Nitroaspirin (NCX-4016), an NO donor, is antiangiogenic through induction of loss of redox-dependent viability and cytoskeletal reorganization in endothelial cells

We recently reported that NCX-4016, a derivative of aspirin containing a nitro moiety that releases nitric oxide (NO) in a sustained fashion in biologic systems, is a potent cytotoxic agent inhibiting the proliferation of cisplatin-resistant human ovarian cancer cells. Therefore, we hypothesize that NCX-4016 possesses antiangiogenic properties. Our study with the bovine lung microvascular endothelial cells (BLMVECs) revealed that NCX-4016 significantly induced the loss of redox-dependent cell viability in a dose- and time-dependent manner, as assayed by the redox-sensitive Alamar blue cell viability assay. Fluorescence microscopy of cells labeled with NO-specific fluorophore (DAF-FM) confirmed that NCX-4016 generated significant levels of intracellular NO. NO donors, including S-nitroso-N-acetylpenicillamine, spermine NONOate, and isosorbide dinitrite, were less effective in causing loss of cell viability. Thiol-protectant, N-acetylcysteine, significantly attenuated the NCX-4016-induced loss of cell viability, suggesting the role of alteration of thiol-redox status therein. NCX-4016 also suppressed oxygen consumption, decreased transendothelial electrical resistance (EC barrier dysfunction), and induced actin cytoskeletal reorganization in BLMVECs. The in vitro assay with human umbilical vein ECs and BLMVECs revealed that NCX-4016, in a dose-dependent manner, significantly inhibited angiogenesis with almost complete inhibition at a 100-microM concentration, suggesting that NCX-4016 can act as an antiangiogenic drug.     

Application of low dose of FK506 in pancreas transplantation model in Wistar-SD rats

Objective: To probe into application of low dose of FK506(Tacrolimus) in pancreas transplantation.

Methods: Effects of low-dose FK506 (Tacrolimus) in pancreas transplantation with examination of ELISA Electron microscopy and TUNEL by method of random control were studied.

Results: Blood glucose concentration in control group is higher than that in treated group A (FK506) and treated group B (CsA) 7 days after transplantation (p?<?0.05). Serum C-peptide and insulin concentrations in control group are less than that in treated group A (FK506) and treated group B (CsA) 7 days after transplantation (p?<?0.05). Blood glucose, serum C-peptide and insulin concentrations are same as that in control group, group A (FK506) and group B (CsA) (p?>?0.05).There are more apoptotic nuclei in control group than that in treated group A (FK506) and treated group B (CsA) (p?<?0.05). There is no significant difference between group A (FK506) and treated group B (CsA) in sum of apoptotic nuclei (p?>?0.05). There is no significant difference among treated group A (FK506) and treated group B (CsA) in electron microscopy fields.

Conclusion: Low-dose FK506 applied in pancreas transplantation could not only be effective for immunosuppressive, but also be safe for islet cells of pancreas.     

Effect of FK506 on the interleukin 15-driven proliferation and apoptosis of anti-CD3-activated umbilical cord blood T cells.

BACKGROUND: Increased susceptibility of umbilical cord blood (CB) T cells to FK506 immunosuppression may contribute to the lessened severity of graft-vs-host disease in CB transplantation. OBJECTIVE: To investigate the FK506 sensitivity of interleukin 15 (IL-15)- and IL-2-driven proliferation and apoptosis of anti-CD3-stimulated CB T cells compared with adult peripheral blood (APB) T cells. METHODS: Surface flow cytometric analysis (CD25 and CD95), carboxyfluorescein diacetae succinimidyl ester staining to track CD3+ T-cell division, and flow cytometric analysis of apoptotic cell death using Annexin V were performed to determine the effect of FK506 on CD3+ T-cell activation and apoptosis after anti-CD3 stimulation in the presence of IL-15 or IL-2. RESULTS: IL-15 is superior to IL-2 in promoting CD25 expression and proliferation of anti-CD3-stimulated CB and APB T cells. Although IL-15-driven proliferation evaluated by carboxyfluorescein diacetae succinimidyl ester staining revealed comparable sensitivity to FK506 in anti-CD3-stimulated CB and APB T cells, IL-15-driven CD25 up-regulation in CB T cells was more sensitive to FK506 inhibition than APB T cells. FK506 down-regulated anti-CD3-induced apoptosis in CB and APB T cells (P < .01). However, the FK506 sensitivity of anti-CD3-induced T-cell apoptosis was lost in IL-15-supplemented CB cultures (P = .51) but not in corresponding APB cultures (P = .002). The IL-15-enhanced Fas expression on CB T cells (CD95) was decreased by FK506, similar to that observed with adults. CONCLUSIONS: We observed differential FK506 sensitivity of IL-15-driven CD25 up-regulation and apoptotic response comparing CB and APB T cells. This finding suggests the potential therapeutic benefit of FK506 in ameliorating graft-vs-host disease by decreasing IL-15-driven donor T-cell proliferation without inhibiting associated activation-induced apoptosis during CB transplantation.     

Beneficial effects of FK506 for experimental temporal lobe epilepsy

FK506, originally classified as an immunosuppressant, may also be implicated in some events in the central nervous system. FK506 elicits both neuroprotective and neurotrophic effects in vitro. FK506 is neuroprotective for focal cerebral ischemia, but it is not clear whether FK506 has neuroprotective effects for other brain diseases. In this study, we investigated possible neuroprotective effects of FK506 in experimental temporal lobe epilepsy (TLE) induced by kainic acid (KA) or trimethyltin (TMT). In rat models, we observed marked protection against seizures, abnormal behaviors, and accompanying delayed neuronal damage in the hippocampus by the systemic injection of FK506.     

Increased osteocyte apoptosis during the development of femoral head osteonecrosis in spontaneously hypertensive rats

We investigated the presence of osteocyte apoptosis in the necrotic trabeculae of the femoral head of spontaneously hypertensive rat (SHR) using the in situ nick end labeling (TUNEL) method and transmission electron microscopy. The occurrence of osteonecrosis and ossification disturbance was significantly higher in SHR compared with Wistar Kyoto (WKY) rats, and Wistar (WT) rats used as control animals (P < 0.01). A high population of TUNEL positive osteocytes was detected mainly in 10- and 15-week-old SHRs. Sectioned examination of the femoral head of SHRs and WKY rats by electron microscopy revealed apoptotic cell appearances such as aggregation of chromatin particles and lipid formation. In contrast, a positive reaction was significantly lower in osteocytes in the femoral heads of WT rats (P < 0.01). Our results indicate that apoptosis forms an important component of the global pathologic process affecting the femoral head of SHR, which leads to osteonecrosis in this region.     

Injections of vehicle, but not cyclosporin A or tacrolimus (FK506), afford neuroprotection following injury in the developing rat brain

Susceptibility of the injured rat brain to seizures depends on the developmental stage at which the injury had been inflicted. Our previous study shows that tacrolimus (FK506) and cyclosporin A (CsA) applied following the injury can also decrease or increase the seizure susceptibility in an age-dependent way. To find possible neuronal substrates of the effects, we examined influences of the agents on the injured brain and on its neuronal population. Rat brains were mechanically injured on postnatal days 6 (P6) or 30 (P30). Twenty minutes and 24 hours following the injury, FK506 or CsA were injected in clinically used pharmaceutical formulations (Prograf or Sandimmun, respectively). The brains were fixed on postnatal day 60 and processed for histological examinations. To detect if negative effects of the injury could be abolished by the treatments, we examined the brain weight, the size of the injured region, and the nerve cell density, including the density of calretinin- and parvalbumin-immunopositive cells. We have found that long-term effects of treatments with the FK506- and CsA-containing pharmaceutical formulations were never better than those of the vehicle alone (Cremophor and ethanol mixture). Moreover, the treatments could even amplify negative consequences of the injury alone. It could, therefore, be concluded that all the neuroprotective effects observed in the present study resulted exclusively from the influence of the vehicle alone. These effects of the brain injury and of subsequent treatments performed at different developmental stages were considered as possible determinants of further increase or decrease in susceptibility to seizures observed in adulthood.     

他克莫司和环孢素A对肾移植后患者炎性细胞因子和血脂的影响

背景：环孢素A与他克莫司是肾移植后临床广泛应用的免疫抑制剂。 目的：观察他克莫司和环孢素A对肾移植后炎性细胞因子和血脂的影响。 方法：选择首次接受同种异体肾移植后患者112例,随机分为环孢素A组和他克莫司组,移植后分别给予环孢素A+吗替麦考酚酯+糖皮质激素三联疗法与他克莫司+吗替麦考酚酯+糖皮质激素三联疗法。 结果与结论：他克莫司组的1年人/肾存活率、治疗逆转率高于环孢素A组(P < 0.05),急性排斥反应发生率低于环孢素A组(P < 0.05);移植后1个月及1年的血清白细胞介素2,6,8和血糖水平高于移植前(P < 0.05),低于环孢素A组(P < 0.05),血清白细胞介素4,10低于移植前(P < 0.05),高于环孢素A组(P< 0.05);移植后1个月的血清总胆固醇、三酰甘油和低密度脂蛋白胆固醇高于移植前(P < 0.05),但低于环孢素A组(P < 0.05);移植后1年的血清总胆固醇和低密度脂蛋白胆固醇低于环孢素A组(P < 0.05)。说明他克莫司可通过抑制肾移植后炎性细胞因子释放,改善糖脂代谢等途径降低患者的排斥反应,提高肾移植的存活率。     

Neuroprotective effects of tacrolimus (FK506) in a model of ischemic cortical cell cultures: role of glutamate uptake and FK506 binding protein 12 kDa

BACKGROUND: The mechanisms underlying the neuroprotective effects of the immunosuppressant tacrolimus, observed in vivo, remain unclear. Here we quantify these effects in vitro, and evaluate the potential involvement of the glutamate and/or immunophilin FK506 binding protein 12 kDa in tacrolimus-induced neuroprotection. METHODS: Primary cultures of neurons and astrocytes from rat cerebral cortex were subjected to transient oxygen-glucose deprivation. Neuronal injury was evaluated by cell counting after immunostaining experiments, lactate dehydrogenase release and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction. The involvement of the immunophilin FK506 binding protein 12 kDa was explored using an anti-FK506 binding protein 12 kDa antibody, (3-3-pyridyl)-1-propyl(2 s)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate and rapamycin. Extracellular glutamate and glutamate uptake were respectively measured by high performance liquid chromatography and l-[3H]glutamate incorporation. RESULTS: When added during either oxygen-glucose deprivation or reoxygenation, FK506 (50-500 pM) offered significant neuroprotection. During oxygen-glucose deprivation, it was able to reverse the oxygen-glucose deprivation-induced increase in extracellular glutamate and decrease in glutamate uptake and this effect was reversed in the presence of threo-3-methyl glutamate, a specific inhibitor of glutamate transporter-1. Blocking FK506 binding protein 12 kDa inhibited the neuroprotection induced by tacrolimus added during either oxygen-glucose deprivation or reoxygenation. Tacrolimus-induced neuroprotection was also reversed in the presence of rapamycin, an immunosuppressant FK506 binding protein 12 kDa ligand devoid of neuroprotective properties and (3-3-pyridyl)-1-propyl(2 s)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate, a non-immunosuppressant ligand of FK506 binding protein 12 kDa, exerteing neuroprotective effects. CONCLUSION: The beneficial effects of tacrolimus during in vitro ischemia/reperfusion seem to indicate the restoration of a glutamate transporter-1-mediated activity and could be mediated by a FK506 binding protein 12 kDa pathway.     

Subject Index to Volume 25

Background: Tacrolimus causes post-transplant diabetes mellitus, however the pathogenetic mechanisms remain controversial. In this study we probed into the mechanisms of tacrolimus-induced diabetes mellitus in rats. Methods: Glucose levels were determined on whole blood samples using a glucose oxidase method. Levels of serum insulin and C-peptide were measured with ELISA. Histological damage of ultra-structure and apoptosis of beta cells of the pancreas were assayed with electric microscope and tunnel methods respectively.—Ultra-structure were assayed with electric microscope and apoptosis of beta cells of the pancreas were assayed with tunel methods. Immunohistochemistry was utilized to detect the sum of insulin receptors of hepatic cells. Results: Compared to control group, insulin and C peptide levels in serum decreased in rats of diabetes mellitus models induced with FK506(P<0.05). Compared to the control group, the sum of apoptosis body in pancreatic islets increased in rats of diabetes mellitus models induced with FK506 (P<0.05). Compared to the control group, electron microscopy showed cytoplasm swelling and vacuolization, and marked decrease or absence of dense-core secretory granules in beta cells in rats with diabetes mellitus induced with FK506.Compared to the control group, expression of insulin receptor of hepatic cell decreased in rats of diabetes mellitus models induced with FK506 (P<0.05). Conclusion: Pathogenetic mechanisms of rats of diabetes mellitus models induced with FK506 including reduction of secretion of insulin in beta cells of pancreatic islets, damages of ultra-structure of beta cells of pancreatic islets, increasing of apoptosis of beta cells of pancreatic islets and decreasing of expression of insulin receptors in hepatic cells.     

UCF-101对大鼠脑缺血再灌注后凋亡抑制蛋白XIAP表达的影响

目的观察UCF-101对大鼠脑缺血再灌注损伤后神经元凋亡及凋亡抑制蛋白XIAP表达的影响,探讨UCF-101对缺血性脑损伤的神经保护作用。方法采用线栓法建立Wistar大鼠大脑中动脉闭塞(MCAO)2h再灌注模型,随机将大鼠分为假手术组、缺血再灌注组及UCF-101处理组,于再灌注后24h取脑,采用TTC法测梗死体积,TUNEL法检测神经元凋亡,免疫组化法观察脑组织神经元XIAP蛋白的表达。结果假手术组未见梗死现象,偶见凋亡神经细胞,神经元中可见棕黄色XIAP颗粒散在分布于核膜周围胞浆中。与假手术组比较,缺血再灌注组可见梗死灶,脑组织凋亡细胞数明显增加,XIAP的表达明显降低(P<0.05);与缺血再灌注组比较,UCF-101处理组梗死体积明显缩小(P<0.05),脑组织凋亡细胞数减少,XIAP的表达均明显增加(P<0.05)。结论 UCF-101神经保护作用可能与上调脑组织神经元XIAP蛋白的表达和抑制神经元的凋亡有关。     

卡托普利预处理对家兔实验性心肌梗塞范围及心肌细胞凋亡的影响

目的:探讨卡托普利预处理对家兔实验性心肌梗塞范围及心肌细胞凋亡的影响。方法:健康家兔,分为(1)假手术对照组(sham-operated control group),(2)急性心梗组(acuteinfarct group),(3)卡托普利预处理组(captopril group,25mg·kg^-1·d^-1,喂饲1周)。结扎冠状动脉左旋支造成急性心肌梗塞模型。测结扎前、结扎后15、30、60min的心功能。结扎前及结扎后1h的全血粘度与血球压积。结扎后6h取心脏,以缺血区重/左室重比为缺血范围,梗塞区重/缺血区重比为梗塞范围。TUNEL法染色检测细胞凋亡,以心肌凋亡阳性细胞数占总心肌细胞数的百分比作为心肌细胞凋亡指数。结果:(1)卡托普利预处理组梗塞范围显著小于心梗组(16.60%±0.94%比36.24%±1.94%,P＜0.05),并改变左室功能及血粘度。(2)梗塞周围可见凋亡心肌细胞,卡托普利预处理该区心肌细胞凋亡指数明显小于急性心梗组(26.37%±0.71%比42.44%±2.32%,P＜0.05)。结论:卡托普利预处理可明显减少梗塞范围及心肌细胞凋亡程度,改善心功能。     

Novel immunosuppressive effect of FK506 by augmentation of T cell apoptosis

We have recently reported the accumulation of oligoclonal activated T cells in the spontaneously developed autoimmune pancreatitis in aly/aly mouse. In this study, we examined the effects of FK506 in this mouse model in preventing autoimmune pancreatitis and investigated its action on calcium signalling apoptosis of alymphoplasia (aly) lymphocytes in vitro. Mice were treated with FK506 from 8 to 25 weeks of age. At the age of 15 weeks, minimal mononuclear cell infiltration was observed in the pancreas in both the FK506 treated group and the control group. Furthermore, a marked cell infiltration associated with destruction of acini and partial fatty changes were observed in 25-week-old control mice. In contrast, FK506 treated mice showed almost no tissue destruction or mononuclear cell infiltration at the age of 25 weeks. Furthermore, at 15 weeks of age, most mononuclear cells in FK506-treated mice were TUNEL positive, whereas only a few were positive in control mice. This augmentation of T cell apoptosis by FK506 was confirmed using naive splenocytes activated by PMA and ionomycin in vitro. Finally, a suppressive effect of FK506 on Bcl-2 production but not on Bax production was confirmed by Western blotting. This unique effect of FK506 on the augmentation of T cell apoptosis is probably one of the mechanisms explaining its beneficial effect on aly autoimmune pancreatitis.     

NGP1-01, a lipophilic polycyclic cage amine, is neuroprotective in focal ischemia

NGP1-01, a member of the pentacycloundecylamine cage compound family, was recently shown to exhibit both NMDA receptor channel blocking and L-type calcium channel antagonism activity. In the present study, focal ischemia was induced in mice by permanent middle cerebral artery occlusion (MCAO) to test for potential neuroprotective properties of the compound. In female CD-1 mice injected 30 min before MCAO, NGP1-01 (20 mg/kg) reduced infarct area by 42.6% (P < 0.05) compared to vehicle-treated controls as visualized by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Concomitantly, NGP1-01 reduced brain swelling by 78.3% (P < 0.001), compared to vehicle (DMSO) treated controls. These data identify NGP1-01 and related compounds as potential lead structures to develop neuroprotective compounds based on a dual mechanism of action.     

Analysis of FK506-mediated protection in an organotypic model of spinal cord damage: heat shock protein 70 levels are modulated in microglial cells

Functional loss after spinal cord injuries is originated by primary and secondary injury phases whose underlying mechanisms include massive release of excitatory amino acids to cytotoxic levels that contribute to neural death. Attenuation of this excitotoxicity is a key point for improving the functional outcome after injury. One of the drugs with potential neuroprotective actions is FK506, a molecule widely used as an immunosuppressant. FK506 may exert neuroprotection via inhibition of calcineurin by binding the FKBP12, or by binding other immunophilins such as FKBP52, leading to modulation of heat shock proteins (Hsp) 90 and 70. In the present study, we used an in vitro model of organotypic culture of rat spinal cord slices to assess whether FK506 is able to protect them against glutamate excitotoxicity. The results showed that FK506 promoted a significant protective effect on the spinal cord tissue at concentrations of 50 and 100 nM. Hsp70 induction was restricted to microglial cells in spinal cord slices treated with either glutamate or FK506. In contrast, the combination of both agents led to a transient reduction in Hsp70 levels in parallel to a marked reduction in IL-1beta precursor production by glial cells. The use of geldanamycin, which promotes persistent induction of Hsp70 in these cells as well as in motoneurons, did not produce tissue neuroprotection. These observations suggest that FK506 might protect spinal cord tissue by targeting on microglial cells and that transient downregulation of Hsp70 on these cells after excitotoxicity is a relevant mechanism of action of FK506.     

Neuroprotective effects of FK506 against excitotoxicity in organotypic hippocampal slice culture

FK506 has been originally classified as an immunosuppressant and is known to exhibit neurotrophic actions in vitro and protective effects on some neurological conditions. We investigated the neuroprotective effects of FK506 on kainic acid (KA)-induced neuronal death in organotypic hippocampal slice cultures (OHSCs). After an 18 h KA (5 μM) treatment, significantly neuronal death was detected in the CA3 region using propidium iodide staining. However, neuronal death was significantly prevented at 24 and 48 h after treatment with 0.1 μM FK506. Using cresyl violet staining, we also observed that an increased number of CA3 neurons survived in the 0.1 μM FK506 group compared to the KA only group. Based on the results of the Western blot analysis, the expressions of 5-lipoxygenase and caspase-3 were reduced 24 h after 0.1 μM FK506 treatment. The levels of superoxide dismutase (SOD) and phospho-Akt expression were increased by treatment with 0.1 μM FK506. These results suggest that FK506 may have a positive role in protecting neurons against cell death in the KA injury model of OHSCs.     

Interleukin-6/soluble interleukin-6 receptor complex reduces infarct size via inhibiting myocardial apoptosis

Apoptosis of cardiomyocytes plays an important role in reperfusion injury following myocardial infarction. Conversely, interleukin-6 (IL-6)--a potent cytokine--inhibits myeloma cell apoptosis by activating GP130 through the IL-6 receptor (IL-6R). We hypothesized that the IL-6/soluble IL-6R complex can inhibit myocardial apoptosis, and limit infarct size in reperfused acute myocardial infarction. Anesthetized rats were randomly divided into five groups: sham, coronary occlusion and reperfusion rats administered IL-6/soluble IL-6R complex, IL-6 alone, soluble IL-6R (sIL-6R) alone, or a control vehicle. Rats were subjected to 30 min occlusion of the left coronary artery followed by 3 h reperfusion. After reperfusion, the hearts were excised. For detection and quantification of apoptosis, gel electrophoresis of extracted genomic DNA and TUNEL method of paraffin sections were performed. The percentage of the infarct area was measured using tetrazolium chloride staining. The cardiomyocyte apoptosis analysis revealed that apoptosis in the reperfused myocardium was inhibited only in the complex group. Furthermore, the percentage of the infarct area out of the area at risk was remarkably reduced in the complex group (23.8+/-1.8%), compared with that in the vehicle (37.9+/-3.7%), the IL-6 (40.7+/-1.0%), or the sIL-6R (37.5+/-2.4%) groups (P=0.0002). No significant differences were observed among the vehicle, IL-6, and sIL-6R groups. The IL-6/soluble IL-6 receptor complex inhibits cardiomyocyte apoptosis in reperfused acute myocardial infarction. It possibly reduces irreversible reperfusion injury.     

Hippocampal CA1 neuron survival and cytosolic FKBP12, the 12 kDa FK506-binding protein,after ischemia and tacrolimus treatment in gerbils

To assess the neuroprotective effect of tacrolimus (FK506) in transient forebrain ischemia models in gerbils, 10.0 mg/kg of FK506 was injected intraperitoneally immediately following reperfusion and at intervals of 1, 3, 6, 9, and 12 h after reperfusion. FK506 produced a significant neuroprotective effect for up to 6 h after 5 min of ischemia. Immunoblot and immunohistochemistry revealed that the amount of FKBP12, the 12-kDa FK506-binding protein, in the cytosol remained unchanged until 12 h after reperfusion. Translocation of FKBP12 from the nucleus to the cytosol was not observed until 24 h after reperfusion. Administration of FK506 did not appear to induce the cytosolic increase in FKBP12. In this study, no correlation was apparent between the post-ischemic therapeutic efficacy of FK506 and the post-ischemic changes in the cytosolic FKBP12.     

Neuroprotective and antioxidant properties of FKBP-binding immunophilin ligands are independent on the FKBP12 pathway in human cells

We focused on immunophilin isoforms in order to clarify the neuroimmunophilins which were identified as targets for the immunophilin ligands to elicit a neuroprotective effect. Although the expressions of five FK506-binding protein (FKBP) mRNAs were detected in both SH-SY5Y (human neuroblastoma) and U251 (human glioma) cell lysates, the FKBP12 mRNA expression was detected in only the SH-SY5Y cells, and not the U251 cells. However, we found that the SH-SY5Y and the U251 cells were equipotent in the intensity of cellular protection of FK506 (an immunosuppressive immunophilin ligand) and GPI1046 (a non-immunosuppressive FK506 analog), indicating that the protective effect and glutathione activation of FK506 and GPI1046 had little need to bind FKBP12. Therefore, we conclude that the neuroprotective and antioxidant properties of immunophilin ligands are independent on the FKBP12 pathway.