Antioxidant status in delayed healing type of wounds

This investigation studied the contribution of antioxidants in delaying healing in excision cutaneous wounds (8 mm) in diabetic, aged and immunocompromised animals. Skin levels of catalase, glutathione (GSH), ascorbic acid (AA) and vitamin E in streptozotocin-induced diabetic rat were lower as compared to nondiabetics. The 7-d wound tissue of diabetic rats showed an increased vitamin E level along with depleted GSH content. In aged rats (18 months old), higher levels of skin superoxide dismutase (SOD), glutathione peroxidase (Gpx) and thiobarbituric acid reactive substances (TBARS) and lower levels of catalase and GSH were found as compared to their values in young rats (3-4 months old). The levels of SOD, GPx, catalase, AA, GSH and vitamin E in 7-d wound tissue of aged rats were significantly lower in comparison to those in young rats. However, TBARS were elevated in these wound tissues. The non-wounded skin of immunocompromised (athymic) mice showed lower levels of SOD, catalase, and TBARS and higher GSH and GPx levels in comparison to those present in normal mouse skin. Surprisingly, the analysis of 7-d wound tissue showed higher levels of SOD, catalase, GPx, and GSH and lower TBARS level in athymic mice compared to the wound tissue of normal mice. Thus low levels of antioxidants accompanied by raised levels of markers of free radical damage play a significant role in delaying wound healing in aged rats. In diabetic rats reduced glutathione levels may have a contributory role in delaying the healing process. However, in immunocompromised mice the antioxidant status following injury showed an adapted response.     

Preconditioning with ozone/oxygen mixture induces reversion of some indicators of oxidative stress and prevents organic damage in rats with fecal peritonitis

Objective and design  Reactive oxygen and nitrogen species are involved in the pathogenesis of sepsis syndrome with peritonitis and the septic shock. The aim of this study was to determine whether ozone oxidative preconditioning (OOP) may exert beneficial effects in the prevention and treatment of sepsis syndrome in rats inoculated by the intraperitoneal route (i.p.) with fecal material and also to determine if antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) may exert protective effects against this systemic inflammatory disorder. Materials and methods  Male Wistar rats were used. SOD and GPx activities were determined in erythrocytes. Thiobarbituric acid reactive substances (TBARS) content as biomarkers of oxidative stress, alanine amino transferase (ALT), aspartate amino transferase (AST) and creatinine (CRE) were measured in blood serum and myeloperoxidase (MPO) in lung tissue as markers of organs damage. Results  In rats submitted to OOP, SOD and GPx activities were significantly increased and it was accompanied by significant decrease of TBARS content in blood serum. OOP also significantly reduced levels of ALT, AST and CRE in blood serum as well as MPO in rat lung. Conclusion  The results support the important role of SOD and GPx in the protective effects of OOP against organ damage induced by fecal peritonitis in rats.     

Donor age negatively impacts adipose tissue-derived mesenchymal stem cell expansion and differentiation

Background

Human adipose tissue is an ideal autologous source of mesenchymal stem cells (MSCs) for various regenerative medicine and tissue engineering strategies. Aged patients are one of the primary target populations for many promising applications. It has long been known that advanced age is negatively correlated with an organism’s reparative and regenerative potential, but little and conflicting information is available about the effects of age on the quality of human adipose tissue derived MSCs (hAT-MSCs).

Methods

To study the influence of age, the expansion and in vitro differentiation potential of hAT-MSCs from young (<30 years), adult (35-50 years) and aged (>60 years) individuals were investigated. MSCs were characterized for expression of the genes p16^INK4a and p21 along with measurements of population doublings (PD), superoxide dismutase (SOD) activity, cellular senescence and differentiation potential.

Results

Aged MSCs displayed senescent features when compared with cells isolated from young donors, concomitant with reduced viability and proliferation. These features were also associated with significantly reduced differentiation potential in aged MSCs compared to young MSCs.

Conclusions

In conclusion, advancing age negatively impacts stem cell function and such age related alterations may be detrimental for successful stem cell therapies.     

硒化合物对人脐带间充质干细胞ROS和NO生成及NOS活性的影响

目的:通过观察不同的硒化合物对人脐带间充质干细胞(MSCs)一氧化氮(NO)、活性氧(ROS)的生成及一氧化氮合酶(NOS)活性的影响,初步探讨硒的免疫调节作用机制。方法:在MSCs培养的过程中,分别添加0.1μmol/L和0.5μmol/L亚硒酸钠[Se(Ⅳ)]、硒代胱氨酸(Se-Cys)、纳米硒(Nano-Se),采用硝酸还原酶法检测24 h和48 h MSCs NO和NOS活性的变化,通过DCFH-DA荧光探针检测细胞内ROS。结果:在24 h,对照组、0.1μmol/L和0.5μmol/L Se(Ⅳ)处理MSCs的NO水平分别是(18.13±6.80)μmol/L、(20.93±5.68)μmol/L和(16.73±5.03)μmol/L。在48 h,0.1μmol/L和0.5μmol/L Se(IV)处理MSCs的NO水平分别是(17.20±9.11)μmol/L(P0.05)和(9.98±4.35)μmol/L(P0.01),明显低于对照组的(26.23±4.35)μmol/L。Se(IV)对ROS和NOS有明显的抑制作用,Se-Cys和Nano-Se对NO、ROS的生成和NOS活性没有明显影响。结论:Se(Ⅳ)对MSCs NO、ROS的生成和NOS活性有明显的抑制效应。     

Selenium supplementation restores the antioxidative capacity and prevents cell damage in bone marrow stromal cells in vitro

Bone marrow stromal cells (BMSCs) and other cell populations derived from mesenchymal precursors are developed for cell-based therapeutic strategies and undergo cellular stress during ex vivo procedures. Reactive oxygen species (ROS) of cellular and environmental origin are involved in redox signaling, cumulative cell damage, senescence, and tumor development. Selenium-dependent (glutathione peroxidases [GPxs] and thioredoxin reductases [TrxRs]) and selenium-independent (superoxide dismutases [SODs] and catalase [CAT]) enzyme systems regulate cellular ROS steady state levels. SODs process superoxide anion to hydrogen peroxide, which is subsequently neutralized by GPx and CAT; TrxR neutralizes other ROS, such as peroxinitrite. Primary BMSCs and telomerase-immortalized human mesenchymal stem cells (hMSC-TERT) express GPx1-3, TrxR1, TrxR2, SOD1, SOD2, and CAT. We show here that in standard cell cultures (5%-10% fetal calf serum, 5-10 nM selenite), the activity of antioxidative selenoenzymes is impaired in hMSC-TERT and BMSCs. Under these conditions, the superoxide anion processing enzyme SOD1 is not sufficiently stimulated by an ROS load. Resulting oxidative stress favors generation of micronuclei in BMSCs. Supplementation of selenite (100 nM) restores basal GPx and TrxR activity, rescues basal and ROS-stimulated SOD1 mRNA expression and activity, and reduces ROS accumulation in hMSC-TERT and micronuclei generation in BMSCs. In conclusion, BMSCs in routine cell culture have low antioxidative capacity and are subjected to oxidative stress, as indicated by the generation of micronuclei. Selenite supplementation of BMSC cultures appears to be an important countermeasure to restore their antioxidative capacity and to reduce cell damage in the context of tissue engineering and transplantation procedures.     

Changes of HSP72-expression in leukocytes are associated with adaptation to exercise under conditions of high environmental temperature

Overexpression of the heat shock protein HSP72 provides thermotolerance. We asked if two consecutive endurance runs 1 week apart (CR1, CR2) and additional environmental heat stress affect HSP72-expression in leukocytes of nonheat-acclimated endurance athletes. Twelve subjects were allocated randomly into two groups. Group HH completed both runs at 28 degrees C ambient temperature, and group NH performed CR1 at 18 degrees C and CR2 at 28 degrees C. HSP72-expression was determined by flow cytometry and RT-PCR before and 0, 24, and 48 h after exercise. Additionally, post-exercise cells were exposed to in vitro heat shock (HS; 2 h, 42 degrees C). The prolonged, high HSP72 protein level after CR1 in HH compared with NH may reflect thermotolerance induced by endurance exercise at high ambient temperature. Adaptation of cardiocirculatory/thermoregulatory capacity after CR2 in HH went along with a more rapid down-regulation of HSP72 compared with CR1. HSP72 mRNA demonstrated temperature-related changes after exercise. The reduced HS response in vitro after CR2 may represent exercise-related adaptation mechanisms. HSP72 concentrations in leukocytes may indicate previous exercise- and temperature-related stress conditions and adaptation in immunocompetent cells.     

Nuclear Ago2/HSP60 contributes to broad spectrum of hATSCs function via Oct4 regulation

AIMS: Argonaute2 (Ago2) plays a fundamental role in microRNA-mediated gene regulation through its intrinsic endonuclease activity. In this study we demonstrate the novel functions and molecular mechanisms by which nuclear Ago2 directly regulates HSP (heat shock protein) 60 expression and stem cell self-renewal. HSP60 is a crucial regulator of ROS (reactive oxygen species), senescence, and apoptotic cell death in several tissues and cell types. RESULTS: HSP60 is regulated via inactivation of p38/JNK and p53 and binds directly to the regulatory regions of the TERT, c-myc, GPx3, p53, and STAT3 genes. Using HSP60 CHIP-PCR experiments, we show that HSP60 binds directly to the Oct4 and Nanog genes and directly regulates Oct4 and other stemness genes involved in human adipose tissue-derived stem cell (hATSC) differentiation. HSP60 also positively regulates ROS-scavenging factors, including GPx3 and TXNL1, which directly modulate cytosolic ROS in hATSCs. Moreover, our study shows that Oct4 regulates HSP60 expression and controls hATSC survival and self-renewal after binding to the HSP60 gene. Furthermore, HSP60-mediated regulation of Oct4 contributes to neuronal and endodermal β-cell differentiation of hATSCs in vitro and in vivo and downregulates mesoderm-specific gene expression. INNOVATION AND CONCLUSION: We show that increased levels of Ago2 or HSP60 effectively induce nuclear localization of HSP60, which directly controls Oct4, c-Myc, p53, TERT, and STAT3 for transdifferentiation programs. Collectively, we suggest a novel model in which nuclear Ago2 controls HSP60 in hATSCs.     

人骨髓胚胎样干细胞向多核肌纤维诱导分化的研究

目的: 比较骨髓来源的胚胎样干细胞(ELSCs)与间充质干细胞(MSCs)的体外成肌分化能力。方法:采用胚胎干细胞扩增用的无血清Knockout-DMEM培养基在明胶包被过的培养瓶中培养人骨髓单个核细胞以分离ELSCs,传统方法从相同骨髓中分离MSCs,倒置相差显微镜下观察细胞形态特征,采用免疫荧光染色鉴定多潜能抗原标志的表达。成肌分化液分别培养ELSCs和MSCs,采用免疫染色法检测肌纤维特异性抗原标志肌球蛋白重链(MHC)、成肌素(myogenin)和MyoD蛋白的表达, RT-PCR检测MHC、myogenin和MyoD mRNA的表达,计算MHC阳性肌纤维的比例以比较ELSCs与MSCs的体外成肌分化能力。结果:无血清培养基可从骨髓中分离到弱表达多潜能抗原标志Oct-4、Nanog-3和Sox-2的ELSCs,体积较小,形态纤细均一,在形态方面不同于相同骨髓来源的MSCs,后者不表达多潜能抗原标志。在成肌分化液中培养,ELSCs和MSCs均可被诱导为在蛋白和mRNA水平表达MHC和myogenin的多核肌纤维,但诱导培养10 d时,ELSCs的MHC蛋白阳性肌纤维的比例为(25.7±4.1)%,MSCs为(15.8±7.6)%,ELSCs的成肌分化能力明显高于MSCs(P<0.05)。 结论:骨髓ELSCs能被诱导为多核肌纤维,并具有比来自相同骨髓的MSCs更强的成肌分化能力,ELSCs是肌病治疗更理想的种子细胞。     

Human umbilical cord blood-derived mesenchymal stem cells undergo cellular senescence in response to oxidative stress

Since human mesenchymal stem cells (MSCs) are therapeutically attractive for tissue regeneration and repair, we examined the physiological responses of human umbilical cord blood-derived MSCs (hUCB-MSCs) to genotoxic stress. We found that that sublethal doses of reactive oxygen species (ROS) and ionizing radiation cause DNA damage and reduce DNA synthesis and cell proliferation in hUCB-MSCs, resulting in cellular senescence. In contrast, these physiological changes were limited in human fibroblast and cancer cells. Our data show that reduced activities of antioxidant enzymes, which may occur due to low gene expression levels, cause hUCB-MSCs to undergo cellular senescence in response to oxidative stress and ionizing radiation. Resistance of hUCB-MSCs to oxidative stresses was restored by increasing the intracellular antioxidant activity in hUCB-MSCs via exogenous addition of antioxidants. Therefore, the proliferation and fate of hUCB-MSCs can be controlled by exposure to oxidative stresses.     

传代培养人胚胎生殖干细胞的生物学鉴定

目的：通过对传代培养至第5代细胞分子标记物的表达分析,鉴定传代培养的细胞为人胚胎生殖干细胞（embrgonic germ stem cells,EGCs）。方法：取5～10周人胚胎的生殖腺嵴和肠背系膜,进行组织块体外培养人ECA2s,挑选传代培养至第5代生长良好的细胞,采用免疫细胞化学法分别检测SSEA-1、SSEA-3、Oct-4、hTERT的表达。结果：组织块体外培养得到的细胞,经免疫细胞化学法显示的单个细胞或细胞集落,均阳性表达SSEA-1、SSEA-3、Oct-4、hTERT。结论：组织块体外传代培养第5代所获得的细胞,为未分化的人EGCs。     

氧化低密度脂蛋白通过氧化应激诱导小鼠造血干细胞衰老

 目的 探讨氧化低密度脂蛋白（ox-LDL）体外诱导造血干细胞(HSCs)衰老的可能机制。方法 用免疫磁性分选法分离纯化小鼠HSC,与ox-LDL共培养,采用β-半乳糖苷酶（SA-β-Gal）染色检测衰老HSC,流式细胞术检测HSC细胞周期分布,混合集落培养(CFU-Mix)检测HSC混合集落形成能力。流式细胞术和免疫荧光检测HSC产生活性氧（ROS）的量,酶学比色法检测HSC培养上清液超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）及丙二醛（MDA）含量。Southern blot和TRAP-PCR法检测HSC端粒长度和端粒酶活性。结果 ox-LDL诱导HSC呈现典型的衰老生物学表现：SA-β-Gal染色阳性细胞率显著增高（P <0.01）; G0/G1 期比例明显增加,S 期显著减少（P <0.01） ;CFU-Mix数量显著减少（P <0.01）。衰老HSC端粒缩短（P <0.05）,端粒酶活性降低（P <0.05）。衰老HSC ROS含量显著增加（P <0.01）,细胞培养上清液中SOD、GSH-Px活力下降、 MDA含量增加（P <0.05）。 结论 ox-LDL能通过氧化应激诱导HSC衰老,其机制可能与ROS的蓄积及抗氧化酶活性受抑引起端粒功能异常有关。     

Peripheral parameters of oxidative stress in patients with infiltrative Graves' ophthalmopathy treated with corticosteroids

Infiltrative ophthalmopathy, which may develop in patients with Graves' disease, is considered an inflammatory disorder of autoimmune background. There is growing evidence that changed reactive oxygen species (ROS) metabolism plays an important role in pathogenesis of autoimmune diseases. Corticotherapy is a principal method of ophthalmopathy treatment, and its therapeutic effect is partially connected with influence on ROS generation systems. This study was undertaken to investigate corticosteroids treatment influence on blood extracellular indices of ROS metabolism in Graves' ophthalmopathy patients. Plasma indices of free radical generation and scavenging were determined in 22 euthyroid patients with active infiltrative Graves' ophthalmopathy initially, after intensive corticotherapy and after completing of steroid treatment. Age- and sex-matched 24 healthy volunteers and 25 euthyroid Graves' patients without overt ophthalmopathy served as controls. In the ophthalmopathy patients hydrogen peroxide (H(2)O(2)), lipid hydroperoxides (ROOH), thiobarbituric acid-reacting substances (TBARS) and ceruloplasmin (CP) levels and superoxide dismutase (SOD) and catalase (CAT) activities were increased, whereas glutathione peroxidase (GPx) and glutathione reductase (GR) activities were reduced. Intensive corticotherapy resulted in normalization (partial for ROOH) of ROS metabolism peripheral markers. After the withdrawal of corticosteroids a reduction of ophthalmopathy clinical activity was present, yet a marked restoration of increased oxidative stress indices was observed, along with activation of antioxidant defence systems (not significant for CAT activity). These data demonstrate that corticosteroids are effective in reduction of peripheral oxidative stress present in infiltrative Graves' ophthalmopathy, but this effect tends to be transient.     

通心络超微粉对猪急性心肌梗死后移植骨髓间充质干细胞的影响

目的探讨通心络超微粉能否改善猪急性心肌梗死(AMI)局部微环境,从而提高移植自体骨髓间充质干细胞(MSCs)在体存活和移植效果。方法28头中华小型猪被分为4组(n=7),分别为对照、通心络超微粉(自AMI前3 d用药直至后4 d)、MSCs移植和通心络超微粉+MSCs联合组。AMI模型由阻断冠状动脉左前降支90 min制成,再灌注后即刻经心肌内注射DAPI标记的自体骨髓MSCs(3×107cells/动物)。在移植后1周(基线)和6周(终点),以核磁共振(MRI)检测心功能。6周后处死动物,检测移植细胞在体存活和分化、梗死周边心肌凋亡和氧化应激指标。结果终点时,与对照组、MSCs组和通心络超微粉组相比,联合组纤维化和炎症细胞浸润显著减轻,并伴较多存活心肌。与MSCs移植组相比,联合组移植细胞的在体存活和成心肌分化能力均显著增强(P<0.0001)。MRI显示在基线时各心功能参数无显著差异(P>0.05);终点时,和对照组相比,仅联合组左室射血分数(LVEF)显著增加(P<0.0001),室壁运动障碍节段数、梗死室壁增厚率、梗死面积和左室质量指数均显著减小(P<0.0001)。终点时,和对照组相比,在通心络超微粉组和联合组梗死周边区的心肌凋亡均显著减轻(P<0.0001),SOD显著增加(P<0.05),MDA显著减少(P<0.05)。结论实验表明AMI再灌注后即刻心肌内移植自体骨髓MSCs生存和分化能力有限,未产生显著功能学获益。而围移植期使用通心络超微粉可显著改善梗死局部微环境,提高移植细胞存活和分化,并产生显著心功能获益。     

一氧化氮调节间充质干细胞免疫抑制功能的机制研究

为探讨一氧化氮(nitric oxide,NO)在间充质干细胞(mesenchymal stem cells,MSC)发挥免疫调节功能中的作用机制。通过全骨髓贴壁法分离培养得到高纯度的MSC,利用炎症因子(TNF-α,IL-1α)和Con A分别刺激MSC制备MSC上清培养液,3 H-TdR掺入法检测MSC及其培养上清液对脾细胞增殖的抑制效应;ELISA方法检测各组培养上清液中前列腺素E-2(prostaglandin E-2,PGE-2)的含量;通过NO抑制剂L-canavanine和NO的直接供体SNP分别作用于MSC,探讨NO对MSC免疫调节功能的影响。结果显示:全骨髓贴壁法可获得较高纯度的MSC;MSC及其上清培养液均能发挥免疫调节功能;MSC分泌大量的PGE-2;抑制NO产生可以下调MSC对PGE-2的分泌而增加NO量可以上调MSC对PGE-2的分泌。研究结果表明,NO通过刺激MSC分泌抗炎因子PGE-2,从而使MSC发挥长效免疫调节功能。本研究阐明了NO介导的MSC长效免疫调节功能的机制,为MSC的临床应用奠定了理论基础。     

Assessment of viability and osteogenic ability of human mesenchymal stem cells after being stored in suspension for clinical transplantation

Human mesenchymal stem cells (MSCs) were suspended in phosphate-buffered saline (PBS) and stored up to 24 h at 4 degrees C, 24 degrees C, and 37 degrees C. More than 80% viability was maintained at any temperature for at least 1 h, then gradually decreased over time. After 24 h, the viabilities at 4 degrees C, 24 degrees C, and 37 degrees C were about 81%, 70%, and 62%, respectively. The MSCs suspended/stored in PBS at 4 degrees C for 24 h also exhibited in vitro osteogenic differentiation capability as evidenced by mineralized matrix formation as well as high alkaline phosphatase activity when cultured in an osteogenic medium. Furthermore, in vivo implantation experiments using the MSCs also demonstrated new bone formation. Because MSCs are known to possess multipotential stem cell characteristics, these data indicate that human MSCs stored in PBS at 4 degrees C could be delivered to distant medical facilities for the purpose of hard tissue and other types of tissue regeneration therapy.     

致敏小鼠骨髓间充质干细胞体外培养及多向分化功能的测定

目的：评价异基因脾细胞输注致敏的小鼠骨髓源性间充质干细胞(MSCs)的体外培养生长能力及其多向分化功能。方法：应用贴壁培养法体外培养间充质干细胞,流式检测其表面标志以及检测其成骨、成脂和成肌多向分化状况;结果：致敏小鼠骨髓源性MSCs与非致敏小鼠骨髓源性MSCs比较,形态学无差异且均表达CD29+、CD105+、CD44+和Sca-1+ ;CD34-、CD11b-;同时在相应的诱导条件下具有向成骨、成脂、成肌多向分化的能力。结论：异基因脾细胞输注致敏的小鼠,其骨髓源性MSCs的形态学和功能与正常小鼠的MSCs比较评估未见异常。     

小鼠骨髓间充质干细胞的分离培养及形态学观察

目的建立一种简单实用的小鼠骨髓间充质干细胞(MSCs)的分离培养方法,通过了解其细胞生物学特性,为MSCs的应用提供实验依据。方法采用差速贴壁法体外分离、扩增MSCs,倒置显微镜观察原代及传代细胞的形态及生长过程。结果在小鼠骨髓间充质干细胞的培养过程中,血液系细胞在换液过程被去除,成纤维细胞污染经差速贴壁法也可去除。获得的骨髓间充质细胞形态较均一,生长状态良好。结论采用差速贴壁培养法可获得一定纯度的MSCs,此法简单、实用,并且获得的细胞生长状态良好,增殖能力强.     

尼古丁对人脐带间充质干细胞增殖和迁移的影响

 目的： 探讨尼古丁对人脐带间充质干细胞（MSCs）一氧化氮（NO）、一氧化氮合酶（NOS）、诱导型一氧化氮合酶（iNOS)、活性氧（ROS）、线粒体膜电位及细胞因子生成的影响。方法： 尼古丁作用MSCs后,硝酸还原酶法测NO的释放情况;分光光度计法测NOS和iNOS活性;流式细胞技术测ROS及线粒体膜电位的变化;ELISA法测培养上清液中细胞间黏附分子 1（ICAM-1）、基质细胞衍生因子 1（SDF-1）、基质金属蛋白酶 9(MMP-9)、金属蛋白酶组织抑制物 1（TIMP-1）、转化生长因子 β1（TGF-β1）、胰岛素样生长因子 I（IGF-I）和碱性成纤维细胞生长因子（bFGF）水平的变化。结果： 在24 h和36 h,NO水平随尼古丁浓度上升而增加（P<0.05）,但在48 h,0.8  g/L与1.0  g/L组,NO水平低于对照组;NOS和iNOS活性随尼古丁浓度上升而逐渐增加;尼古丁可增加MSCs的ROS水平,并降低线粒体膜电位;在尼古丁作用下,MSCs分泌SDF-1、TGF-β1、IGF-I及bFGF水平下降,ICAM-1、MMP-9及TIMP-1表达均上升。结论： 尼古丁通过增加NO、NOS、iNOS和ROS水平,降低线粒体膜电位,使SDF-1、TGF-β1、IGF-I及bFGF分泌下降,ICAM-1、MMP-9及TIMP-1表达上升,可能影响MSCs的增殖、黏附、迁移等特性。     

Mesenchymal stem cells induce endothelial activation via paracine mechanisms.

Mesenchymal stem cells (MSCs) are bone marrow-derived, pluripotent cells that possess the ability to transdifferentiate into various mesenchymal tissues such as bone, endothelium, and (heart) muscle. Therefore, these cells may provide a therapeutic tool, especially for the treatment of myocardial infarction. The interaction of the MSCs with the endothelial barrier and their ability to ultimately leave blood vessels after application are crucial in this context. In this study, the authors focused on the soluble factors produced by MSCs and their effect on the intracellular signal transduction of endothelial cells. The authors performed immunohistochemical measurements on human umbilical vein endothelial cells (HUVECs) treated with conditioned stem cell medium and took measurements of the intracellular nitric oxide (NO) levels and calcium changes. After application of conditioned stem cell medium, the authors detected an increase in endothelial NO synthase (eNOS) activity by translocation (Ca(2+)) and by phosphorylation (increase of pAKT and peNOS1177). Additionally, the authors observed an upregulation of pERK within the same time. The phosphorylated eNOS forms are linked to these findings and the increase of intracellular NO in the DAF measurements. Moreover, conditioned medium also increased intracellular calcium levels in endothelial cells. Concluding, the authors postulate that MSCs emit soluble factors that alter the NO and calcium levels of endothelial cells and may be important for facilitate crossing the endothelial barrier.     

Heat shock and 5-azacytidine inhibit nitric oxide synthesis and tumor necrosis factor-alpha secretion in activated macrophages

To elucidate the role of stress response during macrophage activation, the effects of heat shock and the amino acid analog, 5-azacytidine on nitric oxide (NO) production, tumor necrosis factor-alpha (TNF-alpha) secretion, and heat shock protein (HSP) synthesis have been studied in murine peritoneal macrophages (C57BL/6). Heat shock (1 hr at 43 degrees C) or 5-azacytidine markedly inhibited the release of NO into the medium from interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS)-stimulated macrophages. Although heat shock significantly decreased TNF-alpha secretion only at the initiation stage of macrophage stimulation, 5-azacytidine treatment resulted in a more prolonged reduction in the secretion of TNF-alpha. When heat-shocked cells were stimulated with IFN-gamma plus LPS under normal culture conditions at 37 degrees C, the heat shock-induced inhibition of NO release reversed progressively with increasing recovery time. Although the total amount of cellular HSP72 measured by Western blot increased time-dependently over 7 hr, newly synthesized HSP72 measured by [35S]methionine incorporation was evident only after 1 and 3 hr of recovery time after heat shock treatment. At these time points, the lowest nitrite accumulation and TNF-alpha secretion into the medium was evident. It is concluded that signaling pathways related to newly synthesized HSP such as HSP72 are implicated in the down regulation of NO synthesis and TNF-alpha secretion in macrophages.