ß1-integrin-matrix interactions modulate cerebral microvessel endothelial cell tight junction expression and permeability.

Acutely following focal cerebral ischemia disruption of the microvessel blood-brain barrier allows transit of plasma proteins into the neuropil as edema formation that coincides with loss of microvessel endothelial ß1-integrins. We extend previous findings to show that interference with endothelial ß1-integrin-matrix adhesion by the monoclonal IgM Ha2/5 increases the permeability of primary cerebral microvascular endothelial cell monolayers through reorganization of claudin-5, occludin, and zonula occludens-1 (ZO-1) from inter-endothelial borders. Interference with ß1-integrin-matrix adhesion initiates F-actin conformational changes that coincide with claudin-5 redistribution. ß1-integrin-matrix interference simultaneously increases phosphorylation of myosin light chain (MLC), while inhibition of MLC kinase (MLCK) and Rho kinase (ROCK) abolishes the Ha2/5-dependent increased endothelial permeability by 6 h after ß1-integrin-matrix interference. These observations are supported by concordant observations in the cortex of a high-quality murine conditional ß1-integrin deletion construct. Together they support the hypothesis that detachment of ß1-integrins from abluminal matrix ligands increases vascular endothelial permeability through reorganization of tight junction (TJ) proteins via altered F-actin conformation, and indicate that the ß1-integrin-MLC signaling pathway is engaged when ß1-integrin detachment occurs. These findings provide a novel approach to the research and treatment of cerebral disorders where the breakdown of the blood-brain barrier accounts for their progression and complication.

Effect of N-arachidonoyl-l-serine on human cerebromicrovascular endothelium.

N-arachidonoyl-l-serine (ARA-S) is an endogenous lipid, chemically related to the endocannabinoid, N-arachidonoyl ethanolamine (i.e., anandamide) and with similar physiologic and pathophysiologic functions. Reports indicate that ARA-S possesses vasoactive and neuroprotective properties resembling those of cannabinoids. However, in contrast to cannabinoids, ARA-S binds weakly to its known classical receptors, CB1 and CB2, and is therefore considered to be a 'cannabinoid-like' substance. The originally described ARA-S induced-endothelial-dependent vasorelaxation was not abrogated by CB1, CB2 receptor antagonists or TRPV1 competitive inhibitor. The present report demonstrates that ARA-S enhances the fluorescence staining of both cannabinoid receptors (CB1 and CB2) in human brain endothelial cells (HBEC). This reaction is specific since it was reduced by respective selective receptor antagonist (SR141716A and SR141728A). ARA-S alone or in the presence of ET-1 was shown to alter the cytoskeleton (actin). Both ARA-S stimulated phosphorylation of various kinases (MAPK, Akt, JNK and c-JUN) and alteration of cytoskeleton are mediated via CB1, CB2 and TRPV1 receptors. The findings also showed the involvement of Rho/Rock and PI3/Akt/NO pathways in the ARA-S-induced phosphorylation of kinases and actin reorganization in HBEC. All of the above mentioned ARA-S-induced effects were reduced by the treatment with LY294002 (inhibitor of PI3/Akt kinase), except MAPK kinase. In addition, MAPK, JNK, c-JUN phosphorylation were inhibited by H1152 (inhibitor of Rho/ROCK kinase), except Akt kinase. Furthermore, PI3/Akt pathway was inhibited by pretreatment with l-NAME (inhibitor of NOS). The findings suggest that ARA-S is a modulator of Rho kinase and may play a critical role in the regulation of its activity and subsequent effects on the cytoskeleton and its role in supporting essential cell functions like vasodilation, proliferation and movement.

Synthetic Oligodeoxynucleotides Containing Multiple Telemeric TTAGGG Motifs Suppress Inflammasome Activity in Macrophages Subjected to Oxygen and Glucose Deprivation and Reduce Ischemic Brain Injury in Stroke-Prone Spontaneously Hypertensive Rats.

The immune system plays a fundamental role in both the development and pathobiology of stroke. Inflammasomes are multiprotein complexes that have come to be recognized as critical players in the inflammation that ultimately contributes to stroke severity. Inflammasomes recognize microbial and host-derived danger signals and activate caspase-1, which in turn controls the production of the pro-inflammatory cytokine IL-1ß. We have shown that A151, a synthetic oligodeoxynucleotide containing multiple telemeric TTAGGG motifs, reduces IL-1ß production by activated bone marrow derived macrophages that have been subjected to oxygen-glucose deprivation and LPS stimulation. Further, we demonstrate that A151 reduces the maturation of caspase-1 and IL-1ß, the levels of both the iNOS and NLRP3 proteins, and the depolarization of mitochondrial membrane potential within such cells. In addition, we have demonstrated that A151 reduces ischemic brain damage and NLRP3 mRNA levels in SHR-SP rats that have undergone permanent middle cerebral artery occlusion. These findings clearly suggest that the modulation of inflammasome activity via A151 may contribute to a reduction in pro-inflammatory cytokine production by macrophages subjected to conditions that model brain ischemia and modulate ischemic brain damage in an animal model of stroke. Therefore, modulation of ischemic pathobiology by A151 may have a role in the development of novel stroke prevention and therapeutic strategies.

Preservation and enumeration of endothelial progenitor and endothelial cells from peripheral blood for clinical trials.

AIMS: Endothelial progenitor cells (EPCs) are markers of vascular repair. Increased numbers of circulating endothelial cells (ECs) are associated with endothelial damage. MATERIALS & METHODS: We enumerated EPC-EC by using Enrichment kit with addition of anti-human CD146-PE/Cy7 from peripheral blood mononuclear cell (PBMC) isolated either by red blood cell (RBC) lysing solution or by Ficoll centrifugation, and from fresh and preserved samples. PBMCs were quantified by flow cytometry. RESULTS: RBC lysis yielded higher percentage of PBMC (p = 0.0242) and higher numbers of PBMC/ml (p = 0.0039) than Ficoll. Absolute numbers of CD34(+)CD133(+)VEGFR2(+) and CD146(+)CD34(+)VEGFR2(+) were higher (p = 0.0117 for both), when isolated by RBC lysis than by Ficoll, when no difference in other subsets was found. Cryopreservation at -160°C and -80°C and short-term preservation at room temperature decreased EPC-EC. CONCLUSIONS: Our data support use of fresh samples and isolation of PBMC from human blood by RBC lysis for enumeration of EPC and EC.

A new role for downstream Toll-like receptor signaling in mediating immediate early gene expression during focal cerebral ischemia.

To better understand the role of downstream Toll-like receptor (TLR) signaling during acute cerebral ischemia, we performed cDNA microarrays, on brain RNA, and cytokine arrays, on serum, from wild type (WT), MyD88-/- and TRIF-mutant mice, at baseline and following permanent middle cerebral artery occlusion (pMCAO). The acute stress response pathway was among the top pathways identified by Ingenuity Pathway Analysis of microarray data. We used real-time polymerase chain reaction to confirm the expression of four immediate early genes; EGR1, EGR2, ARC, Nurr77, in this pathway, and insulin degrading enzyme (IDE). Compared to WT, baseline immediate early gene expression was increased up to10-fold in MyD88-/- and TRIF-mutant mice. However, following pMCAO, immediate early gene expression remained unchanged, from this elevated baseline in these mice, but increased up to 12-fold in WT. Furthermore, expression of IDE, which also degrades ß-amyloid, decreased significantly only in TRIF-mutant mice. Finally, sE-Selectin, sICAM, sVCAM-1, and MMP-9 levels were significantly decreased only in MyD88-/- compared with WT mice. We thus report a new role for downstream TLR signaling in immediate early gene expression during acute cerebral ischemia. We also show that the TRIF pathway regulates IDE expression; a major enzyme that clears ß-amyloid from the brain.

Poly(ADP-ribose) polymerase-1 and its cleavage products differentially modulate cellular protection through NF-kappaB-dependent signaling.

Poly(ADP-ribose) polymerase-1 (PARP-1) and its cleavage products regulate cell viability and NF-kappaB activity when expressed in neurons. PARP-1 cleavage generates a 24 kDa (PARP-1(24)) and an 89 kDa fragment (PARP-1(89)). Compared to WT (PARP-1WT), the expression of an uncleavable PARP-1 (PARP-1(UNCL)) or of PARP-1(24) conferred protection from oxygen/glucose deprivation (OGD) or OGD/restoration of oxygen and glucose (ROG) damage in vitro, whereas expression of PARP-1(89) was cytotoxic. Viability experiments were performed in SH-SY5Y, a human neuroblastoma cell line, as well as in rat primary cortical neurons. Following OGD, the higher viability in the presence of PARP-1UNCL or PARP-1(24) was not accompanied with decreased formation of poly(ADP-riboses) or higher NAD levels. PARP-1 is a known cofactor for NF-kappaB, hence we investigated whether PARP-1 cleavage influences the inflammatory response. All PARP-1 constructs mimicked PARP-1WT in regard to induction of NF-kappaB translocation into the nucleus and its increased activation during ischemic challenge. However, expression of PARP-1(89) construct induced significantly higher NF-kB activity than PARP-1WT; and the same was true for NF-kappaB-dependent iNOS promoter binding activity. At a protein level, PARP-1UNCL and PARP-1(24) decreased iNOS (and lower levels of iNOS transcript) and COX-2, and increased Bcl-xL The increased levels of NF-kB and iNOS transcriptional activities, seen with cytotoxic PARP-189, were accompanied by higher protein expression of COX-2 and iNOS (and higher levels of INOS transcript) and lower protein expression of Bcl-xL Taken together, these findings suggest that PARP-1 cleavage products may regulate cellular viability and inflammatory responses in opposing ways during in vitro models of "ischemia".

Downstream Toll-like receptor signaling mediates adaptor-specific cytokine expression following focal cerebral ischemia.

BACKGROUND: Deletion of some Toll-like receptors (TLRs) affords protection against cerebral ischemia, but disruption of their known major downstream adaptors does not. To determine whether compensation in the production of downstream effectors by one pathway when the other is disrupted can explain these findings, we examined cytokine/chemokine expression and inflammatory infiltrates in wild-type (WT), MyD88(-/-) and TRIF-mutant mice following permanent middle cerebral artery occlusion (pMCAO). METHODS: Cytokine/chemokine expression was measured with a 25-plex bead array in the serum and brains of all three groups of mice at baseline (no surgery/naïve) and at 3 hours and 24 hours following pMCAO. Brain inflammatory and neutrophil infiltrates were examined 24 hours following pMCAO. RESULTS: IL-6, keratinocyte chemoattractant (KC), granulocyte colony-stimulating factor (G-CSF) and IL-10 were significantly decreased in MyD88(-/-) mice compared to WT mice following pMCAO. Significantly, decreased levels of the neutrophil chemoattractants KC and G-CSF corresponded with a trend toward fewer neutrophils in the brains of MyD88(-/-) mice. IP-10 was significantly decreased when either pathway was disrupted. MIP-1 α was significantly decreased in TRIF-mutant mice, consistent with TRIF-dependent production. MyD88(-/-) mice showed elevations of a number of Th2 cytokines, such as IL-13, at baseline, which became significantly decreased following pMCAO. CONCLUSIONS: Both MyD88 and TRIF mediate pathway-specific cytokine production following focal cerebral ischemia. Our results also suggest a compensatory Th2-type skew at baseline in MyD88(-/-) mice and a paradoxical switch to a Th1 phenotype following focal cerebral ischemia. The MyD88 pathway directs the expression of neutrophil chemoattractants following cerebral ischemia.

Reversal of postischemic hypoperfusion by tempol: endothelial signal transduction mechanism.

This report entails in vivo and in vitro studies concerned with free radical species involved in brain ischemia. The participation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the early manifestation of cerebral ischemia/reperfusion was investigated in gerbils exposed to transient global ischemia using 4-OH-2,2,6,6-tetramethylpiperidine-1-oxyl (TPL), a well-known antioxidant. TPL treatment reversed cerebral postischemic hypoperfusion and tissue edema in these animals. The findings are consistent with ROS/RNS participation in tissue injury and the reduction of cerebromicrovascular blood flow (CBF) during postischemic recirculation. The activation/deactivation of signal transduction pathway by oxidation/antioxidation [i.e., using hydrogen peroxide (H2O2)/TPL] was evaluated in cultured human brain endothelial cells (HBEC) to assess the involvement of endothelial-dependent mechanisms. The data showed that H2O2 activates various "stress" kinases and vasodilalator-stimulated phosphoprotein (VASP); activation of this pathway was reduced by inhibitors of Rho- or IP-3 kinases, as well as TPL. H2O2 also induced cytoskeleton (actin) rearrangements in HBEC; this effect was prevented by inhibitors of Rho/IP3 kinase or TPL. The observed activation/deactivation of H2O2-induced "stress" kinase is in agreement with the reported capacity of ROS/RNS to stimulate the oxidative signal transduction pathway. The noted TPL reduction of H2O2-induced phosphorylation of kinase strongly suggests that the beneficial effect of TPL implicates the stress signal transduction pathway. This may represent a mechanism for the cerebral postischemic manifestations observed by in vivo experiments.

Circulating CD133+CD34+ progenitor cells inversely correlate with soluble ICAM-1 in early ischemic stroke patients.

BACKGROUND AND PURPOSE: Both endothelial progenitor cells (EPC) and markers of neuroinflammation are candidate biomarkers for stroke severity and outcome prediction. A relationship between EPC and neuroinflammatory markers in early stroke is not fully elucidated. The objectives were to investigate correlations between EPC and neuroinflammation markers (adhesion molecules ICAM-1, VCAM-1, E-selectin, tumor necrosis factor (TNF)-α, interleukin (IL)-6, endothelin (ET)-1, markers of tissue injury (matrix metalloproteinases (MMP)-9 and tissue inhibitor of matrix metalloproteinases (TIMP)-1) in early stroke patients. METHODS: We prospectively recruited symptomatic patients with ischemic cerebrovascular disease. We assessed stroke severity by using of acute (diffusion-weighted imaging (DWI) and final lesion volumes (fluid attenuated inversion recovery (FLAIR). We measured serum soluble ICAM-1, VCAM-1, E-selectin, MMP-9, TIMP-1 and plasma TNF-α, IL-6, ET-1 by ELISA, and quantified EPC in mononuclear fraction of peripheral blood on days 1 and 3 in 17 patients (mean(SD) age 62(14), with admission National Institutes of Health Stroke Scale (NIHSS) 10(8)) selected from 175 patients with imaging confirmed ischemic stroke. Non-parametric statistics, univariate and multivariate analysis were used. RESULTS: Only ICAM-1 inversely correlated with EPC subset CD133+CD34+ on day 1 (Spearman r = -0.6, p < 0.01) and on day 3 (r = -0.967, p < 0.001). This correlation remained significant after adjustment for age and NIHSS (beta -0.992, p < 0.004), for glucose and systolic blood pressure (beta -0.86, p < 0.005), and for white blood cells and hematocrit (beta -1.057, p < 0.0001) on day 3. MMP-9 (r = 0.509, p < 0.04) and MMP-9/TIMP-1 (r = 0.59, p < 0.013) on day 1 correlated with acute lesion volume. Both IL-6 (r = 0.624, p < 0.01) and MMP-9/TIMP-1 (r = 0.56, p < 0.02) correlated with admission NIHSS. CONCLUSION: Our study showed that high ICAM-1 is associated with low CD133+CD34+subset of EPC. Biomarkers of neuroinflammation may predict tissue injury and stroke severity in early ischemia.

Interendothelial claudin-5 expression depends on cerebral endothelial cell-matrix adhesion by ß(1)-integrins.

The hypothesis tested by these studies states that in addition to interendothelial cell tight junction proteins, matrix adhesion by ß(1)-integrin receptors expressed by endothelial cells have an important role in maintaining the cerebral microvessel permeability barrier. Primary brain endothelial cells from C57 BL/6 mice were incubated with ß(1)-integrin function-blocking antibody (Ha2/5) or isotype control and the impacts on claudin-5 expression and microvessel permeability were quantified. Both flow cytometry and immunofluorescence studies demonstrated that the interendothelial claudin-5 expression by confluent endothelial cells was significantly decreased in a time-dependent manner by Ha2/5 exposure relative to isotype. Furthermore, to assess the barrier properties, transendothelial electrical resistance and permeability measurements of the monolayer, and stereotaxic injection into the striatum of mice were performed. Ha2/5 incubation reduced the resistance of endothelial cell monolayers significantly, and significantly increased permeability to 40 and 150 kDa dextrans. Ha2/5 injection into mouse striatum produced significantly greater IgG extravasation than the isotype or the control injections. This study demonstrates that blockade of ß(1)-integrin function changes interendothelial claudin-5 expression and increases microvessel permeability. Hence, endothelial cell-matrix interactions via ß(1)-integrin directly affect interendothelial cell tight junction claudin-5 expression and brain microvascular permeability.

Stromal-derived factor-1[alpha] correlates with circulating endothelial progenitor cells and with acute lesion volume in stroke patients.

BACKGROUND AND PURPOSE: Endothelial progenitor cells (EPC) are important participants of neovascularization and are mobilized through signaling with stromal-derived factor (SDF-1α), vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor, and stem cell factor. The association between EPC levels and these growth factors (GF) in acute stroke has not been previously established. We aimed to determine the association between EPC and these GF, and to elucidate a relationship between these GF and stroke severity in acute stroke patients. METHODS: Seventeen patients were selected from 175 patients with imaging-confirmed acute ischemic stroke. EPC were quantified using CD34, CD133, and VEGF-R2 markers. Plasma VEGF, SDF-1α, granulocyte colony-stimulating factor, and stem cell factor were determined by enzyme-linked immunosorbent assay on days 1 and 3, and brain MRI was performed at baseline and on days 1 and 5 after the stroke onset. RESULTS: Levels of SDF-1α strongly (r=0.6) correlated with the numbers of EPC subsets CD133(+)VEFG-R2(+) (P<0.004), CD34(+)VEGF-R2(+) (P<0.01), and CD34(+)CD133(+)VEGF-R2(+) (P<0.01) on day 1. Stem cell factor strongly (r=0.5) correlated with CD133(+)VEGF-R2(+) (P<0.05). SDF-1α moderately inversely (r=-0.49) correlated with baseline diffusion-weighted imaging lesion volumes (P<0.04). Median levels of SDF-1α (1561 pg/mL) increased (P<0.04) on day 3 compared to day 1 (1379 pg/mL). Similarly, VEGF at day 3 (95 pg/mL) increased (P<0.03) compared to day 1 (64 pg/mL). CONCLUSIONS: These results indicate that SDF-1α and stem cell factor correlate with an increase in EPC early in ischemic stroke patients.

Increased plasma and tissue MMP levels are associated with BCSFB and BBB disruption evident on post-contrast FLAIR after experimental stroke.

In this study, we examined the relationship between tissue and blood levels of matrix metalloproteinase (MMP)-2 and MMP-9 through gelatin zymography at multiple time points after experimental stroke. We additionally investigated the association between these levels and the evidence of blood-cerebrospinal fluid (CSF) barrier (BCSFB) and blood-brain barrier (BBB) disruption on post-contrast fluid-attenuated inversion-recovery (FLAIR) imaging. Increased plasma MMP-9 was associated with BCSFB disruption at 1h post-reperfusion. Ventricular enhancement ipsilateral to the stroke was 500+/-100%, significantly higher than sham, 24, and 48 h groups. Increased tissue MMP-2 and MMP-9 were associated with BBB disruption at 48 h post-reperfusion. Parenchymal enhancement was 60+/-20% for a volume equivalent to 260+/-80 mm(3). Although the percent enhancement was comparable across groups, the volume of enhancing lesion was significantly higher at 48 h (260+/-80 mm(3), 100%) in comparison to 1 h (8+/-3 mm(3), 3%) and 24 h (51 mm(3), 18%). These findings support the use of imaging markers of BCSFB and BBB status as indirect measures of MMP regulation in the blood and brain tissue. The methods presented herein should be useful in understanding the link between MMPs, barrier integrity, and subsequent hemorrhagic transformation.

Hypertension-induced vascular remodeling contributes to reduced cerebral perfusion and the development of spontaneous stroke in aged SHRSP rats.

Stroke in spontaneously-hypertensive, stroke-prone (SHRSP) rats is of particular interest because the pathogenesis is believed to be similar to that in the clinical setting. In this study, we employed multi-modal MRI-ASL, DWI, T(2), GRE, T(1) (pre/post contrast)-to investigate the natural history of spontaneous cerebral infarction and the specific role of cerebral perfusion in disease development. Twelve female SHRSP rats (age: approximately 1 year) were imaged within 1 to 3 days of symptom onset. The distribution of ischemic lesions was the following: 28.1% visual, 21.9% striatal, 18.8% motorsensory, 12.5% thalamic, 12.5% auditory, 3.1% frontal/prelimbic, and 3.1% multiple areas. Ischemic lesions had significantly reduced blood flow in comparison with healthy tissue. Ischemic lesions were characterized by hyperplastic, thrombosed, and compressed vessels. These findings suggest that ischemic lesion development is related to hypertension-induced vascular remodeling and persistent hypoperfusion. This model should be useful for studying the relationship between chronic hypertension and subsequent stroke, both in terms of primary and secondary prevention.

Past and recent BBB studies with particular emphasis on changes in ischemic brain edema: dedicated to the memory of Dr. Igor Klatzo.

The blood-brain barrier (BBB) functions to protect the environment of the brain through endothelial cells and their interactions with other cells and components of the cerebral vasculature and the brain parenchyma. Alterations in the BBB as a result of injuries (i.e., brain ischemia and traumatic brain injury) play a crucial role in the pathophysiological response.The following is a brief review of the BBB and the mechanisms by which its cellular elements participate in barrier disruptions such as those associated with ischemia and resulting brain edema formation.

Mucosal tolerization to E-selectin protects against memory dysfunction and white matter damage in a vascular cognitive impairment model.

Vascular cognitive impairment (VCI) is the second most prevalent type of dementia in the world. The white matter damage that characterizes the common subcortical ischemic form of VCI can be modeled by ligating both common carotid arteries in the Wistar rat to induce protracted cerebral hypoperfusion. In this model, we find that repetitive intranasal administration of recombinant E-selectin to induce mucosal tolerance and to target immunomodulation to activating blood vessels potently suppresses both white matter (and possibly gray matter) damage and markers of vessel activation (tumor necrosis factor and E-selectin); it also preserves behavioral function in T-maze spontaneous alternation, T-maze spatial discrimination memory retention, and object recognition tests. Immunomodulation may be an effective novel strategy to prevent progression of VCI.

The rapid decrease in astrocyte-associated dystroglycan expression by focal cerebral ischemia is protease-dependent.

During focal cerebral ischemia, the detachment of astrocytes from the microvascular basal lamina is not completely explained by known integrin receptor expression changes. Here, the impact of experimental ischemia (oxygen-glucose deprivation (OGD)) on dystroglycan expression by murine endothelial cells and astrocytes grown on vascular matrix laminin, perlecan, or collagen and the impact of middle cerebral artery occlusion on alphabeta-dystroglycan within cerebral microvessels of the nonhuman primate were examined. Dystroglycan was expressed on all cerebral microvessels in cortical gray and white matter, and the striatum. Astrocyte adhesion to basal lamina proteins was managed in part by alpha-dystroglycan, while ischemia significantly reduced expression of dystroglycan both in vivo and in vitro. Furthermore, dystroglycan and integrin alpha6beta4 expressions on astrocyte end-feet decreased in parallel both in vivo and in vitro. The rapid loss of astrocyte dystroglycan during OGD appears protease-dependent, involving an matrix metalloproteinase-like activity. This may explain the rapid detachment of astrocytes from the microvascular basal lamina during ischemic injury, which could contribute to significant changes in microvascular integrity.

Responses of endothelial cell and astrocyte matrix-integrin receptors to ischemia mimic those observed in the neurovascular unit.

BACKGROUND AND PURPOSE: Apposition of endothelial cells and astrocyte foot processes to the basal lamina matrix is postulated to underlie the cerebral microvessel permeability barrier. Focal cerebral ischemia induces rapid loss of select matrix-binding integrins from both cell compartments in the nonhuman primate. This study is the first to examine the conditions underlying integrin loss from these cell-types during ischemia in vitro and their relation to the changes in vivo. METHODS: The impact of normoxia or standardized oxygen-glucose deprivation on integrin expression by murine primary cerebral endothelial cells and astrocytes grown on matrix substrates (collagen IV, laminin, and perlecan) of the basal lamina were quantitatively assessed by flow cytometry. RESULTS: Endothelial cell expression of the beta1 and alpha 5 subunits significantly increased on all matrix ligands, whereas astrocytes displayed modest significant decreases in alpha 5 and alpha 6 subunits. Oxygen-glucose deprivation produced a further significant increase in subunit beta1 expression by both cell types, but a clear decrease in both alpha1 and alpha 6 subunits by murine astrocytes. CONCLUSIONS: Ischemia in vitro significantly increased endothelial cell beta1 expression, which is consistent with the increase in beta1 transcription by microvessels peripheral to the ischemic core. The loss of alpha1 and alpha 6 integrins from murine astrocytes is identical to that seen in the nonhuman primate in vivo. These findings establish both isolated murine cerebral endothelial cells and astrocytes as potential integrin response cognates of microvascular cells of the neurovascular unit in primates, and allow determination of the mechanisms of their changes to ischemia.

Human brain endothelium: coexpression and function of vanilloid and endocannabinoid receptors.

The arachidonic acid derivative, 2-arachidonoyl-glycerol (2-AG), was initially isolated from gut and brain; it is also produced and released from blood and vascular cells. Many of the 2-AG-induced cellular responses (i.e., neuromodulation, cytoprotection and vasodilation) are mediated by cannabinoid receptors CB1 and CB2. The findings presented here demonstrate the expression of CB1, CB2 and TRPV1 receptors on cerebromicrovascular endothelial cells (HBEC). The expression of TRPV1, CB1 and CB2 receptor mRNA and proteins were demonstrated by RT-PCR and polyclonal antibodies, respectively. The endocannabinoid 2-AG, and other related compounds [anandamide (ANA), methanandamide (m-ANA), N-(4-hydroxyphenyl-arachidonyl-ethanolamide) (AM404) and capsaicin] dose-dependently stimulated Ca2+ influx in HBEC. The selective TRPV1 receptor antagonist (capsazepine), CB1 receptor antagonist (SR141716A) and CB2 receptor antagonist (SR144528) inhibited these responses. The effects of capsaicin, a specific agonist for TRPV1 receptors, were inhibited by capsazepine, but only weakly by CB1 or CB2 receptor antagonists. 2-AG also induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP); this response was mediated by VR1 receptors. These studies clearly indicate that 2-AG and other related compounds may function as agonists on VR1 receptors, as well as CB1 and CB2 receptors, and implicated these factors in various HBEC functions.

Mucosal tolerance to E-selectin provides cell-mediated protection against ischemic brain injury.

We have demonstrated that induction of mucosal tolerance to E-selectin, a cytokine-inducible adhesion molecule restricted to activating blood vessels, prevents ischemic and hemorrhagic stroke in spontaneously hypertensive, genetically stroke-prone (SHR-SP) rats. We now examine whether mucosal tolerance to E-selectin has protective effects in ischemic brain damage after permanent middle cerebral artery occlusion (MCAO) in SHR-SP rats and whether these effects are related to generation of regulatory T cells. Rats were exposed to intranasal administration of E-selectin every other day for 10 days (single tolerization group) or on two tolerization schedules separated by 11 days (booster tolerization group). Control groups received PBS on corresponding schedules. MCAO was performed 48 h after the last dose of E-selectin or PBS. There were 45.8% and 37.9% (P < 0.05) decreases of infarction volume in the E-selectin booster group compared with the PBS group at 6 and 48 h, respectively. Single tolerization with E-selectin had only a slight trend toward a decrease in infarction volume (6.3%). CD8-positive cells were decreased in brains of E-selectin booster animals (46.6%, P < 0.01) compared with controls; splenocyte-culture supernatant levels of IL-10 were increased (59.3%, P < 0.05) in E-selectin booster animals. A decrease of infarction volume (34%, P < 0.05) was also observed in SHR-SP rats subjected to MCAO after adoptive transfer of splenocytes from E-selectin-tolerized compared with PBS-tolerized donors. The results indicate that, in addition to preventing stroke, mucosal tolerance to E-selectin is cytoprotective. Thus, immunomodulation targeted to activated blood vessel segments can both reduce stroke occurrence and attenuate brain damage if a stroke supervenes.

Induction of mucosal tolerance to E-selectin prevents ischemic and hemorrhagic stroke in spontaneously hypertensive genetically stroke-prone rats.

BACKGROUND AND PURPOSE: Inflammatory and immune mechanisms can precipitate cerebrovascular thrombosis and hemorrhage. Immunologic tolerance can be induced to a specific antigen by intranasal instillation of that antigen. Lymphocytes tolerized in this way provide local immunosuppression on restimulation with the same antigen. This study tests whether tolerization of lymphocytes to E-selectin can suppress local vessel activation and prevent stroke. METHODS: Spontaneously hypertensive genetically stroke-prone rats (n=113) were distributed among the following studies: comparison of ischemic infarcts/intraparenchymal hemorrhages after single or repetitive tolerization schedules with ovalbumin, E-selectin, or PBS; comparison of E-selectin tolerization- and PBS tolerization-induced suppression of delayed-type hypersensitivity in animals subsequently sensitized to E-selectin; and comparison of PBS-, ovalbumin-, and E-selectin-tolerized groups (after intravenous lipopolysaccharide to activate vessels) regarding transforming growth factor-beta1-positive splenocyte counts, plasma interferon-gamma levels, anti-human E-selectin antibodies, endothelial intercellular adhesion molecule-1, and anti-endothelial cell antibodies. RESULTS: Nasal instillation of E-selectin, which is specifically expressed on activated endothelium, potently inhibited the development of ischemic and hemorrhagic strokes in spontaneously hypertensive stroke-prone rats with untreated hypertension. Repeated schedules of tolerization were required to maintain the resistance to stroke. Suppression of delayed-type hypersensitivity to E-selectin and increased numbers of transforming growth factor-beta1-positive splenocytes showed that intranasal exposure to E-selectin induced immunologic tolerance. E-selectin tolerization also reduced endothelial activation and immune responses after intravenous lipopolysaccharide, as shown by marked suppression of intercellular adhesion molecule-1 expression, anti-endothelial cell antibodies on luminal endothelium, and plasma interferon-gamma levels compared with the control condition. CONCLUSIONS: The novel findings in this study support further investigation of immunologic tolerance as applied to the prevention of stroke.