Erythropoietin and cerebral vascular protection： role of nitric oxide

Cerebral vasospasm after subarachnoid hemorrhage (SAH) is a major clinical problem causing cerebral ischemia and infarction. The pathogenesis of vasospasm is related to a number of pathological processes including endothelial damage and alterations in vasomotor function leading to narrowing of arterial diameter and a subsequent decrease in cerebral blood flow. Discovery of the tissue protective effects of erythropoietin (EPO) stimulated the search for therapeutic application of EPO for the prevention and treatment of cerebrovascular disease. Recent studies have identified the role of EPO in vascular protection mediated by the preservation of endothelial cell integrity and stimulation of angiogenesis. In this review, we discuss the EPO-induced activation of endothelial nitric oxide (NO) synthase and its contribution to the prevention of cerebral vasospasm.     

Erythropoietin administration in vivo increases vascular nitric oxide synthase expression

This study was designed to determine whether recombinant human erythropoietin (rHuEpo) administration increases vascular nitric oxide (NO) production in healthy rats. We hypothesized that rHuEpo hypertension is associated with increased endothelial expression of nitric oxide synthase and augmented NO-dependent vasodilation. Male rats were instrumented with pulsed Doppler flow probes around their ascending aorta and with arterial and femoral catheters. Rats were treated for 14 days with rHuEpo (2 U/d) or vehicle. rHuEpo elevated hematocrit and increased mean arterial pressure (142 +/- 3 versus 116 +/- 4 mm Hg). Thoracic aorta segments from rHuEpo rats had a modest increase in NO-dependent relaxation assessed by acetylcholine (10(-10) to 10(-5) mol/L) relaxation of phenylephrine (PE) (10(-6) mol/L) contracted arteries. Relaxation to NO-donor, s-nitrosyl acetylpenicillamine, and PE contraction were not different from control arteries. The NO synthase inhibitor, N-omega-nitro-L-arginine, increased blood pressure and total peripheral resistance more in rHuEpo rats at both 10 and 30 mg/kg. NOS expression in rHuEpo aorta and plasma NOx concentrations were increased compared with control. Thus, it appears that vascular eNOS expression is increased and causes basal vasodilation in rHuEpo hypertensive rats.     

Erythropoietin prevents haloperidol treatment-induced neuronal apoptosis through regulation of BDNF.

Functional alterations in the neurotrophin, brain-derived neurotrophic factor (BDNF) have recently been implicated in the pathophysiology of schizophrenia. Furthermore, animal studies have indicated that several antipsychotic drugs have time-dependent (and differential) effects on BDNF levels in the brain. For example, our previous studies in rats indicated that chronic treatment with the conventional antipsychotic, haloperidol, was associated with decreases in BDNF (and other neurotrophins) in the brain as well as deficits in cognitive function (an especially important consideration for the therapeutics of schizophrenia). Additional studies indicate that haloperidol has other deleterious effects on the brain (eg increased apoptosis). Despite such limitations, haloperidol remains one of the more commonly prescribed antipsychotic agents worldwide due to its efficacy for the positive symptoms of schizophrenia and its low cost. Interestingly, the hematopoietic hormone, erythropoietin, in its recombinant human form rhEPO has been reported to increase the expression of BDNF in neuronal tissues and to have neuroprotective effects. Such observations provided the impetus for us to investigate in the present study whether co-treatment of rhEPO with haloperidol could sustain the normal levels of BDNF in vivo in rats and in vitro in cortical neuronal cultures and further, whether BDNF could prevent haloperidol-induced apoptosis through the regulation of key apoptotic/antiapoptotic markers. The results indicated that rhEPO prevented the haloperidol-induced reduction in BDNF in both in vivo and in vitro experimental conditions. The sustained levels of BDNF in rats with rhEPO prevented the haloperidol-induced increase in caspase-3 (p<0.05) and decrease in Bcl-xl (p<0.01) protein levels. Similarly, in vitro experiments showed that rhEPO prevented (p<0.001) the haloperidol-induced neuronal cell death as well as the decrease in Bcl-xl levels (p<0.01). These findings may have significant implications for the development of neuroprotective strategies to improve clinical outcomes when antipsychotic drugs are used chronically.     

Acetyl-L-carnitine cytoprotection against 1-methyl-4-phenylpyridinium toxicity in neuroblastoma cells

Acetyl-L-carnitine (ALCAR) plays an integral role in the transport of long chain fatty acids across the inner mitochondrial membrane for oxidative phosphorylation. In non-human primates, administration of ALCAR was reported to prevent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurological injury to the substantia nigra. The present study investigates the effects of ALCAR against the toxicity of 1-methyl-4-phenylpyridinium (MPP(+)), the neurotoxic metabolite of MPTP, in murine brain neuroblastoma cells. MPP(+), a potent mitochondrial toxin, induced a dose-dependent reduction in mitochondrial oxygen consumption and cell viability, corresponding to an accelerated rate of cellular glucose utilization. Treatment with ALCAR, but not L-carnitine, prevented MPP(+) toxicity and partially restored intracellular ATP concentrations, but did not reverse the MPP(+)-induced loss of mitochondrial oxygen consumption. These data indicate that protective effects are independent of oxidative phosphorylation. ALCAR had a substantial glucose sparing effect in both controls and MPP(+)-treated groups, demonstrating a potential role in enhancing glucose utilization through glycolysis. Antagonizing the entry of fatty acids into the mitochondria, with either insulin or malonyl CoA, did not interfere with ALCAR protection against MPP(+). On the contrary, insulin potentiated the protective effects of ALCAR. In conclusion, these data indicate that ALCAR protects against MPP(+) toxicity, independent of mitochondrial oxidative capacity or beta-oxidation of fatty acids. In contrast, the protective effects of ALCAR appear to involve potentiation of energy derived from glucose through anaerobic glycolysis.     

Ascorbic acid-dependent cytoprotection of ovarian cells by leukocyte and nonleukocyte peroxidases

Luteal cells contain high levels of ascorbic acid that is secreted by stimulation with agents like luteinizing hormone (LH) and prostaglandin F2 alpha (PGF2 alpha). One role for interstitial ascorbic acid, we propose, may be the detoxification of H2O2 by regeneration of catalytically active peroxidase. By serving as a preferred secondary substrate, ascorbic acid regenerates the catalytically active peroxidase that is inhibited irreversibly by H2O2 alone. To test this hypothesis, luteal cells were incubated in the absence and presence of peroxidases and H2O2, and the maximal cyclic AMP and steroidogenic response to LH was examined. In luteal cells, H2O2 is known to severely inhibit LH-sensitive cyclic AMP accumulation and steroidogenesis, and the addition of lactoperoxidase, myeloperoxidase, eosinophil peroxidase, or ascorbic acid (1 mM) alone had no effect on these responses to H2O2. However, co-incubation of ascorbic acid and the peroxidases completely reversed the inhibition of cyclic AMP accumulation and steroidogenesis produced by H2O2. These findings and the results that show direct oxidation of ascorbic acid in the presence of peroxidase and H2O2, but not with H2O2 alone, support the conclusion that ascorbic acid released from cells may detoxify H2O2 by regenerating the catalytically active state of peroxidases.     

Erythropoietin prevents vascular inflammation and oxidative stress in subtotal nephrectomized rat aorta beyond haematopoiesis

1. Recombinant human erythropoietin (rHuEPO) has been used for the management of renal anaemia. Recent studies suggest pleiotropic properties of rHuEPO in various tissues. The aim of the present study was to investigate the vasoprotective effects of rHuEPO in renal failure rats. 2. Rats subjected to 5/6 and 17/18 nephrectomy (5/6Nx and 17/18Nx rats, respectively) were treated with rHuEPO (75 U/kg, s.c.) three times a week for 2 weeks. 3. Administration of rHuEPO to 5/6Nx or 17/18Nx rats had no effect on systolic blood pressure or decreased haematocrit. However, rHuEPO treatment normalized proteinuria and creatinine clearance in 5/6Nx, but not in 17/18Nx, rats. 4. Vasodilation in response to acetylcholine in aortic rings was impaired in 5/6Nx and 17/18Nx rats and improved by rHuEPO in both groups. Immunohistochemical analysis revealed that macrophage infiltration into adventitial areas and the expression of osteopontin were enhanced in aortas from 5/6Nx and 17/18Nx rats, but that rHuEPO suppressed these effects. In addition, rHuEPO attenuated medial hyperplasia and NADPH oxidase‐derived superoxide production in 5/6Nx and 17/18Nx rats. 5. Activation of the Akt signalling pathway was evident in rHuEPO‐treated rats as the increased expression of phosphorylated Akt and glycogen synthase kinase‐3β. Treatment with rHuEPO restored the expression of phosphorylated endothelial nitric oxide synthase in the aorta and urinary excretion of NO_x in nephrectomized rats. 6. These results suggest that a low dose of rHuEPO results in the normalization of endothelial function, vascular inflammation and oxidative stress in rats with renal ablation beyond haematopoiesis. In addition, these vasoprotective effects are observed even in a state of deteriorating renal dysfunction.     

重组人促红细胞生成素对卵巢癌细胞EPOR表达及肿瘤增殖的影响

目的通过检测卵巢癌细胞中的促红细胞生成素受体（EPOR）表达，以及r—HuEPO对卵巢癌细胞增值的影响，探讨r—HuEPO在癌性贫血治疗中的意义。方法RT—PCR方法检测人卵巢癌细胞株HO-8910EPOR mRNA的表达及卵巢癌细胞增值影响。结果卵巢癌细胞表面有EPOR的表达。r—HuEPO干预后卵巢癌细胞增值受到抑制（P〈0．01）。结论EPOR在卵巢癌细胞株HO8910有表达，r—HuEPO可抑制卵巢癌细胞的增值发生，提示r—HuEPO可用于卵巢癌伴发的贫血的治疗。     

Clonidine accumulation in human neuronal cells

After transport across several epithelial barriers including the blood–brain barrier, clonidine interacts with ₂-adrenergic receptors and imidazoline binding sites in the brain. We hypothesized that neuronal cells take up clonidine thereby removing the drug from the extracellular fluid compartment. Uptake of [³H]clonidine into SH-SY5Y neuroblastoma cells was linear for up to 1 min, unaffected by inside directed Na⁺ or Cl⁻ gradients but strongly inhibited by an outside pH of 6.0. The cells accumulated [³H]clonidine 50–70-fold uphill against a concentration gradient. Unlabeled clonidine, guanabenz, imipramine, diphenhydramine, maprotiline, quinine and the endogenous monoamine phenylethylamine (2 mM) strongly inhibited the [³H]clonidine uptake by 60–95%. Tetraethylammonium, choline and N-methyl-4-phenylpyridinium had no effect. The accumulation at pH 7.5 was saturable with an apparent Michaelis–Menten constant (K_t) of 0.7 mM. We conclude that SH-SY5Y cells not only bind clonidine to extracellular receptors but also take up the drug rapidly by a specific and concentrative mechanism.     

Immunocytochemical localization of beta-citryl-L-glutamate in primary neuronal cells and in the differentiation of P19 mouse embryonal carcinoma cells into neuronal cells

The immunocytochemical localization of beta-citryl-L-glutamate (beta-CG) in primary neuronal cells and in the differentiation of P19 cells was examined. 1: Cells with the morphological features of neurons in the primary culture were specifically stained with the anti-beta-CG antibody both in neurites and in the cell body. 2: The neuronal cells differentiated from P19 cells were distinctly stained with the anti-beta-CG antibody both in neurites and in the cell body, while the non-neuronal cells were not. 3: The concentration of beta-CG was low in the P19 cells, but increased significantly with the differentiation of P19 cells into neurons. It was shown that beta-CG was localized exclusively in neurons. These findings suggest that beta-CG plays functional roles in the differentiation and growth of neuron.     

Ulcer formation and cytoprotection by acetazolamide

Acetazolamide, a carbonic anhydrase inhibitor, was administered orally and subcutaneously to rats. Acetazolamide increased the gastric ulcerogenicity of indomethacin, but inhibited gastric ulcers produced by acidified aspirin. When administered alone to fasted rats, it did not produce gastric ulcers. Acetazolamide was also cytoprotective for the stomach (it reduced dose dependently the number of gastric necrotic lesions caused by absolute ethanol given orally) and for the small intestine (it prevented dose dependently intestinal lesions produced by administration of a high dose of indomethacin). Acetazolamide did not prevent the antiulcer effect of PGE2 (against aspirin-induced ulcers) nor the cytoprotective effect of 16,16-dimethyl PGE2 (against ethanol-induced gastric lesions). The degree of gastric cytoprotection increased with time after a single administration of acetazolamide; the optimal effect occurred 60 and 90 min after oral and subcutaneous administration, respectively. Pretreatment with indomethacin completely prevented the cytoprotective effect of acetazolamide; this suggests that the cytoprotective effect may be mediated by endogenous release of prostaglandins by the stomach. All the effects of acetazolamide reported here were observed after either oral or subcutaneous administration. The mechanism by which acetazolamide influences ulcer formation and is cytoprotective is unknown.     

促红细胞生成素抗神经元凋亡的信号传导通路

凋亡是许多神经系统疾病中神经元损伤的主要死亡形式,促红细胞生成素(erythropoietin,Epo)可促进造血前体细胞的增殖和分化,调节红细胞的生成。近来研究发现,Epo及其功能受体(erythropoietinreceptor,Epo-R)在中枢神经系统中也有表达,且对神经元的凋亡有保护作用,该文就Epo抗神经元凋亡的信号传导通路方面略做一综述。     

Erythropoietin: new horizon in cardiovascular medicine

Erythropoietin (EPO), a renal cytokine, regulates proliferation, differentiation and maturation of erythroid cells. Recombinant human erythropoietin (rH-EPO) is well known to correct anemia in patients with chronic renal failure undergoing dialysis. Recent studies have reported several non-hematopoietical effects of EPO. Erythropoietin receptors have been discovered in a variety of tissues, including the cardiovascular system. Recently published data including recent patent documented an enhancement of cardiac function in patients with heart failure receiving EPO treatment. Furthermore, experiments carried out in animal models of ischemia/reperfusion (IR) injury have shown a significant reduction in infarct size following EPO treatment. Other beneficial effects of EPO are related to its pro-angiogenic action on endothelial cells, which might be of potential value in patients with ischemic heart disease. Taken together, these findings suggest that EPO may be clinically useful as an adjunct in the treatment of different cardiovascular conditions, besides the simple correction of anemia. This review will focus on the pleiotropic effects of EPO in the cardiovascular system and its promising novel applications.     

Organoprotection and cytoprotection of histamine differ in rats

Aims  The aim of the present study was to compare the organoprotective (in vivo) and cytoprotective (in vitro) effects of histamine. Methods  In vivo, gastric mucosal damage was produced by intragastric (ig) administration of 1 ml 96% ethanol (EtOH) in Sprague-Dawley rats. The animals were sacrificed 1 h after EtOH administration, when the gastric mucosal damage was measured. Histamine was given subcutaneously (sc) 30 min before administration of EtOH with and without PGI₂Na (5 μg/kg sc). Gastric acid secretion was also measured 1 h after pylorus ligation in control (saline-), histamine- and PGI₂-treated animals. The affinity, intrinsic activity curves and the values of pD₂ and pA₂ were determined in EtOH-treated and in PGI₂-treated animals. For the in-vitro studies, a mixed population of rat gastric mucosal cells was isolated by pronase digestion. Cells were preincubated for 60 min with histamine (10^♪−8-10⁻⁶ mol/L) with or without PGI₂Na (10⁻⁴ mol/L). At the end of this incubation period, cells were treated with 15% EtOH with or without 10⁻⁶-10³ mol/L indomethacin (IND) for 5 min. Cell viability was tested by trypan blue exclusion test and succinic dehydrogenase activity. Results  

Adenosine, oxidative stress and cytoprotection

Adenosine, a metabolite of ATP, serves a number of important physiological roles in the body. These actions contribute to sedation, bradycardia, vasorelaxation, inhibition of lipolysis and regulation of the immune system and are mediated, in part, through activation of three distinct adenosine receptor (AR) subtypes. To date, four receptor types have been cloned: A1, A2A, A2B and A3. It is becoming increasing clear that adenosine contributes significantly to cytoprotection, a function mediated principally by the A1AR and A3AR. In this review, we survey the literature on the role of adenosine and the mechanisms underlying cytoprotection and ischemic preconditioning, a process characterized by cytoprotection derived from repeated brief ischemic challenges. An important recent observation is that the expression of several AR subtypes could be regulated by oxidative stress to provide a greater cytoprotective role. Thus, like other proteins known to be regulated during ischemia, the A1AR and A3AR can be considered as being inducible receptors.     

Cyanide-induced neurotoxicity: calcium mediation of morphological changes in neuronal cells

Calcium channel blockade decreases the elevation of brain calcium as well as the tremors produced by cyanide in mice. To determine if cyanide-induced morphological changes could also be inhibited by calcium channel blockade, the effect of diltiazem was studied in cultured rat pheochromocytoma (PC12) cells, a neuronal model. Incubation with KCN (1 to 10 mM for 1 to 2 hr) caused depletion of secretory granules, alignment of remaining granules along the plasma membrane, and mitochondrial swelling. All these effects were inhibited by pretreatment with 0.01 mM diltiazem. Scanning electron microscopy revealed that cyanide (1 to 10 mM for 1 to 2 hr) produced loss of microvilli and bleb formation in PC12 cells. These changes were partially inhibited by preincubation with 0.01 mM diltiazem. Incubation of cells with 10 mM cyanide increased release of lactic dehydrogenase (LDH) into the culture media at 60 and 120 min. A decrease in cell viability, as determined by trypan blue dye exclusion, paralleled the release of LDH. At 120 min of cyanide incubation, 24% of the cells excluded dye. Both the release of LDH and decreased cell viability were attenuated by pretreatment with diltiazem. The results indicate that the influx of extracellular calcium is an important factor mediating cyanide-induced morphologic changes in neuronal cells.     

骨髓间充质干细胞向神经细胞分化及移植研究进展

骨髓间充质干细胞(mesenchymalstemcells,MSCs)又称骨髓基质细胞(bonemarrowstromal cells,BMSCs),是干细胞研究的又一新的发现,是目前干细胞研究的热点和前沿。MSCs具有方便获取、易于分离培养和扩增纯化,在体外培养条件下多代扩增后仍保持多向分化潜能的特点,且遗传背景稳定,