Cilostazol ameliorates collagenase-induced cerebral hemorrhage by protecting the blood-brain barrier.

Intracranial hemorrhage remains a devastating disease. Among antiplatelet drugs, cilostazol, a phosphodiesterase 3 inhibitor, was recently reported to prevent secondary hemorrhagic stroke in patients in a clinical trial. The aim of this study was to evaluate whether pre-treatment with cilostazol could decrease the intracranial hemorrhage volume and examine the protective mechanisms of cilostazol. We evaluated the pre-treatment effects of the antiplatelet drug cilostazol on the collagenase-induced intracranial hemorrhage volume and neurological outcomes in mice. To estimate the mechanism of collagenase injury, we evaluated various vascular components in vitro, including endothelial cells, vascular smooth muscle cells, pericytes, and a blood-brain barrier model. Cilostazol pre-treatment reduced the intracranial hemorrhage volume with sufficient inhibition of platelet aggregation, and motor function was improved by cilostazol treatment. Blood-brain barrier permeability was increased by collagenase-induced intracranial hemorrhage, and cilostazol attenuated blood-brain barrier leakage. Terminal deoxynucleotidyl transferase dUTP nick-end labeling and western blot analysis showed that cilostazol prevented pericyte cell death by inducing cyclic adenosine monophosphate-responsive element-binding protein phosphorylation. Cilostazol also prevented endothelial cell death and protected collagen type 4, laminin, and vascular endothelial- and N-cadherins from collagenase injury. In conclusion, cilostazol reduced collagenase-induced intracranial hemorrhage volume by protecting the blood-brain barrier.

Protective Effect of Bendavia (SS-31) Against Oxygen/Glucose-Deprivation Stress-Induced Mitochondrial Damage in Human Brain Microvascular Endothelial Cells.

Mitochondria play a key role in cell survival by perfoming functions such as adenosine tri-phosphate (ATP) synthesis, regulation of apoptotic cell death, calcium storage. Hypoxic conditions induce mitochondrial dysfunction, which leads to endothelial injury in cerebral ischemia. Functional disorders include the following: collapse of mitochondrial membrane potential, reduction of ATP synthesis, and generation of reactive oxygen species (ROS). Bendavia, a novel tetra-peptide, has been reported to restrict the uncoupling of the mitochondrial membrane chain, protect the synthesis of ATP, and inhibit ROS generation. In the present study, we investigated whether bendavia protects mitochondria under hypoxic and starved conditions by using human brain microvascular endothelial cells (HBMVECs). After pre-treatment with bendavia, we exposed HBMVECs to oxygen glucose deprivation (OGD) for 6 h. We then assessed cell viability, the level of caspase-3/7 activity, ROS generation, mitochondrial membrane potential, ATP contents, and the number of mitochondria. Bendavia recovered cell viability and reduced the caspase-3/7 activity induced by OGDinduced damage. Bendavia also recovered mitochondrial functions. These results suggest that bendavia protects mitochondrial function against OGD-induced injury and inhibits apoptosis in HBMVECs. Consequently, our findings indicate that bendavia might become the new therapeutic drug of choice to target mitochondria in case of cerebral ischemia.

The phosphodiesterase III inhibitor cilostazol ameliorates ethanolinduced endothelial dysfunction.

Intracranial hemorrhage (ICH) remains a devastating disease, and heavy alcohol consumption is an underlying risk factor. The aim of this study was to study the mechanism of ethanol-induced endothelial cell damage and to evaluate the protective effect of cilostazol against ethanol-induced damage. We first evaluated transendothelial electrical resistance (TEER) and cell viability of human brain microvascular endothelial cells at the ethanol concentration shown to cause mild-to-moderate intoxication in the clinic. We also assessed the permeability of fluorescein isothiocyanate (FITC)- dextran and the change in tight junction proteins. Furthermore, we studied the potential of cilostazol to protect endothelial cells from ethanol-induced dysfunction. Concentration- and time-dependent effects of ethanol on cell viability and TEER showed that TEER was reduced at each concentration of ethanol tested during exposures of >2 h, but cell viability was not changed. Permeability of FITC-dextran was enhanced, and both tight junction and adherens junction proteins were reduced by 3-h ethanol treatment. The permeability of FITC-dextran was ameliorated by administration of cilostazol in a concentration-dependent manner. The protective effect of cilostazol was obstructed by administration of a protein kinase A inhibitor. Using gelatin zymography, we found that the protective effect of cilostazol was by reducing matrix metalloproteinase 9 (MMP-9) activation, but it had no effect on reactive oxygen spices (ROS). Our results indicate that cilostazol protected endothelial cells against ethanol-induced endothelial dysfunction by inhibiting ROS-mediated activation of MMP-9.

Diabetes mellitus aggravates hemorrhagic transformation after ischemic stroke via mitochondrial defects leading to endothelial apoptosis.

Diabetes is a crucial risk factor for stroke and is associated with increased frequency and poor prognosis. Although endothelial dysfunction is a known contributor of stroke, the underlying mechanisms have not been elucidated. The aim of this study was to elucidate the mechanism by which chronic hyperglycemia may contribute to the worsened prognosis following stroke, especially focusing on mitochondrial alterations. We examined the effect of hyperglycemia on hemorrhagic transformation at 24 hours after middle cerebral artery occlusion (MCAO) in streptozotocin (STZ) -induced diabetic mice. We also examined the effects of high-glucose exposure for 6 days on cell death, mitochondrial functions and morphology in human brain microvascular endothelial cells (HBMVECs) or human endothelial cells derived from induced pluripotent stem cells (iCell endothelial cells). Hyperglycemia aggravated hemorrhagic transformation, but not infarction following stroke. High-glucose exposure increased apoptosis, capase-3 activity, and release of apoptosis inducing factor (AIF) and cytochrome c in HBMVECs as well as affected mitochondrial functions (decreased cell proliferation, ATP contents, mitochondrial membrane potential, and increased matrix metalloproteinase (MMP)-9 activity, but not reactive oxygen species production). Furthermore, morphological aberration of mitochondria was observed in diabetic cells (a great deal of fragmentation, vacuolation, and cristae disruption). A similar phenomena were seen also in iCell endothelial cells. In conclusion, chronic hyperglycemia aggravated hemorrhagic transformation after stroke through mitochondrial dysfunction and morphological alteration, partially via MMP-9 activation, leading to caspase-dependent apoptosis of endothelial cells of diabetic mice. Mitochondria-targeting therapy may be a clinically innovative therapeutic strategy for diabetic complications in the future.

Involvement of Mincle and Syk in the changes to innate immunity after ischemic stroke.

Accumulating evidence shows that post-ischemic inflammation originated by Toll-like receptors (TLR) plays critical roles in ischemic stroke. However, the functions of other innate immune receptors are poorly understood in cerebral ischemia. Macrophage-inducible C-type lectin, Mincle, is one of the innate immune receptor C-type lectin-like receptor (CLR) to response against dying cells. In the present study, we showed that Mincle, its ligand SAP130, and its downstream phospho-Syk/Syk were upregulated after ischemia, and that Mincle is expressed in immune and non-immune cells in the ischemic brains of mice and human. We treated mice with piceatannol, a Syk inhibitor, and consequently the infarct volume and swelling were suppressed by piceatannol. The levels of phospho-Syk, MMP9 and ICAM-1 were downregulated, and the level of Claudin5 was uplegurated in piceatannol-treated groups. These data indicate that innate immune system, such as Mincle and Syk plays a pivotal role in the pathogenesis after the ischemia and reperfusion.

The growth factor progranulin attenuates neuronal injury induced by cerebral ischemia-reperfusion through the suppression of neutrophil recruitment.

BACKGROUND: To improve the clinical outcome of patients who suffered ischemic stroke, cerebral ischemia-reperfusion (I/R) injury is one of the major concerns that should be conquered. Inflammatory reactions are considered a major contributor to brain injury following cerebral ischemia, and I/R exacerbates these reactions. The aim of this study was to investigate the possible ameliorative effects of progranulin (PGRN) against I/R injury in mice. METHODS: In vivo I/R was induced in four-week-old male ddY mice by 2 h of MCAO (middle cerebral artery occlusion) followed by 22 h of reperfusion. We evaluate expression of PGRN in I/R brain, efficacy of recombinant-PGRN (r-PGRN) treatment and its therapeutic time-window on I/R injury. Two hours after MCAO, 1.0 ng of r-PRGN or PBS was administered via intracerebroventricular. We assess neutrophil infiltration, expression of tumor necrosis factor (TNF)-α, matrix metalloproteinase-9 (MMP-9) and phosphorylation of nuclear factor-κB (NF-κB) by immunofluorescense staining and Western blotting. We also investigate neutrophil chemotaxis and intercellular adhesion molecule-1 (ICAM-1) expression in vitro inflammation models using isolated neutrophils and endothelial cells. RESULTS: We found that expression of PGRN was decreased in the I/R mouse brain. r-PGRN treatment at 2 h after MCAO resulted in a reduction in the infarct volume and decreased brain swelling; this led to an improvement in neurological scores and to a reduction of mortality rate at 24 h and 7 d after MCAO, respectively. Immunohistochemistry, Western blotting, and gelatin zymography also confirmed that r-PGRN treatment suppressed neutrophil recruitment into the I/R brain, and this led to a reduction of NF-κB and MMP-9 activation. In the in vitro inflammation models, PGRN suppressed both the neutrophil chemotaxis and ICAM-1 expression caused by TNF-α in endothelial cells. CONCLUSIONS: PGRN exerted ameliorative effects against I/R-induced inflammation, and these effects may be due to the inhibition of neutrophil recruitment into the I/R brain.

Cilostazol ameliorates warfarin-induced hemorrhagic transformation after cerebral ischemia in mice.

BACKGROUND AND PURPOSE: Although long-term treatment with the oral anticoagulant warfarin is widely used to prevent cardioembolic ischemic stroke, it has been reported that warfarin can exacerbate hemorrhagic transformation (HT) after cerebral ischemia. We investigated whether cilostazol, a phosphodiesterase-III inhibitor, suppressed the warfarin-induced HT after cerebral ischemia in mice. METHODS: Male ddY mice were treated with oral warfarin before 3-hour middle cerebral artery occlusion followed by 21-hour reperfusion to induce HT. The duration of warfarin pretreatment was determined by measurement of prothrombin time-international normalized ratio value. Cilostazol or vehicle was administered by intraperitoneal injection immediately after reperfusion. The infarct volume, brain swelling, and brain hemoglobin content were evaluated at 24 hours after middle cerebral artery occlusion. We also evaluated the survival rate of each treated group for 7 days after surgery. To investigate the mechanism underlying cilostazol's effects, the proteins involved in vascular endothelial integrity were investigated using Western blotting. RESULTS: HT volume was exacerbated by warfarin treatment, and cilostazol (3 mg/kg, i.p.) suppressed this exacerbation (sham, mean±SD, 29.2±13.4 mg/dL; vehicle, 33.3±11.9 mg/dL; warfarin, 379.4±428.9 mg/dL; warfarin+cilostazol 1 mg/kg, 167.5±114.2 mg/dL; warfarin+cilostazol 3 mg/kg, 116.9±152.3 mg/dL). Furthermore, cilostazol improved survival rate and upregulated the expression of tight junction proteins and vascular endothelial cadherin. CONCLUSIONS: Cilostazol reduced the warfarin-related risk of HT after ischemia by protecting the vascular endothelial cells. This result suggested that cilostazol administration in patients with acute ischemic stroke might reduce HT.

Tissue plasminogen activator prevents restoration of tight junction proteins through upregulation of angiopoietin-2.

We examined the temporal profiles of changes in the expressions of tight junction proteins (TJPs; namely, claudin-5, occludin, and ZO-1) after focal cerebral ischemia/reperfusion in mice. We also examined the effects of delayed treatment with tissue plasminogen activator (tPA) on the expressions of TJPs and angiopoietin (Ang) -1/2/Tie2. Mice subjected to a 6-h filamental middle cerebral artery (MCA) occlusion were treated with tPA (10 mg/kg, intravenously, just after the start of reperfusion) or vehicle. The expressions of TJPs were significantly decreased in the early phase of ischemia/reperfusion, and then gradually recovered. A delayed treatment with tPA decreased the expressions of TJPs when examined at 42 h after reperfusion. In contrast, delayed tPA treatment markedly increased Ang-2, but not Ang-1 expression, when examined at 30 h after reperfusion. Treatment with tPA at 300 µg/ml also significantly decreased Ang- 2, but not Tie2 expression, in an in vitro monolayer model generated using human brain microvascular endothelial cells subjected to serum-deprivation. These findings suggest that delayed tPA treatment prevents recovery of TJPs following focal cerebral ischemia/reperfusion, partially via upregulation of Ang-2.

Pharmacological inhibition of TLR4-NOX4 signal protects against neuronal death in transient focal ischemia.

Recent data have shown that TLR4 performs a key role in cerebral ischemia-reperfusion injury which serves as the origin of the immunological inflammatory reactions. However, the therapeutic effects of pharmacological inhibitions of TLR4 and its immediate down-stream pathway remain to be uncovered. In the present study, on mice, intracerebroventricular injection of resatorvid (TLR4 signal inhibitor; 0.01 µg) significantly reduced infarct volume and improved neurological score after middle cerebral artery occlusion and reperfusion. The levels of phospho-p38, nuclear factor-kappa B, and matrix metalloproteinase 9 expressions were significantly suppressed in the resatorvid-treated group. In addition, NOX4 associates with TLR4 after cerebral ischemia-reperfusion seen in mice and human. Genetic and pharmacological inhibitions of TLR4 each reduced NOX4 expression, leading to suppression of oxidative/nitrative stress and of neuronal apoptosis. These data suggest that resatorvid has potential as a therapeutic agent for stroke since it inhibits TLR4-NOX4 signaling which may be the predominant causal pathway.

The conditioned medium of murine and human adipose-derived stem cells exerts neuroprotective effects against experimental stroke model.

This study investigated the possible ameliorative effects of adipose-derived stem cells-conditioned medium (ASC-CM) on experimental ischemic stroke. In vivo ischemic stroke was induced in mice after 2h of middle cerebral artery occlusion (MCAO) followed by 22 h reperfusion. Culture of SH-SY5Y human neuroblastoma cells with 100 µM glutamate for 24h was used as an in vitro neuronal apoptosis model. Intracerebroventricular (i.c.v.) administration of 30- and 100-fold concentrated murine ASC-CM 1h prior to MCAO resulted in a dose-dependent reduction in the infarct volume and the brain swelling. The administration of murine ASC-CM immediately after MCAO was also effective, but administration 2h after MCAO was not. Neuroprotective effects of murine ASC-CM were also confirmed in an in vitro model. Pretreatment with 100-fold concentrated murine ASC-CM at 10% of the total culture volume significantly reduced glutamate-induced excitotoxicity in the SH-SY5Y cells. Similar reduction in the MCAO-induced infarction volume was seen following i.c.v. administration of 100-fold concentrated human ASC-CM or murine ASC-CM. In conclusion, ASC-CM appears to exert ameliorative effects on experimental ischemic stroke i\n both in vivo and in vitro models. These findings suggest the feasibility of ASC-CM administration as a therapy for acute stage stroke.

Blockade of phosphodiesterase-III protects against oxygen-glucose deprivation in endothelial cells by upregulation of VE-cadherin.

We recently reported that a phosphodiesterase-III inhibitor, cilostazol, prevented the hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tissue plasminogen activator (tPA) and that it reversed tPA-induced cell damage by protecting the neurovascular unit, particularly endothelial cells. However, the mechanisms of cilostazol action are still not clearly defined. The adheren junction (AJ) protein, VE-cadherin, is a known mediator of endothelial barrier sealing and maintenance. Therefore, we tested whether cilostazol might promote expression 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 compared cilostazol with aspirin treatments and examined 2 representative AJ proteins: VE-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM-1). A protein kinase A (PKA) inhibitor, LY294002 (a PI3-K inhibitor), db-cAMP, and RP-cAMPS were 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 not PECAM-1. Aspirin did not prevent cell damage. P13-K inhibition by LY294002 had no influence on the effects of cilostazol, but inhibition of cAMP/PKA with PKA inhibitor and Rp-cAMPS suppressed cilostazol-induced inhibition of cell 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-dependent pathways in brain endothelial cells; thus, cilostazol effects on adhesion molecule signaling may provide protection against OGD stress in endothelial cells.

Phosphodiesterase-III inhibitor prevents hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tPA.

The purpose of the present study was to investigate whether cilostazol, a phosphodiesterase-III inhibitor and antiplatelet drug, would prevent tPA-associated hemorrhagic transformation. Mice subjected to 6-h middle cerebral artery occlusion were treated with delayed tPA alone at 6 h, with combined tPA plus cilostazol at 6 h, or with vehicle at 6 h. We used multiple imaging (electron microscopy, spectroscopy), histological and neurobehavioral measures to assess the effects of the treatment at 18 h and 7 days after the reperfusion. To further investigate the mechanism of cilostazol to beneficial effect, we also performed an in vitro study with tPA and a phosphodiesterase-III inhibitor in human brain microvascular endothelial cells, pericytes, and astrocytes. Combination therapy with tPA plus cilostazol prevented development of hemorrhagic transformation, reduced brain edema, prevented endothelial injury via reduction MMP-9 activity, and prevented the blood-brain barrier opening by inhibiting decreased claudin-5 expression. These changes significantly reduced the morbidity and mortality at 18 h and 7 days after the reperfusion. Also, the administration of both drugs prevented injury to brain human endothelial cells and human brain pericytes. The present study indicates that a phosphodiesterase-III inhibitor prevents the hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tPA.