Jingjing Su

Blockade of bradykinin B2 receptor more effectively reduces postischemic blood-brain barrier disruption and cytokines release than B1 receptor inhibition.

Blood-brain barrier disruption and brain edema are detrimental in ischemic stroke. The kallikrein-kinin system appears to play an important role in the regulation of vascular permeability and is invoked in edema formation. The effects of kinins are mediated by bradykinin receptors B1R and B2R. However, little is known about the exact roles of bradykinin receptors in the early stage of cerebral ischemia. In this study, we demonstrated that ischemia upregulated the level of B1R and B2R at 24h after reperfusion by immunofluorescence assays, mainly expressed in astrocytes and neurons, respectively, in the ischemic penumbra. Moreover, B2R inhibition more effectively reduced neurological severity scores, blood-brain barrier permeability and cytokines release than B1R inhibition did. Additionally, B2R inhibition also significantly suppressed B1R protein level. Therefore, blockade of B2R may be a more effective strategy for the treatment of ischemic brain injury than B1R inhibition within 24h after reperfusion.

Glutathione Prevents Free Fatty Acids-Induced Oxidative Stress and Apoptosis in Human Brain Vascular Endothelial Cells Through Akt Pathway

The damage of human brain vascular endothelial cells (HBVECs) is the key pathogenesis of diabetes‐associated cerebral vascular complications. The aim of this study was to elucidate the effects of glutathione (GSH) on free fatty acids (FFAs)‐induced HBVECs apoptosis, oxidative stress, and the involved possible signaling pathway.

Tissue kallikrein protects neurons from hypoxia/reoxygenation-induced cell injury through Homer1b/c.

Previous studies have demonstrated that human tissue kallikrein (TK) gene delivery protects against mouse cerebral ischemia/reperfusion (I/R) injury through bradykinin B2 receptor (B2R) activation. We have also reported that exogenous TK administration can suppress glutamate- or acidosis-induced neurotoxicity through the extracellular signal-regulated kinase1/2 (ERK1/2) pathway. To further explore the neuroprotection mechanisms of TK, in the present study we performed immunoprecipitation analysis and identified a scaffolding protein Homer1b/c using MALDI-TOF MS analysis. Here, we tested the hypothesis that TK reduces cell injury induced by oxygen and glucose deprivation/reoxygenation (OGD/R) through activating Homer1b/c. We found that TK increased the expression of Homer1b/c in a concentration- and time-dependent manner. Moreover, TK facilitated the translocation of Homer1b/c to the plasma membrane under OGD/R condition by confocal microscope assays. We also observed that overexpression of Homer1b/c showed the neuroprotection against OGD/R-induced cell injury by enhancing cell survival, reducing LDH release, caspase-3 activity and cell apoptosis. However, the knockdown of Homer1b/c by small interfering RNA showed the opposite effects, indicating that Homer1b/c had protective effects against OGD/R-induced neuronal injury. More interestingly, TK exerted its much more significantly neuroprotective effects after Homer1b/c overexpression, whereas it exerted its reduced effects after Homer1b/c knockdown. In addition, TK pretreatment increased the phosphorylation of the ERK1/2 and Akt-GSK3β through Homer1b/c activation. The beneficial effects of Homer1b/c were abolished by the ERK1/2 or PI3K antagonist. Therefore, we propose novel signaling mechanisms involved in the anti-hypoxic function of TK through activation of Homer1b/c-ERK1/2 and Homer1b/c-PI3K-Akt signaling pathways.

G-CSF Protects Human Brain Vascular Endothelial Cells Injury Induced by High Glucose, Free Fatty Acids and Hypoxia through MAPK and Akt Signaling

Granulocyte-colony stimulating factor (G-CSF) has been shown to play a neuroprotective role in ischemic stroke by mobilizing bone marrow (BM)-derived endothelial progenitor cells (EPCs), promoting angiogenesis, and inhibiting apoptosis. Impairments in mobilization and function of the BM-derived EPCs have previously been reported in animal and human studies of diabetes where there is both reduction in the levels of the BM-derived EPCs and its ability to promote angiogenesis. This is hypothesized to account for the pathogenesis of diabetic vascular complications such as stroke. Here, we sought to investigate the effects of G-CSF on diabetes-associated cerebral vascular defect. We observed that pretreatment of the cultured human brain vascular endothelial cells (HBVECs) with G-CSF largely prevented cell death induced by the combination stimulus with high glucose, free fatty acids (FFA) and hypoxia by increasing cell viability, decreasing apoptosis and caspase-3 activity. Cell ultrastructure measured by transmission electron microscope (TEM) revealed that G-CSF treatment nicely reduced combination stimulus-induced cell apoptosis. The results from fluorescent probe Fluo-3/AM showed that G-CSF greatly suppressed the levels of intracellular calcium ions under combination stimulus. We also found that G-CSF enhanced the expression of cell cycle proteins such as human cell division cycle protein 14A (hCdc14A), cyclinB and cyclinE, inhibited p53 activity, and facilitated cell cycle progression following combination stimulus. In addition, activation of extracellular signal-regulated kinase1/2 (ERK1/2) and Akt, and deactivation of c-Jun N terminal kinase (JNK) and p38 were proved to be required for the pro-survival effects of G-CSF on HBVECs exposed to combination stimulus. Overall, G-CSF is capable of alleviating HBVECs injury triggered by the combination administration with high glucose, FFA and hypoxia involving the mitogen-activated protein kinases (MAPK) and Akt signaling cascades. G-CSF may represent a promising therapeutic agent for diabetic stroke.

The role of aquaporin 4 in apoptosis after intracerebral hemorrhage

BackgroundWe previously reported that aquaporin-4 deletion (AQP4-/-) in mice increased edema and altered blood-brain barrier integrity following intracerebral hemorrhage (ICH). To date, little is known about the role of AQP4 in apoptosis after ICH. The purpose of this study was to examine the role of AQP4 in apoptosis and its mechanisms after ICH using AQP4-/- mice.MethodsWe compared the survival rate and neurological deficits in wild-type (AQP4+/+) mice with those in AQP4-/- mice following ICH. Histological changes were detected with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and Hoechst staining. The cell types involved were determined by immunocytochemical studies. We also measured activated caspase-3, caspase-9, caspase-8, Bax, and Bcl-2 with Western blotting at 1, 3, and 7 days after ICH. A cytokine protein assay was used to detect cytokines in AQP4+/+ and AQP4-/- mice following ICH, and the results were verified by ELISA.ResultsWe found more apoptotic cells in AQP4-/- mice following ICH; the cell types involved were predominantly neurons and astrocytes. Western blotting showed that the expression of activated caspase-3 and caspase-8 was significantly increased (P <0.05). Moreover, we demonstrated a greater enhancement in the release of TNF-Α and IL-1Β, as well as their receptors, in AQP4-/- mice following ICH than in AQP4+/+ mice by cytokine protein assay and Western blotting (P <0.05). The inhibitors of TNF-Α and IL-1Β reduced apoptotic cells after ICH in AQP4-/- mice compared with wild-type mice (P <0.05).ConclusionsAQP4 deletion increases apoptosis following ICH, and the underlying mechanism may be through cytokines, especially TNF-Α and IL-1Β, initiating the apoptotic cascade, as well as activation of caspase-3 and caspase-8.

HCdc14A is involved in cell cycle regulation of human brain vascular endothelial cells following injury induced by high glucose, free fatty acids and hypoxia

Cell cycle processes play a vital role in vascular endothelial proliferation and dysfunction. Cell division cycle protein 14 (Cdc14) is an important cell cycle regulatory phosphatase. Previous studies in budding yeast demonstrated that Cdc14 could trigger the inactivation of mitotic cyclin-dependent kinases (Cdks), which are required for mitotic exit and cytokinesis. However, the exact function of human Cdc14 (hCdc14) in cell cycle regulation during vascular diseases is yet to be elucidated. There are two HCdc14 homologs: hCdc14A and hCdc14B. In the current study, we investigated the potential role of hCdc14A in high glucose-, free fatty acids (FFAs)-, and hypoxia-induced injury in cultured human brain vascular endothelial cells (HBVECs). Data revealed that high glucose, FFA, and hypoxia down-regulated hCdc14A expression remarkably, and also affected the expression of other cell cycle-related proteins such as cyclin B, cyclin D, cyclin E, and p53. Furthermore, the combined addition of the three stimuli largely blocked cell cycle progression, decreased cell proliferation, and increased apoptosis. We also determined that hCdc14A was localized mainly to centrosomes during interphase and spindles during mitosis using confocal microscopy, and that it could affect the expression of other cycle-related proteins. More importantly, the overexpression of hCdc14A accelerated cell cycle progression, enhanced cell proliferation, and promoted neoplastic transformation, whereas the knockdown of hCdc14A using small interfering RNA produced the opposite effects. Therefore, these findings provide novel evidence that hCdc14A might be involved in cell cycle regulation in cultured HBVECs during high glucose-, FFA-, and hypoxia-induced injury.

Regulation of acid-sensing ion channel 1a function by tissue kallikrein may be through channel cleavage

Recently, we have demonstrated that serine protease tissue kallikrein (TK) can protect cortical neurons against ischemia-acidosis/reperfusion-induced injury, and that this effect might be mediated by acid-sensing ion channels (ASICs). However, little is known about how TK regulates the function of ASICs. Here we provided evidence that the regulation of ASIC1a function by TK was probably correlated with its cleavage. High concentration of TK (3μM) partially cleaved the extracellular loop of ASIC1a, followed by a marked decrease of LDH release and an increase of cell survival at pH 6.2. Pretreatment with a protease inhibitor aprotinin inhibited the cleavage of ASIC1a and prevented functional regulation by TK. However, the cleavage of ASIC2a, which was not functionally modified by TK, was not observed. Therefore, we propose that the limited proteolysis of extracellular loop within ASIC1a might be one of the potential regulatory mechanisms of ASIC1a function by TK.

Acid‐sensing Ion Channels Activation and Hypoxia Upregulate Homer1a Expression

Recent studies have indicated that dynamic alterations in the structure of postsynaptic density (PSD) are involved in the pathogenesis of many central nervous system disorders, including ischemic stroke. Homer is the newly identified scaffolding protein located at PSD and regulates synaptic function. Homer1a, an immediate early gene, has been shown to be induced by several stimulations, such as glutamate, brain‐derived neurotrophic factor, and trauma. However, whether acidosis mediated by acid‐sensing ion channels (ASICs) and hypoxia during cerebral ischemia can change Homer1a expression remains to be determined.

Progressive Hemorrhagic Transformation Following Dual Antiplatelet Therapy

Dual antiplatelet therapy (DAT) is often used after endovascular interventional treatment to prevent the recurrence of a stroke. However, DAT may lead to cerebral hemorrhage. We describe two stroke patients with subacute progressive cerebral hemorrhagic transformation (HT) following DAT with aspirin and Plavix. One patient experienced HT 3 weeks after carotid artery stenting that was performed 3 days after an acute stroke; the other patient developed progressive HT within 4 weeks following emergent thrombectomy for acute occlusion of the inferior M2 branch of the right middle cerebral artery (MCA). The first patient was a 54-year-old male with a history of hypertension and smoking. He was admitted for acute cerebral infarction in the left temporoparietal lobe and received intravenous thrombolysis (Figure 1A). He was found to have significant stenosis in the proximal portion of the left internal carotid artery (ICA) (Figure 1B). Three days after the stroke, the patient underwent successful angioplasty (4 9 20 mm Sterling Monorail balloon, Boston Scientific, Natick, MA) and stenting (7 9 30 mm Wallstent, Boston Scientific, Natick, MA) of the stenotic left carotid artery (Figure 1C). One week after the procedure, the patient was discharged with partial motor aphasia, right facial palsy, and right limb hemiplegia (upper, grade 0/5; lower, grade 4/5). Aspirin (100 mg qd), Plavix (75 mg qd), and atorvastatin (20 mg qn) were used for secondary prevention. Three weeks after the procedure, the patient complained of dizziness, headache, nausea, and vomit(A) (B) (C)

Delayed Shortening and Shifting of Carotid Wallstent

Carotid angioplasty and stenting (CAS) is a minimally invasive technique that is a reasonable alternative to carotid endarterectomy (CEA), especially in patients at high risk for surgery [1]. Although CAS is technically simple and is considered to be a low risk procedure, intracranial hemorrhage, distal embolization, transient hypotension, hyperperfusion syndrome, and other complications have been reported [2,3]. However, there have been few reported cases of delayed shortening of the Carotid Wallstent (Boston Scientific Corporation, Natick, MA, USA) used in the procedure. We report a case of delayed Carotid Wallstent shortening and shifting and discuss the possible causes of this complication. A 64-year-old male was admitted for inarticulacy and limb weakness of 1 month. The patient had a history of hypertension for 15 years (up to 205/130 mmHg) but was usually taking Adalat with good blood pressure control. He also had a history of smoking for more than 40 years (20 cigarettes/day) and drinking for decades (0.5 kg/day). On physical examination, the patient had mild facial and lingual paralysis of the right side; the rest of the neurological examination was normal. Brain MRI showed an acute cerebral infarct lesion in the right basal ganglia. Contrastenhanced magnetic resonance (MR) angiography showed a significant stenosis of the proximal segment of the right internal carotid artery (Figure 1A). A CAS procedure was performed under local anesthesia with embolic protection devices. Stent deployment was performed with a 7 9 30 mm self-expandable Carotid Wallstent (Figure 1B), and postdilatation was performed with a 4-mm balloon (Sterling, Boston Scientific, Natick, MA, USA) because of residual stenosis (Figure 1C). Three weeks after the operation, the patient developed diplopia; on examination, he had limited adduction of the left eye on right gaze. He had persistent mild facial and lingual paralysis of the right side. The patient did not have limb weakness, and the left Babinski sign was positive. Bultrasonography showed no stenosis or occlusion of either carotid artery. The patient was considered to be having another stroke. Medication for secondary prevention was continued. Ten months after the operation, the patient underwent cranial digital subtraction angiography (DSA) follow-up; the right Carotid Wallstent was found to have shortened and shifted to the common carotid artery (CCA), and there was mild intimal hyperplasia within the stent (Figure 1D). Shortening and shifting of the carotid artery stent have rarely been reported. The only previous report is from Yoon et al. [4] who reported that four patients who had received Carotid Wallstent implantation experienced delayed shortening and shifting of the Wallstents and restenosis. The Carotid Wallstent can be shortened or elongated to comply with the vessel diameter during implantation; however, such a phenomenon has rarely occurred after implantation. There are two possible causes for shortening of the Wallstent in this case. Firstly, the marked mismatch of diameters between the CCA and the internal carotid artery (ICA) may have induced the shortening of the stent. In our case, the CCA was approximately 7.7 mm in diameter, whereas the ICA diameter was 3.8 mm. This resulted in a rather conical-shaped Wallstent implantation. A slow further expansion of the Wallstent at the CCA level could have