Current Neurovascular Research (v.8, #2)

Blockade of Phosphodiesterase-III Protects Against Oxygen-Glucose Deprivation in Endothelial Cells by Upregulation of VE-Cadherin by Mitsunori Ishiguro, Yukiya Suzuki, Keisuke Mishiro, Mamoru Kakino, Kazuhiro Tsuruma, Masamitsu Shimazawa, Shinichi Yoshimura, Toru Iwama, Hideaki Hara (86-94).
We recently reported that a phosphodiesterase-III inhibitor, cilostazol, prevented the hemorrhagictransformation induced by focal cerebral ischemia in mice treated with tissue plasminogen activator (tPA) and that itreversed tPA-induced cell damage by protecting the neurovascular unit, particularly endothelial cells. However, themechanisms of cilostazol action are still not clearly defined. The adheren junction (AJ) protein, VE-cadherin, is a knownmediator of endothelial barrier sealing and maintenance. Therefore, we tested whether cilostazol might promoteexpression of adhesion molecules in endothelial cells, thereby preventing deterioration of endothelial barrier functions.Human brain microvascular endothelial cells were exposed to 6-h oxygen-glucose deprivation (OGD). We comparedcilostazol with aspirin treatments and examined 2 representative AJ proteins: VE-cadherin and platelet endothelial celladhesion molecule-1 (PECAM-1). A protein kinase A (PKA) inhibitor, LY294002 (a PI3-K inhibitor), db-cAMP, and RpcAMPSwere used to assess the roles of cAMP, PKA, and PI3-K signaling, respectively, in cilostazol-induced responses.Cilostazol and db-cAMP prevented OGD-stress injury in endothelial cells by promoting VE-cadherin expression, but notPECAM-1. Aspirin did not prevent cell damage. P13-K inhibition by LY294002 had no influence on the effects ofcilostazol, but inhibition of cAMP/PKA with PKA inhibitor and Rp-cAMPS suppressed cilostazol-induced inhibition ofcell damage and promotion of VE-cadherin expression. In contrast, OGD stress had no detectable effects on VEGF,VEGF receptor, or angiopoietin-1 levels. Cilostazol promotes VE-cadherin expression through cAMP/PKA-dependentpathways in brain endothelial cells; thus, cilostazol effects on adhesion molecule signaling may provide protection againstOGD stress in endothelial cells.

Brain-Targeting Form of Docosahexaenoic Acid for Experimental Stroke Treatment: MRI Evaluation and Anti-Oxidant Impact by Fabien Chauveau, Tae-Hee Cho, Magali Perez, Michel Guichardant, Adrien Riou, Pierre Aguettaz, Madeleine Picq, Michel Lagarde, Yves Berthezene, Norbert Nighoghossian, Marlene Wiart (95-102).
Epidemiologic studies report cardiovascular protection conferred by omega-3 fatty acids, in particulardocosahexaenoic acid (DHA). However, few experimental studies have addressed its potential in acute stroke treatment.The present study used multimodal MRI to assess in vivo the neuroprotection conferred by DHA and by a brain-targetingform of DHA-containing lysophosphatidylcholine (AceDoPC) in experimental stroke. Rats underwent intraluminalmiddle cerebral artery occlusion (MCAO) and were treated at reperfusion by intravenous injection of i) saline, ii) plasmafrom donor rats, iii) DHA or iv) AceDoPC, both solubilized in plasma. Twenty-four hours after reperfusion, animalsunderwent behavioral tests and were sacrificed. Multiparametric MRI (MRA, DWI, PWI, T2-WI) was performed at H0,during occlusion, and at H24, before sacrifice. Brain tissue was used for assay of F2-isoprostanes as lipid peroxidationmarkers. Initial lesion size and PWI/DWI mismatch were comparable in the four groups. Between H0 and H24, lesion sizeincreased in the saline group (mean±s.d.: +18%±20%), was stable in the plasma group (-3%±29%), and decreased in theDHA (-17%±15%, P=0.001 compared to saline) and AceDoPC (-34%±27%, P=0.001 compared to saline) groups.Neuroscores in the AceDoPC group tended to be lower than in the other groups (P=0.07). Treatments (pooled DHA andAceDoPC groups) significantly decreased lipid peroxidation as compared to controls (pooled saline and vehicle) (P=0.03).MRI-based assessment demonstrated the neuroprotective effect of DHA in the MCAO model. Results further highlightedthe therapeutic potential of engineered brain-targeting forms of omega-3 fatty acids for acute stroke treatment.

Multiple complications can ensue in the cardiovascular, renal, and nervous systems during diabetes mellitus(DM). Given that endothelial cells (ECs) are susceptible targets to elevated serum D-glucose, identification of novelcellular mechanisms that can protect ECs may foster the development of unique strategies for the prevention andtreatment of DM complications. Erythropoietin (EPO) represents one of these novel strategies but the dependence of EPOupon Wnt1 and its downstream signaling in a clinically relevant model of DM with elevated D-glucose has not beenelucidated. Here we show that EPO can not only maintain the integrity of EC membranes, but also prevent apoptoticnuclear DNA degradation and the externalization of membrane phosphatidylserine (PS) residues during elevated Dglucoseover a 48-hour period. EPO modulates the expression of Wnt1 and utilizes Wnt1 to confer EC protection duringelevated D-glucose exposure, since application of a Wnt1 neutralizing antibody, treatment with the Wnt1 antagonistDKK-1, or gene silencing of Wnt1 with Wnt1 siRNA transfection abrogates the protective capability of EPO. EPOthrough a novel Wnt1 dependent mechanism controls the post-translational phosphorylation of the pro-apoptoticforkhead member FoxO3a and blocks the trafficking of FoxO3a to the cell nucleus to prevent apoptotic demise. EPO alsoemploys the activation of protein kinase B (Akt1) to foster phosphorylation of GSK-3β that appears required for EPOvascular protection. Through this inhibition of GSK-3β, EPO maintains β-catenin activity, allows the translocation of β-catenin from the EC cytoplasm to the nucleus through a Wnt1 pathway, and requires β-catenin for protection againstelevated D-glucose since gene silencing of β-catenin eliminates the ability of EPO as well as Wnt1 to increase ECsurvival. Subsequently, we show that EPO requires modulation of both Wnt1 and FoxO3a to oversee mitochondrialmembrane depolarization, cytochrome c release, and caspase activation during elevated D-glucose. Our studies identifycritical elements of the protective cascade for EPO that rely upon modulation of Wnt1, Akt1, FoxO3a, GSK-3β, β-catenin,and mitochondrial apoptotic pathways for the development of new strategies against DM vascular complications.

The Renin Prosequence Enhances Constitutive Secretion of Renin and Optimizes Renin Activity by Noureddine Brakch, Flore Allemandou, Irene Keller, Juerg Nussberger (121-130).
Renin is cleaved from its precursor prorenin into mature renin. We investigated the impact of the reninproregion on the generation and secretion of enzymatically active renin.We compared the effects of the following sequences of human prorenin with wild type prorenin[1-383]: prosequence [1-43], hinge sequence [1-62], Des[1-43]prorenin (renin), Des[1-62]prorenin and prorenin[N260]. These sequences wereindividually expressed in CV1 cells (constitutive pathway model) and AtT20 cells (regulated and constitutive pathwaysmodel), and Des[1-43]prorenin was also coexpressed together with the different prosequences. Renin concentration andactivity were measured in cell extracts and culture media.Deletion of the prosequence reduces renin activity in both cell types, but it leaves (total) renin concentration unchanged.Coexpression of the prosequence with renin enhances renin secretion in both cell types: Constitutively secreted renin isenhanced by coexpression of renin together with any of the prosequence containing molecules [1-43], [1-62] orprorenin[N260].Immunofluorescence in AtT20 cells shows lysosomal typical labeling of prorenin and Des[1-43]prorenin. In AtT20 cellsexpressing prorenin[1-383], stimulation of regulated secretion increases prorenin but not renin release.The renin prosequence [1-43] optimizes renin activity possibly through appropriate protein folding and it enhances theconstitutive secretion of (pro)renin. The major part of generated renin may be targeted to lysosomes.

A Hyperlipidemic Diet Induces Structural Changes in Cerebral Blood Vessels by Elena Constantinescu, Florentina Safciuc, Anca V. Sima (131-144).
The cerebrovascular pathology is an important contributor to the death rate presently. Hyperlipidemia, anestablished risk factor for cardiovascular diseases, is also incriminated in the neurodegenerative disorders. The aim of thisstudy was to evaluate the effect of a hyperlipidemic (HL) diet on the morphology of the cerebral vessels and on theamyloid deposition in the HL hamster, an accepted model of atherosclerosis. Hamsters fed a HL diet were testedperiodically for serum parameters and sacrificed after 3 and 6 months. The methods used were: paraffin embedding,thioflavin S amyloid staining, fluorescence and electron microscopy (EM). Increased serum cholesterol and triglyceridescharacterized the HL hamsters. The carotid arteries developed fatty streaks after 3 months and atherosclerotic plaquesafter 6 months HL diet. The brain cortex comprised irregularly shaped microvessels with large perivascular spaces,enlarged endothelial cells (EC) and occasionally a lumen full of lipoprotein particles. The thioflavin S reaction revealed adiscreet staining of the capillaries walls; the EC cytoplasm and basal lamina contained a fibrillar material, with a patternsimilar to an incipient amyloid deposit. Some large meningeal vessels from animals with serum cholesterol over1000mg/dl presented an intense autofluorescence in the adventitia; EM examination identified lipid-loaded perivascularcells in these areas. In conclusion, the detected morphological changes induced by the HL diet could represent a seriousimpairment for the normal brain function. These data may contribute to the better understanding of the risks ofhyperlipidemia for the mental health, and its reversal could become a new therapeutic approach for neurodegenerativedisorders.

Cannabinoid Receptor Type 2 Activation Yields Delayed Tolerance to Focal Cerebral Ischemia by Lei Ma, Zhenghua Zhu, Yu Zhao, Lihong Hou, Qiang Wang, Lize Xiong, Xiaoling Zhu, Ji Jia, Shaoyang Chen (145-152).
We demonstrated in our previous research that pretreatment with electroacupuncture (EA) induces rapid (2hafter EA) and delayed (24h after EA) tolerance to focal cerebral ischemia. We further elucidate the endocannabinoid andcannabinoid receptor type 1(CB1) are involved in the rapid ischemic tolerance induced by EA pretreatment. The presentstudy aimed at investigating the involvement of the cannabinoid receptor type 2 (CB2) in the neuroprotection conferred byEA pretreatment. Focal cerebral ischemia was induced by middle cerebral artery occlusion for 120 min at 2h and 24hfollowing EA pretreatment in male Sprague-Dawley rats, respectively. Cerebral ischemic injury was evaluated byneurobehavioral scores and infarction volume percentages 72h after reperfusion in the presence or absence of AM251, aselective CB1 receptor antagonist, and AM630, a selective CB2 receptor antagonist. The expression of CB1 and CB2receptor in the striatum of ischemic hemisphere was also evaluated. The rapid and delayed ischemic tolerance induced byEA pretreatment was reversed by AM251 and AM630 respectively. CB2 receptor expression was up-regulated in thestriatum of rat brains at 24h after EA stimuli. These results indicate that CB2 receptor contributed to the delayedneuroprotective effect whereas CB1 receptor to the rapid ischemic tolerance induced by EA pretreatment against focalcerebral ischemia in rats.

Elevated Levels of Bilirubin and Long-Term Exposure Impair Human Brain Microvascular Endothelial Cell Integrity by Ines Palmela, Filipa L. Cardoso, Michael Bernas, Leonor Correia, Ana R. Vaz, Rui F. M. Silva, Adelaide Fernandes, Kwang S. Kim, Dora Brites, Maria A. Brito (153-169).
The pathogenesis of encephalopathy by unconjugated bilirubin (UCB) seems to involve the passage of highlevels of the pigment across the blood-brain barrier (BBB) and the consequent damage of neuronal cells. However, itremains to be clarified if and how the disruption of BBB occurs by UCB. We used confluent monolayers of human brainmicrovascular endothelial cells (HBMEC) to explore the sequence of events produced by UCB. A cell line and primarycultures of HBMEC were exposed to 50 or 100 iM UCB, in the presence of 100 iM human serum albumin, to mimicmoderate and severe jaundice, for 1-72 h. UCB caused loss of cell viability in a concentration-dependent manner. UCBinhibited the secretion of interleukin-6, interleukin-8, monocyte chemoattractant protein-1 and vascular endothelialgrowth factor at early time points, but enhanced their secretion later on. Upregulation of mRNA expression, particularlyby 100 iM UCB, preceded cytokine secretion. Other early events include the disruption of glutathione homeostasis andthe increase in endothelial nitric oxide synthase expression followed by nitrite production. Prolonged exposure to UCBupregulated the expression of β-catenin and caveolin-1. In conclusion, elevated concentrations of UCB affect the integrityof HBMEC monolayers mediated by oxidative stress and cytokine release. UCB also induced increased expression ofcaveolin-1, which has been associated with BBB breakdown, and β-catenin, probably as an attempt to circumvent thatimpairment. These findings provide a basis for target-directed therapy against brain endothelial injury caused by UCB.

Protein-energy malnutrition (PEM) affects ~16% of patients at admission for stroke. We previously modeledthis in a gerbil global cerebral ischemia model and found that PEM impairs functional outcome and influencesmechanisms of ischemic brain injury and recovery. Since this model is no longer reliable, we investigated the utility of therat 2-vessel occlusion (2-VO) with hypotension model of global ischemia for further study of this clinical problem. Male,Sprague-Dawley rats were exposed to either control diet (18% protein) or PEM induced by feeding a low protein diet (2%protein) for 7d prior to either global ischemia or sham surgery. PEM did not significantly alter the hippocampal CA1neuron death (p = 0.195 by 2-factor ANOVA) or the increase in dendritic injury caused by exposure to global ischemia.Unexpectedly, however, a strong trend was evident for PEM to decrease the consistency of hippocampal damage, asshown by an increased incidence of unilateral or no hippocampal damage (p = 0.069 by chi-square analysis). AlthoughPEM caused significant changes to baseline arterial blood pH, pO2, pCO2, and fasting glucose (p < 0.05), none of thesevariables (nor hematocrit) correlated significantly with CA1 cell counts in the malnourished group exposed to 2-VO (p >0.269). Intra-ischemic tympanic temperature and blood pressure were strictly and equally controlled between ischemicgroups. We conclude that co-existing PEM confounded the consistency of hippocampal injury in the 2-VO model.Although the mechanisms responsible were not identified, this model of brain ischemia should not be used for studyingthis co-morbidity factor.