bicuculline in anesthetized and pancuronium‐immobilized piglets. For selective head cooling, ice packs were placed around the skull during seizures. Head cooling decreased core temperature from 37.2 ± 0.2 to 35.3 ± 0.40 C and reduced pial arteriolar diameter and heart rate, whereas blood pressure was not altered. After 48 h of recovery, cerebral vascular responses to endothelium‐dependent dilators (bradykinin, glutamate, hemin) were assessed using cranial window microscopy. In the normal temperature group, apoptotic changes in cerebral vessels detected by TUNEL staining were accompanied by reduced vasodilator responses. In the head cooling group, no apoptotic cerebral vascular cells were detected, and cerebral vascular reactivity was preserved during postictal period. Overall, head cooling during seizures prevents apoptosis and sustained loss of cerebral vascular function. Cerebroprotective effects of head cooling counteract loss of cerebral blood flow regulation and may be beneficial for neuronal survival in neonatal seizures. The study is supported by NIH/NINDS.

Influence of oxidative stress on disease development

There is ever increasing data indicating the vmast contribution of oxidative stress to the pathogenesis of numerous diseases (atherosclerosis, hypertension, heart failure, diabetes mellitus, stroke, rheumatoid arthritis, and others). Thus, in the pathogenesis of atherosclerosis the primary role is held by reactive oxygen species that are synthetized by endothelial cells of arterial blood vessels, leukocytes and macrophages. Furthermore, native particles of lipoproteins of small density become atherogenic through oxidation caused by reactive oxygen species. The oxidation of small-density lipoproteins stimulates the inflammatory process, and it in turn steps up adhesion and the inflow of monocytes and affects the synthesis and release of numerous proinflammatory cytokines involved in the further course of the process. One of the reasons for the development of arterial hypertension is the simultaneous activation of NAD(P)H oxidase and 12/15-lipoxygenase, since it results in the stepped up production of reactive oxygen species. These stimulate the production of matrix metalloproteinase 2, which lead to vascular remodelling and to increased apoptosis of heart muscle cells. Stepped up apoptosis is linked with myocardial infarction, cardiomyopathies and the development of heart failure. The sensitivity of ?-cells of the endocrine part of the pancreas to reactive oxygen species favor the naturally low concentrations of the collectors of free radicals in them, as well as an increase in the concentration of proinflammatory cytokines, glucosis and lipids that induce a reduction in the mass and function of ?-cells. Hyperglycemia in diabetes mellitus causes tissue damage through non-enzyme glycosylation of intracellular and extracellular proteins, which results in: reduced enzyme activity, damaged nucleic acid, disrupted natural decomposition of proteins, and activation of cytotoxic pathways. These processes are the basis of the pathogenesis of numerous complications of diabetes mellitus. Since inducible nitrogen-oxide synthesis launches processes that stimulate apoptosis of cerebral endothelial cells, and superoxide-anion radicals, hypochloric acid and hydrogen peroxide damage the parenchyma of an ischemic brain and biomacromolecules (causing lipid peroxidation, oxidation of proteins and deoxyribonucleic acid), brain damage occurs during cerebral ischemia and reperfusion.

Amyloid beta oligomers trigger death receptors‐mediated apoptosis in cerebral endothelial cells

Vascular deposition of Aβ in sporadic and familial Alzheimer's disease, through poorly understood molecular mechanisms, leads to alterations in cerebral blood flow, focal ischemia, and cerebral micro‐/macro‐hemorrhages, significantly contributing to cognitive impairment. We aimed to determine the molecular mechanisms triggering apoptosis of vessel wall cells in presence of Aβ40 or its vasculotropic variants E22Q or L34V. Challenging human brain microvascular endothelial cells with both variants and wild‐type Aβ40, we showed that TRAIL death receptors DR4 and DR5 specifically mediate oligomeric Aβ induction of extrinsic apoptotic pathways. Caspase‐8 activation preceded activation of caspase‐9. The caspase‐8 inhibitor cFLIP was downregulated, and mitochondrial paths were engaged through BID cleavage. Up‐regulation of DR4 and DR5 and co‐localization with Aβ at the cell membrane suggested their involvement as initiators of the apoptotic machinery. Direct binding assays using receptor chimeras confirmed the specific interaction of oligomeric Aβ with DR4 and DR5 whereas apoptosis protection achieved through RNA silencing of both receptors highlighted their active role in downstream apoptotic pathways unveiling new targets for therapeutic intervention.Support: NIH AG30539, NS051715, American Heart and Alzheimer's Associations.

Inhibition of HSP90‐dependent telomerase activity in amyloid β‐induced apoptosis of cerebral endothelial cells

Although apoptosis induced by amyloid β (Aβ) has been identified, the effect of Aβ on telomerase activity in relation to apoptosis induction remains unclear. In the present study, Aβ(1-40) and Aβ(25-35), but not Aβ(1-16) and Aβ(35-25), reduce the viability of primary cerebral endothelial cells (CECs) in accordance with apoptosis induction. Increases in caspase 3 and PARP protein cleavage with reductions of the Bcl-2/Bax protein ratio accompanied by a loss in the mitochondria membrane potential were identified in Aβ(1-40) and Aβ(25-35)-treated CECs. A significant decrease in intracellular telomerase activity by Aβ(1-40) and Aβ(25-35) was detected; meanwhile, reduced telomerase activity by telomerase reverse transcriptase (TERT) siRNA enhanced the cytotoxic effect of Aβ. The addition of serum might block the Aβ(25-35)-induced cytotoxic effect via elevated telomerase activity in according with stimulating phospho-AKT protein expression, which was blocked by adding AKT inhibitor LY294002. Decreases in heat shock protein 90 (HSP90) and its client proteins including TERT, AKT, p53, CDK4 were observed in Aβ(1-40) and Aβ(25-35), but not Aβ(1-16) and Aβ(35-25), -treated CECs. The knockdown of HSP90 gene expression by HSP90 siRNA significantly inhibits telomerase activity with decreasing TERT protein expression. The application of HSP90 activity inhibitor geldanamycin (GA) and radicicol (RD) potentiates the telomerase inhibition and apoptosis induction of Aβ in CECs. An increase in protein ubiquitination by Aβ(25-35), but not Aβ(35-25), treatment was examined, and Aβ-inhibited HSP90 and TERT protein expression and telomerase activity was reversed by adding proteasome inhibitor, MG132. Additionally, increased TERT protein ubiquitination by Aβ(25-35) was detected in CECs via immunoprecipitation/Western blotting analysis. The data of the present study firstly demonstrates that telomerase inhibition contributes to the apoptosis induction of Aβ in CECs.

Oxidized low-density lipoprotein induces apoptotic insults to mouse cerebral endothelial cells via a Bax–mitochondria–caspase protease pathway

Cerebral endothelial cells (CECs) are crucial components of the blood-brain barrier. Oxidized low-density lipoprotein (oxLDL) can induce cell injuries. In this study, we attempted to evaluate the effects of oxLDL on mouse CECs and its possible mechanisms. Mouse CECs were isolated from brain tissues and identified by immunocytochemical staining of vimentin and Factor VIII. oxLDL was prepared from LDL oxidation by copper sulfate. Exposure of mouse CECs to oxLDL decreased cell viability in concentration- and time-dependent manners. oxLDL time-dependently caused shrinkage of cell morphologies. Administration of oxLDL to CECs induced DNA fragmentation in concentration- and time-dependent manners. Analysis of the cell cycle revealed that oxLDL concentration- and time-dependently increased the proportion of CECs which underwent apoptosis. Analysis of confocal microscopy and immunoblot revealed that oxLDL significantly increased cellular and mitochondrial Bax levels as well as the translocation of this proapoptotic protein from the cytoplasm to mitochondria. In parallel with the increase in the levels and translocation of Bax, oxLDL time-dependently decreased the mitochondrial membrane potential. Exposure of mouse CECs to oxLDL decreased the amounts of mitochondrial cytochrome c, but enhanced cytosolic cytochrome c levels. The amounts of intracellular reactive oxygen species were significantly augmented after oxLDL administration. Sequentially, oxLDL increased activities of caspase-9, -3, and -6 in time-dependent manners. Pretreatment with Z-VEID-FMK, an inhibitor of caspase-6, significantly decreased caspase-6 activity and the oxLDL-induced DNA fragmentation and cell apoptosis. This study showed that oxLDL induces apoptotic insults to CECs via signal-transducing events, including enhancing Bax translocation, mitochondrial dysfunction, cytochrome c release, increases in intracellular reactive oxygen species, and cascade activation of caspase-9, -3, and -6. Therefore, oxLDL can damage the blood-brain barrier through induction of CEC apoptosis via a Bax-mitochondria-caspase protease pathway.

Protein Phosphatase 2A RegulatesbimExpression via the Akt/FKHRL1 Signaling Pathway in Amyloid-β Peptide-Induced Cerebrovascular Endothelial Cell Death

Amyloid-beta peptide (Abeta)-induced death in cerebral endothelial cells (CECs) is preceded by mitochondrial dysfunction and signaling events characteristic of apoptosis. Mitochondria-dependent apoptosis engages Bcl-2 family proteins, especially the BH3-only homologues, which play a key role in initiating the apoptotic cascade. Here, we report that the expression of bim, but not other BH3-only members, was selectively increased in cerebral microvessels isolated from 18-month-old APPsw (Tg2576) mice, a model of cerebral amyloid angiopathy (CAA), suggesting a pivotal role for Bim in Abeta-induced cerebrovascular degeneration in vivo. A similar expression profile was observed in Abeta-treated CECs. Furthermore, Abeta induction of bim expression involved a pro-apoptotic transcription factor, FKHRL1. FKHRL1 bound to a consensus sequence in the bim promoter region and was activated by Abeta before bim expression. FKHRL1 activity was negatively regulated by phosphorylation catalyzed by Akt, an anti-apoptotic kinase. Akt upregulation by adenoviral gene transfer inhibited Abeta-induced FKHRL1 activation and bim induction. In addition, Abeta increased the activity of protein phosphatase 2A (PP2A), a ceramide-activated protein phosphatase. Suppression of PP2A activity by RNA interference or a specific inhibitor, okadaic acid, effectively suppressed Abeta-induced Akt inactivation and FKHRL1 activation, leading to an attenuation of bim expression and cell death in CECs. Coimmunoprecipitation experiments revealed that Abeta enhanced the binding of the PP2A regulatory subunit PP2ACalphabeta to Akt. These results implicate PP2A as an early regulator of Abeta-induced bim expression and CEC apoptosis via the Akt/FKHRL1 signaling pathway. We raise the possibility that this pathway may play a role in cerebrovascular degeneration in CAA.

Pravastatin Attenuates Ceramide‐Induced Cytotoxicity in Mouse Cerebral Endothelial Cells with HIF‐1 Activation and VEGF Upregulation

Ceramide is a pro-apoptotic lipid messenger that induces oxidative stress and may mediate apoptosis in cerebral endothelial cells (CECs) induced by TNF-alpha/cycloheximide, lipopolysaccharide, oxidized LDL, IL-1, and amyloid peptide. Exposure of CECs to C2 ceramide for 12 h caused cell death in a concentration-dependent manner, with a LC50 of 30 microM. Statins are inhibitors of 3-hydroxyl-3-methyl coenzyme A reductase which is the rate-limiting enzyme for cholesterol biosynthesis. Pretreatment with pravastatin at 20 microM for 16 h substantially attenuated ceramide cytotoxicity in mouse CECs. Increases in vascular endothelial growth factor (VEGF) expression were detected within 1-3 h after pravastatin treatment. This pravastatin action was accompanied by the activation of hypoxia-inducible factor-1 (HIF-1), a transcription factor known to activate VEGF expression. These results raise the possibility that pravastatin may protect CECs against ceramide-induced death via the HIF-VEGF cascade.

Cytotoxicity of cytokines in cerebral microvascular endothelial cell

Objective: Several studies reported that the levels of proinflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-8 are elevated in the cerebrospinal fluid (CSF) of patients after subarachnoid hemorrhage (SAH). Cytokines in CSF may contribute to the development of vasospasm and cerebral ischemia. In the present study, we investigated the possible cytotoxic effects of these cytokines on cultured cerebral microvascular endothelial cells. Method: The effects of TNF-α, IL-1β, IL-6, and IL-8 were tested using cell viability assay, DNA fragmentation analysis (DNA laddering), Western blot analysis (Anti-poly-(ADP-ribose) polymerase [PARP] antibody), and caspase-3 activity. Results: TNF-α and IL-1β, but not IL-6 or IL-8, caused cell detachment in a dose-dependent manner ( p<0.05). TNF-α (200 pg/ml) and IL-1β (150 pg/ml) produced DNA ladders at 24–72 h. TNF-α but not IL-1β cleaved the PARP from 116- to 85-kDa fragments and enhanced caspase-3 activity at 24–72 h after incubation with endothelial cells. Caspase-3 inhibitor at 10 μmol/l significantly prevented TNF-α-induced cell detachment ( p<0.05). Discussion: TNF-α induces apoptosis in cultured cerebral endothelial cells through the cleavage of caspase-3. IL-1β decreases the adherent cells, produces DNA ladders, but fails to cleave PARP or increase caspase-3 activity. IL-1β may induce apoptosis in cerebral endothelial cells through different pathway from that of TNF-α.

Substitutions at codon 22 of Alzheimer's abeta peptide induce diverse conformational changes and apoptotic effects in human cerebral endothelial cells.

Cerebral amyloid angiopathy is commonly associated with normal aging and Alzheimer's disease and it is also the principal feature of hereditary cerebral hemorrhage with amyloidosis Dutch type, a familial condition associated to a point mutation G to C at codon 693 of the amyloid beta (Abeta) precursor protein gene resulting in a Glu to Gln substitution at position 22 of the Abeta (E22Q). The patients carrying the AbetaE22Q variant usually present with lobar cerebral hemorrhages before 50 years of age. A different mutation described in several members of three Italian kindred who presented with recurrent hemorrhagic strokes late in life, between 60 and 70 years of age, also associated with extensive cerebrovascular amyloid deposition has been found at the same position 22, this time resulting in a Glu to Lys substitution (E22K). We have compared the secondary structure, aggregation, and fibrillization properties of the two Abeta40 variants and the wild type peptide. Using flow cytometry analysis after staining with propidium iodide and annexin V, we also evaluated the cytotoxic effects of the peptides on human cerebral endothelial cells in culture. Under the conditions tested, the E22Q peptide exhibited the highest content of beta-sheet conformation and the fastest aggregation/fibrillization properties. The Dutch variant also induced apoptosis of cerebral endothelial cells at a concentration of 25 micrometer, whereas the wild type Abeta and the E22K mutant had no effect. The data suggest that different amino acids at position 22 confer distinct structural properties to the peptides that appear to influence the onset and aggressiveness of the disease rather than the phenotype.

Enhancement of apoptosis in cerebral endothelial cells by selected inflammatory signals.

Acute inflammation is characterized by (1) microvascular injury attracting acute inflammatory cells (mostly polymorphonuclear neutrophils (PMNs) and macrophages), (2) plasma extravasation leading to swelling, and (3) edema in the inflamed site and tissue destruction.' The accumulation of PMNs and macrophages in the acutely inflamed tissue results from activation and injury of endothelial cells (ECs). PMN-EC and/or macrophage-EC interaction perpetuates progressive vascular injury, edema formation, and tissue injury. In inflammatory reactions, the potential pathogenetic roles of ECs have been increasingly recognized.? Interaction between ECs and PMNs/macrophages in acute inflammation is mediated by a number of inflammatory mediator^.^,' The recruitment of circulating PMNs and macrophages into the inflammatory foci involves the expression of adhesive molecules on ECs, PMNs, and macrophages. Interaction between PMNs/macrophages and ECs engage such inflammatory mediators as cytokines, free radicals, eicosanoids, kinins, and others (e.g., histamines and serotonin). Nitric oxide (NO) has emerged to be a key inflammatory mediator. Proinflammatory actions of NO are at least partly related to the formation of free radicals, such as per~xynitrite.~,~ In this communication, we summarize recent findings from our laboratory on the effects of inflammatory mediators, especially the free radicals and cytokines, on endothelial function in vitro.