高表达HSP70鼠脑胶质瘤细胞瘤苗的体外抗瘤作用

目的 观察高表达热休克蛋白70（HSP70）鼠脑胶质瘤细胞（C6细胞）瘤苗在体外的主动免疫抗瘤效应。并探讨其可能的抗瘤机理。方法 用灭活经诱导高表达HSP70的C6细胞作瘤苗，体外刺激SD大鼠脾细胞，进行肿瘤杀伤试验。用MTT法检测其杀伤活性。FCM及电子显微镜观察杀伤瘤细胞的变化。结果 1，经C6细胞瘤苗刺激的大鼠脾细胞较直接用灭活C6细胞刺激的大鼠脾细胞或新鲜大鼠脾细胞对C6细胞的杀伤率显增高，其中经C6瘤苗刺激6d后的大鼠脾细胞的杀伤率最高，它对热休克型和野生型C6细胞的杀伤率分别达99.25%和63.10%(E:T=20:1)。这种杀伤活性可被鼠HSP70单抗所阻断，2，杀伤试验时，FCM检测到亚二倍体峰，电镜发现靶细胞出现凋亡小体。结论 高表达HSP70的C6细胞瘤苗，在体外有显的免疫原性。可激活大鼠脾细胞对同源C6细胞产生有效的杀伤，通过使靶细胞凋亡可能是这种瘤苗主动免疫抗瘤效应的一个重要的杀伤机制。     

高免疫原性胶质瘤细胞疫苗体外诱导Th-1漂移研究

目的 观察高免疫原性即膜型热休克蛋白70(HSP70)及主要组织相容性抗原复合体Ⅰ类分子(MHC-Ⅰ)双高表达人多形性胶质母细胞瘤U251(GBM U251)细胞疫苗在体外诱导Th-1漂移现象.方法 GBM U251分别以500 U/mL IFN-γ诱导48 h、43℃热休克2 h、500 U/mLIFN-γ诱导48 h+43℃热休克2 h诱导其MHC-Ⅰ类分子、膜型HSP70高表达,随后经丝裂霉素(MMC)灭活制成细胞疫苗.体外刺激健康捐献者外周血单个核细胞(PBMCs)作为效应细胞,进行肿瘤特异性杀伤试验;FCM检测疫苗刺激前后PBMCs CD4<'+>、CD8<'+>T淋巴细胞比例变化;ELISA法检测效应细胞攻击靶细胞后的IFN-γ、IL-2的分泌情况.结果 体外刺激后,HSP70和MHC-Ⅰ类分子双高表达的U251细胞疫苗CD4<'+>、CD8<'+>比例相对于MHC-Ⅰ类分子单高表达或HSP70分子单高表达细胞疫苗组明显增加,体外IFN-γ和IL-2分泌量亦增加,差异均有统计学意义(p<0.05).结论高免疫原性即HSP70和MHC-Ⅰ类分子双高表达U251细胞疫苗体外诱导产生Th-1漂移现象,推测是其体外抗瘤作用的重要机制之一.     

亚低温对大鼠脑缺血再灌注损伤后HSP70mRNA表达及损伤神经细胞凋亡的影响

目的 探讨亚低温对大鼠脑缺血再注损伤后HSP70mRNA、HSP70（热休克蛋白70）表达及损伤神经细胞凋亡的影响。方法 采用大鼠局灶性脑缺血再灌注损伤模型,大脑中动脉阻塞2小时,再灌注损伤10小时,用逆转录聚合酶链反应（RT－PCR）技术、免疫组织化学法和原位缺口末端标记（TUNEL）法分别检测假手术组、对照组和亚低温组HSP70mRNA、HSP70表达水平和凋亡细胞百分率。结果 亚低温组HSP70mRNA、HSP70表达水平较对照组显著升高（P＜0．05）,而凋亡细胞百分率明显低于对照组（P＜0．05）。结论 亚低温上调大鼠脑缺血再灌注损伤后HSP70mRNA、HSP70表达水平可能与其抗损伤神经细胞凋亡作用有关。     

bFGF对脑缺血再灌流后HSP70及凋亡的影响

目的：研究外源性碱性成纤维细胞生长因子（bFGF)对脑缺血再灌流后HSP70表达与细胞凋亡的影响,方法：线栓法制成大鼠大脑中动脉闭塞及再灌流模型,用免疫组化及原位末端标记的方法观察大鼠大脑中动脉闭塞2h后再通1－72h脑组织HSP70表达与凋亡细胞的分布,侧脑室注射外源性bFGF,并观察对它们的影响。结果：bFGF组与生理盐水组相比于6－48h各时间点的HSP70表达增高（P＜0．05）,凋亡细胞数明显减少（P＜0．05,P＜0．01）。结论：外源性bFGF可能诱导脑缺血再灌流后HSP70表达和抑制细胞凋亡。     

替普瑞酮诱导AD模型大鼠海马HSP70表达及其神经保护作用的研究

目的 研究替普瑞酮（Geranylgeranylacetone，GGA）诱导阿尔茨海默病（Alzheimer disease，AD）模型大鼠海马热休克蛋白70（heat shock protein70，HSP70）表达及其对AD大鼠的神经保护作用。方法 90只SD大鼠随机分为替普瑞酮组、模型组与生理盐水组，双侧海马注射Aβ1-42。建立阿尔茨海默病大鼠模型，替普瑞酮组给予替普瑞酮800mg·kg^-1·d^-1灌胃，余两组给予等量生理盐水灌胃；术后1d、7d、14d、21d并于Y迷宫行为学测试后处死大鼠；采用western-blot方法检测各组大鼠海马HSP70表达的变化，HE染色观察海马神经元形态学改变，TUNEL法检测海马神经元凋亡。结果术后14d、21d模型组大鼠学习记忆能力较生理盐水组明显减退（P〈0．05），替普瑞酮组较模型组明显改善（P〈0．05）；术后7d、14d、21d模型组大鼠海马HSP70表达较生理盐水组逐渐减少（P〈0．05），替普瑞酮组HSP70表达明显增加（P〈0．05）；术后21d模型组大鼠海马CA1区神经元结构紊乱，细胞减少，凋亡细胞较生理盐水组明显增加（P〈0．01），替普瑞酮组凋亡细胞较模型组显著减少（P〈0．05），细胞形态学改变减轻。结论替普瑞酮能够诱导AD模型大鼠海马HSP70表达，减少大鼠海马神经元凋亡而产生神经保护作用，改善大鼠的学习记忆能力。     

冷冻治疗对大鼠C6脑胶质瘤细胞凋亡和p21表达的影响

目的研究冷冻治疗对大鼠C6脑胶质瘤细胞凋亡及其调控机制的影响。方法建立C6大鼠脑胶质瘤动物模型,于冷冻治疗后第15天留取标本,以末端标记法(TUNEL)、流式细胞仪(FCM)检测肿瘤细胞凋亡,以免疫组化技术检测p21蛋白的表达。结果冷冻可诱导C6大鼠脑胶质瘤细胞凋亡,上调p21的表达,二者呈显著正相关(P<0.01)。FCM分析显示G1峰前出现典型的亚二倍体峰。结论诱导细胞凋亡可能是冷冻杀伤脑胶质瘤细胞的另一重要机制;p21基因可能参与了这种细胞凋亡的调控。     

亚低温对大鼠弥漫性脑损伤后海马CA3区HSP70表达及细胞凋亡的影响

目的 观察亚低温对大鼠弥漫性脑损伤(DBI)后海马CA3区HSP70在蛋白质和mRNA水平的表达及细胞凋亡上的影响,探讨亚低温脑保护分子生物机制。方法 将大鼠随机分成空白对照、假手术、单纯DBI和DBI后亚低温治疗四组,按Marmarou氏方法制作大鼠DBI模型,采用免疫组化法、逆转录聚合酶链反应(RT-PCR)及流式细胞仪(FCM),分别观察各组动物脑海马CA3区HSP70在蛋白质和mRNA水平的表达及细胞凋亡率。结果 与对照组相比,大鼠DBI后海马CA3区HSP70表达水平及细胞凋亡率均升高(P<0.05);亚低温治疗后,大鼠脑海马CA3区HSP70表达水平较单纯DBI组显著增高(P<0.01),而细胞凋亡率则明显降低(P<0.05)。结论 亚低温对创伤性脑损伤的脑保护机制可能与促进HSP70表达,并减少神经细胞凋亡有关。     

热休克预处理对实验性自身免疫性脑脊髓炎神经细胞凋亡的影响及其机制研究

目的观察热休克预处理（HSP）后实验性自身免疫性脑脊髓炎（EAE）大鼠热休克蛋白70（HSP70）和核因子-KB（NF-KB）的表达及对神经细胞凋亡的影响，探讨其对EAE模型的神经保护作用。方法36只Wistar大鼠随机分为对照组（CON），EAE组和HSP组。EAE组制作成EAE模型；HSP组先给予HSP，24h后再制作成EAE模型；CON组不行特殊处理。观察大鼠神经症状．进行神经功能评分。于免疫后14-17d处死动物，取脊髓行HE染色，检测HSP70、NFKB免疫组化表达及神经细胞的凋亡。结果CON组大鼠没有发病。与EAE组大鼠比较，HSP组大鼠发病率显著降低，起病时间显著延迟，神经功能评分显著降低（均P〈0．05）。EAE组体重增加值较CON组明显减低，HSP后大鼠体重增加值较EAE组显著增加（p〈0．05）。脊髓病理显示HSP组炎性病灶数较EAE组显著减少（p〈0．05）。HSP组与EAE组相比，脊髓内HSP70阳性细胞数显著增加，NF-KB阳性细胞数显著减少，神经细胞凋亡显著抑制（均P〈0．01）。结论HSP对EAE大鼠具有一定的神经保护作用，其机理可能与HSP70表达增加，NF—KB表达受抑制从而导致神经细胞凋亡减少有关。     

脑缺血再灌流后HSP70蛋白的表达及其与凋亡的关系

目的 :研究脑缺血再灌流后 HSP70蛋白的表达及其与凋亡的关系。方法 :应用免疫组化的方法观察大鼠局灶性脑缺血再灌流后脑组织 HSP70蛋白的表达和细胞凋亡的分布。结果 :缺血 2 h再灌流 0 h即可见 HSP70表达和凋亡细胞 ,HSP70至 2 4h达高峰 ,凋亡细胞数于再灌流后 2 4～ 48h达高峰。结论 :脑缺血诱导 HSP70蛋白的表达和细胞凋亡 ,脑缺血后 HSP70蛋白的表达可能参与抑制细胞凋亡     

声动力疗法对C6胶质瘤细胞的生物效应研究

目的探讨声动力化学疗法（SDT）治疗脑胶质瘤的可能性：方法采用四唑盐比色法，即MTT法测定C6胶质瘤细胞托SDT组、超声组、血啉甲醚（HMME）组、对照四组6h的抑瘤率，流式细胞学（FCM）检测四组6h的凋亡率，透射电镜观察四组6h亚细胞结构的变化。结果SDT组抑瘤率为77．61％、凋亡率为38．34％，显著增高；超声组也有一定的抑瘤率、凋亡率：HMME组抑瘤率、凋亡率与对照组无明显差别.透射电镜观察对照组、HMME组C6细胞无明显变化，超声组部分细胞呈早期凋亡表现，SDT组细胞呈现凋亡或坏死状态。结论SDT能显著杀伤体外C6胶质瘤细胞，并诱导其凋亡。     

Alterations in cytokinetics and heat shock protein (70 kDa) expression of glial cell by hyperthermia]

Expression of major heat shock and stress-induced protein, HSP70, is known to be under complex regulation in tumor cells. In this study, we investigated the alternations of cytokinetics and HSP70 expression by hyperthermia in the in vitro experimental systems, using two rat glioma cell lines, two human glioblastoma cell lines and rat glioblast cells. For hyperthermal treatment the flasks were placed in water baths warmed up at 41 -45 degrees C for 15 min. To determine the effect of hyperthermia on the cell cycle progression, the changes in the DNA distribution of the cell population were studied by flow cytometry (FCM). The levels of HSP70 protein were determined by immunoblot analysis. The relationship between cell cycle and HSP70 expression was investigated by FCM using PI and FITC-labelled HSP70 double staining technique. These results were as follows: 1) Compared with the control, hyperthermic treatment at 42 degrees C or 44 degrees C caused both 354A and T98G cells to accumulate in S phase 18 hours after treatment and G2/M phase after 6-18 hours. 2) Hyperthermic treatment at 42 degrees C caused C6 cells to accumulate in S phase 6 hours after treatment, whereas heat treatment at 44 degrees C caused C6 cells to accumulate in S phase after 18 hours and G2/M phase after 6 hours. 3) A172 cells were accumulated only in G2/M phase by hyperthermia. 4) Glioblast cells did not show the alterations of cytokinetics by heat treatment remarkably. 5) HSP70 protein synthesis were enhanced under hyperthermic conditions in all type of cells, whether primary glioblast or permanent glioma cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of ECr on HSP70 expression and morphologic changes following transient cerebral lschemia in rats

OBJECTIVE To investigate the effect of Cudrania tricuspidata root extract (ECr) on ischemic cerebral damage and the expression of the 70KDa heat shock protein (HSP70) following transient focal ischemia. BACKGROUND The role that ECr plays in cerebral ischemia has not been studied. METHODS Healthy wistar rats were randomized to the normal control group(Group A,n=4),the sham-operated control group(Group B,n=4),the ischemia and reperfusion group (Group C,n=24),the ischemia and reperfiusion after ECr pre- administration group(Group D,n=24).The rats of Group C and Group D were subjected to transient left middle cerebral artery occlusion (MCAO)as described by Zea Longa for 1 hour. Brain sections at the level of striatum were performed for HSP70 immunohistochemistry and morphology.HSP70 positive reactions and morphologic changes were semiquantiratively analyzedl. RESLULTS In the rats of Group A and Group B, there were scarcely HSP70 immunoreactivities either in cortex or in striatal neurons. In the ischemic brain regions for Group C rats,the HSP70 was induced in morphologically intact neurons and endothelial cells at hour 6 after recirculation, increased at hour12,peaked at day I, decreased at day 3,and HSP70 expression only occured in endothelial cells at day7. In Group D rats, the HSP70 was induced in the neurons of left MCA distribution at hour 1 after reperfusion, The changes in expression of HSP70 at different time points in Group D rats was in accord whth in Group C, but the number of HSP70 positive cells in Group D increased more, and HSP70 irnmunoreactivity in the HSP70-postive cells were more intense than in Group C. Histopathological study with HE staining showed no neuron pyknosis in Group A or in Group B. While pykrotic cells were present in the ipsilateral cortex and striatal neurons of MCA territory of Group C rats beginning at 6 hours after roper fusion. The change of histopathology in Group D was lighter at every time point. The number of pyknotic neurons in left MCA distribution was less than in Group C and there was no evident cell damage at 3 days and 7 days of reperfusion in Group D rats. CONCLUSION Our study demonstrated that transient focal cerebral ischenia could induce the HSP70 expression and induce neurons pyknosis.While ECr pre-treatment before transient focal ischemia in rats could increase the expression of HSP70 and reduce neuronal injury. These data suggests that might be able to enhance neuronal ischemic tolerance of the rats and might have prophylactic neuroprotective effect on ischemic cerebral damage in rats.     

红藻氨酸诱导癫痫发作鼠脑HSP70表达与组织细胞凋亡关系的研究

目的 :探讨红藻氨酸诱导癫痫发作鼠脑 HSP70表达与组织细胞凋亡的关系。方法 :用流式细胞术及免疫印迹法分别检测红藻氨酸诱导癫痫发作鼠脑海马结构组织细胞不同时间的凋亡率及 HSP70表达量。结果 :红藻氨酸注射后 3小时 ,HSP70表达量即开始增高 ,6小时凋亡率始升高 ,至 4 8小时 ,二者同时达到高峰 (P<0 .0 1) ,此后开始下降。结论 :HSP70是调节红藻氨酸诱导癫痫发作鼠脑细胞凋亡的重要分子之一     

Effect of heat stress on serotonin-2A receptor-mediated intracellular calcium mobilization in rat C6 glioma cells

Summary. This study was conducted to investigate an effect of heat stress at 44°C for 30 min on intracellular Ca²⁺ signaling system and on heat shock protein (HSP)-70 expression. 5-HT-induced Ca²⁺ mobilization was reduced 1, 3 and 6 hrs after heat stress, and recovered to the control level 12 and 24 hrs after heat stress. One hr after heat stress, Ca²⁺ rise was significantly decreased when the cells were stimulated by any concentration of 5-HT. Thrombin-induced Ca²⁺ increase was also markedly reduced 1 hr after heat stress. HSP-70 level was increased 6 and 9 hr after heat stress. In HSP synthesis inhibitor quercetin-treated cells, HSP-70 expression was not enhanced after heat stress, and Ca²⁺ rise in response to 5-HT did not return to the control level. However, the Ca²⁺ rise induced by 5-HT was not restored to the control level after stress in Ac-Asp-Glu-Val-Asp-H (DEVD)-exposed cells while DEVD had little effect on heat stress-induced synthesis of HSP-70. Dexamethasone did not alter the change in HSP-70 expression or Ca²⁺ response after heat stress. These results indicate that heat stress attenuated 5-HT-induced Ca²⁺ mobilization and that HSP-70 expression played an important role in recovery from Ca²⁺ impairment, possibly via protease activity in C6 cells. Received October 16, 1998; accepted December 3, 1999     

Effects of haloperidol and risperidone on the expression of heat shock protein 70 in MK-801-treated rat C6 glioma cells

Non-competitive N-methyl-d-aspartate (NMDA) receptor antagonists such as dizocilpine (MK-801) produce schizophrenia-like psychosis in humans and induce the expression of heat shock protein 70 (HSP70) in rats. The present study examines the effects of antipsychotic drugs, haloperidol and risperidone, on the expression of HSP70 produced by MK-801 in rat C6 glioma cells. After pretreating with haloperidol and risperidone for 1 h, 6 h, 24 h and 72 h, respectively, C6 glioma cells were cultivated again in MK-801 for 6 h, and then, the extent of HSP70 expression was measured by immunoblotting using anti-HSP70 monoclonal antibody. The expression of HSP70 induced by MK-801 significantly decreased as the duration of haloperidol pretreatment was extended (p = 0.002). Risperidone also increasingly attenuated the expression of HSP70 produced by MK-801 as the duration of pretreatment grew longer (p = 0.003). The present findings show that haloperidol and risperidone decrease the HSP70 expression in MK-801-treated rat C6 glioma cells. These results suggest that HSP70 and NMDA receptors may play a significant role in the pathophysiology of schizophrenia.     

Correlation between HSP70 and c-F0S expression and apoptosis in rats undergolng transient focal cerebral ischemla and reperfusion

OBJECTIVE To compare the induction of c-Fos and HSP70 and the presence of apoptosis and the influence of Ginkgo biloba extract (EGb761) in transient focal cerebral ischemic reperfusion rats.BACKGROUND Proto ancogene activation and induction of heat shock protein (HSP) occur in response to cerebral ischemia, but the correlation between these proteins and apoptosis remains uncertain. METHODS Healthy wistar rats were randomized to the normal control group(Group A, n=4), the Sham-operated control group(Group B, n=4), the ischemia and reperfusion group(Group C, n=24), the EGb761 pre-treated ischemia and reperfusion group(Group D, n=24). The rats of Group C and Group D were subjected to transient left middle cerebral artery occlusion (MCAO) as described by Zea longa for 1 hour. RESULTS Immunohistochemical analysis revealed no c-Fos or HSP70-immunoreactivity in Group A and Group B rats. However, in Group C rats, c-Fos was expressed in ipsilateral superficial cortical layers at 1 hour after reperfusion. At 6 hours, c-Fos immunoreactivities were increased in the ipsilateral cortex and were present in the contralateral cortex, while HSP70 were induced beginning in the ipsilateral neurons of MCA distribution. At 12 hours, the expression of c-Fos reached top in superficial cortical layers. At 24 hours HSP70 immunoreactivities reached top both in ipsilateral cortex and in ipsilateral striatum. At 3 days after recirculation, HSP70 expression decreased. c-Fos expression disappeared at day 7 and HSP70 expression only occured in endothelial cells. TUNEL staining showed that there was no cell apoptosis in Gronp A or in Group B. However, in Group C, TUNEL-positive neurons were observed in the border of the penumbra-like area that surrounds the ischemic core at 6 hours following reperfusion and then the number of TUNEL-positive cells reduced gradually. The changes in expression of HSP70 and c-Fos at different time points in Group D was in accord with in Group C, but the number of positive cells and the immunoreactivities in Group D were more intense than in Group C. We found only several TUNEL-positive cells at 6 hours following reperfusion in Group D and no TUNE;L-positive cells were present at other time points.CONCLUSION The present results indicate that c-Fos was expressed from an earlier stage of reperfusion and the expression occured in bilateral cerebral cortex. In contrast, HSP70 induction began later and only occured in ipsilateral neurons of MCA distribution. Our results indicated that EGb761 could increase the expression of c Fos and HSP70 and reduce the apoptosis of neurons.     

The Effects of Venlafaxine and Dexamethasone on the Expression of HSP70 in Rat C6 Glioma Cells

Objective

The present study aimed to determine the intracellular action of the antidepressant, venlafaxine, in C6 glioma cells using heat shock protein 70 (HSP70) immunocytochemistry and HSP70 Western blots; HSP70 is known to be associated with stress and depression.

Methods

The extent of HSP70 expression was measured after rat C6 glioma cells were treated with 1) dexamethasone only, 2) venlafaxine only, 3) simultaneous venlafaxine and dexamethasone, or 4) dexamethasone after venlafaxine pretreatment. Dexamethasone (10 µM, 6 hours) did not affect the level of HSP70 expression relative to control.

Results

Short-term (1 hour) venlafaxine treatment significantly increased the level of HSP 70 expression. Simultaneous long-term (72 hours) venlafaxine and dexamethasone treatment significantly reduced the level of HSP70 expression. Dexamethasone treatment administered following long-term (24 and 72 hours) pretreatment with venlafaxine also significantly reduced the level of HSP70 expression.

Conclusion

Short-term treatment with venlafaxine increases the expression of HSP70, but prolonged treatment with dexamethasone suppresses the venlafaxine-induced expression of HSP70. These findings suggest that HSP70 and dexamethasone play a significant role in the pathophysiology of depression.     

Heat shock protein 72 restores cyclic AMP accumulation after heat shock in N18TG2 cells

Although there are several reports on the alteration of intracellular signal transduction during heat shock in somatic cells, the long term effects of heat shock on neuronal cells remain unknown. In this report, we investigated cyclic AMP (cAMP) accumulation and the expression of heat shock proteins following heat shock in mouse neuroblastoma N18TG2 cells. Basal cAMP accumulation, or that stimulated by serotonin (10 μM), cholera toxin (1 μg/ml), and forskolin (1 μM) was suppressed at 0, 3, and 6 h following heat shock (45°C for 30 min). The cAMP levels were restored at 15 and 24 h after heat shock, corresponding with the expression of stress-induced heat shock protein 72 (HSP72). Quercetin, an inhibitor of HSP expression, decreased the expression of HSP72 and inhibited the recovery of cAMP levels 24 h after heat shock. Quercetin also decreased the basal expression of the constitutive heat shock cognate protein 70 (HSC70) and suppressed cAMP accumulation in non-heat shocked cells. These results suggest that stress-induced HSP72 restores cAMP accumulation to control levels following heat shock and that constitutive HSC70 is related to cAMP levels in non-stress conditions.     

Functional interdependence and colocalization of endothelial nitric oxide synthase and heat shock protein 90 in cerebral arteries.

Heat shock protein 90 (HSP90), an essential component of several signal transduction systems, participates in the activation of endothelial nitric oxide synthase (eNOS) in cells. The objective of the current study was to determine if HSP90 and eNOS were functionally interdependent and colocalized in the cerebral circulation. The authors used isometric force recording, cyclic 3'5'-guanosine monophosphate (cGMP) radioimmunoassay (RIA), and immunogold electron microscopy (EM) to study canine basilar artery. They found that geldanamycin (0.1 to 10 microg/mL), a selective HSP90 inhibitor, caused concentration-dependent contractions in arterial rings (n = 6 dogs). Contractions to geldanamycin were unaffected by a cyclooxygenase inhibitor, indomethacin (10 micromol/L; P < 0.05, n = 6). Functional evidence for interaction between HSP90 and nitric oxide (NO)-mediated signaling included observations that the contractile effect of geldanamycin was the following: (1) endothelium-dependent, (2) abolished by Ng-nitro-L-arginine methylester (L-NAME; 0.3 mmol/L), and (3) non-additive with the contractile effect of this NOS inhibitor (P < 0.01, n = 6 for each). Furthermore, RIA showed significant reduction in cGMP levels in arteries treated with geldanamycin (3 microg/mL; P < 0.02, n = 8), whereas immunogold EM demonstrated areas of colocalization of HSP90 and eNOS selectively in the cytoplasm of endothelial cells. The current findings suggest that in cerebral arteries, endothelial HSP90 plays an important role in modulation of basal NO-mediated signaling. This interaction may be particularly important in stress-induced up-regulation of HSP90 with subsequent alteration of vasomotor function.     

Pharmacologic heat shock protein 70 induction confers cytoprotection against inflammation in gliovascular cells

The inhibition of the 90‐kDa heat shock protein (HSP90) leads to upregulation of the 70‐kDa‐inducible HSP70. HSP70 has been previously shown to be neuroprotective and anti‐inflammatory. Geldanamycin (GA) and other HSP90 inhibitors have emerged as promising therapeutic agents in cancer, presumably owing to their ability to upregulate HSP70. However, the effects of HSP90 inhibition in brain inflammation are still unclear. We investigate the effect of a panel of HSP90 inhibitors on endotoxin‐activated microglia and eventual protection from brain‐derived endothelial cells. Prior studies have shown that GA protects brain cells from oxidative stress. We show here that when astrocytes or microglial BV2 cells were pretreated with GA or other HSP90 inhibitors, endotoxin‐induced cell death was reduced in cocultures of BV2 microglia and brain‐derived endothelial cells (bEND.3). Endotoxin‐stimulated BV2 cells led to increased nitric oxide (NO) and inducible nitric oxide synthase which was prevented by treatment with all HSP90 inhibitors. HSP90 inhibitors also prevented lipopolysaccharide (LPS)‐induced BV2 cell death. We also found that HSP90 inhibition blocked nuclear translocation of nuclear factor kappa B and attenuated IκBα degradation, and inhibited LPS‐activated JAK‐STAT phosphorylation. We show that pharmacologic inhibition of HSP90 with subsequent HSP70 induction protects cells that comprise the cerebral vasculature against cell death owing to proinflammatory stimuli. This approach may have therapeutic potential in neurological conditions with an inflammatory component. GLIA 2015;63:1200–1212