Cytoprotection by pyruvate through an anti-oxidative mechanism in cultured rat calvarial osteoblasts

Although we have previously shown drastic cell death by pyruvate deficiency in osteoblasts at the proliferative stage, the exact mechanism remains unclear so far. Cell survivability was significantly decreased in rat calvarial osteoblasts cultured for 0 to 3 days in vitro (DIV) following replacement of the eutrophic alpha-modified minimum essential medium (alpha-MEM) with Dulbecco's modified eagle medium (DMEM) for cultivation. The addition of pyruvate enriched in alpha-MEM, but not in MEM, entirely prevented cell death induced by the medium replacement throughout a culture period from 0 to 3 DIV. Both cysteine and reduced glutathione protected cell death in cells cultured for 3 DIV without significantly affecting that in cells cultured for 1 DIV, however, while none of lactate, acetate and insulin significantly prevented the cell death irrespective of the culture period up to 3 DIV. A marked increase was detected in intracellular reactive oxygen species (ROS) levels 4 h after the medium replacement. In osteoblasts cultured in alpha-MEM for 3 DIV, but not in those for 7 DIV, hydrogen peroxide (H2O2) markedly decreased cell survivability when exposed for 2 to 24 h. Furthermore, H2O2 was effective in significantly decreasing cell survivability in osteoblasts cultured in DMEM for 7 DIV. Pyruvate at 1 mM not only prevented cell death by H2O2, but also suppressed the generation of intracellular ROS in osteoblasts exposed to H2O2. These results suggest that pyruvate could be cytoprotective through a mechanism associated with the anti-oxidative property rather than an energy fuel in cultured rat calvarial osteoblasts.     

Enhancement of catalase activity by repetitive low-grade H2O2 exposures protects fibroblasts from subsequent stress-induced apoptosis

Exposure of Chinese hamster V79 fibroblasts to mild and repetitive H2O2 doses in culture for 15 weeks produced no change in lipid peroxidation status, GSH/GSSG ratio and glutathione peroxidase activity of these cells (VST cells). In contrast, in VST cells catalase levels underwent a prominent increase which could be significantly inhibited and brought down to control levels after treatment with the catalase inhibitor 3-aminotriazole (3-AT). When control (VC) cells were exposed to UV radiation (UVC 5 J/m2) or H2O2 (7.5mM, 15 min), intracellular reactive oxygen species (ROS) levels rose prominently with significant activation of caspase-3. Marked nuclear fragmentation and lower cell viability were also noted in these cells. In contrast, VST cells demonstrated a significantly lower ROS level, an absence of nuclear fragmentation and an unchanged caspase-3 activity after exposure to UVC or H2O2. Cell viability was also significantly better preserved in VST cells than VC cells after UV or H2O2 exposures. Following 3-AT treatment of VST cells, UVC radiation or H2O2 brought about significantly higher elevations in intracellular ROS, increases in caspase-3 activity, significantly lowered cell viability and marked nuclear fragmentation, indicating the involvement of high catalase levels in the cytoprotective effects of repetitive stress. Therefore, upregulation of the antioxidant defense after repetitive oxidative stress imparted a superior ability to cope with subsequent acute stress and escape apoptotic death and loss of viability.     

A Novel Technique for Culture of Human Dermal Microvascular Endothelial Cells under either Serum-Free or Serum-Supplemented Conditions: Isolation by Panning and Stimulation with Vascular Endothelial Growth Factor

Several physiological and pathophysiological events involving vascular endothelium occur at the microvascular level. Studies on human microvasculature require homogenous primary cultures of microvascular endothelial cells. However, procedures available for isolating and culturing human dermal microvascular cells (HDMEC) result in significant contamination with fibroblasts. To eliminate contamination with fibroblasts or other cells, we developed a procedure to isolate HDMEC from neonatal human foreskin by panning the cells using EN4, an anti-endothelial cell monoclonal antibody. Panned cells uniformly expressed von Willebrand factor and CD36, confirming their microvascular endothelial characteristics, whereas cells cultured without panning showed a significant degree of contamination with fibroblasts. In the presence of vascular endothelial growth factor (VEGF), HDMEC could be cultured under serum-free conditions. VEGF stimulated the growth of HDMEC in a dose-dependent manner in serum-free medium or in media supplemented with either human serum or newborn calf serum. Since differences exist between large vessel endothelial cells and microvascular endothelial cells, we compared the response to VEGF stimulation of HDMEC with human umbilical vein endothelial cells (HUVEC). The dose response of the two cell types to VEGF was different. This effect of VEGF on endothelial cells may be mediated by the VEGF receptorkdr,since mRNA forkdrwas detected using RT–PCR in both HDMEC and HUVEC. The procedure described in this study will make possible the culture of highly enriched HDMEC without contamination with fibroblasts and facilitate studies with these cells under defined assay conditions in a serum-free environment.     

Oxidative stress is a mediator of glucose toxicity in insulin-secreting pancreatic islet cell lines

Pancreatic beta cells secrete insulin in response to changes in the extracellular glucose. However, prolonged exposure to elevated glucose exerts toxic effects on beta cells and results in beta cell dysfunction and ultimately beta cell death (glucose toxicity). To investigate the mechanism of how increased extracellular glucose is toxic to beta cells, we used two model systems where glucose metabolism was increased in beta cell lines by enhancing glucokinase (GK) activity and exposing cells to physiologically relevant increases in extracellular glucose (3.3-20 mm). Exposure of cells with enhanced GK activity to 20 mm glucose accelerated glycolysis, but reduced cellular NAD(P)H and ATP, caused accumulation of intracellular reactive oxygen species (ROS) and oxidative damage to mitochondria and DNA, and promoted apoptotic cell death. These changes required both enhanced GK activity and exposure to elevated extracellular glucose. A ROS scavenger partially prevented the toxic effects of increased glucose metabolism. These results indicate that increased glucose metabolism in beta cells generates oxidative stress and impairs cell function and survival; this may be a mechanism of glucose toxicity in beta cells. The level of beta cell GK may also be critical in this process.     

Altered cell growth and induction of an interferon-responsive gene in fibroblast lines derived from patients with familial Alzheimer's disease

Cell growth and response to interferon (IFN) have been used to assess alteration in properties of cultured skin fibroblasts from patients with familial Alzheimer's Disease (AD). A comparison of the proliferative characteristics between "low" (7-11) and "high" (18-21) passage diploid fibroblasts from control and AD individuals shows that AD cells become severely restricted in their proliferative potential earlier than matched controls as a result of "in vitro aging". In addition, while control cells grow adequately in a serum-free, chemically-defined media (Nutridoma) (90-95% compared to FBS-supplemented media), AD cells grow poorly in Nutridoma (45-53% compared to FBS-supplemented media). An interferon (IFN)-inducible enzyme, 2'5'-oligoadenylate (2-5A) synthetase, is significantly reduced in IFN-treated AD cells compared to age-matched control cells in two cell passage-dependent manner. These data suggest that an intrinsic property of AD cells is the progressive reduced ability to interact with normal biologic signals provided by proteins such as interferons which in turn may contribute to the pathogenesis of AD.     

Serum modulates the intracellular calcium response of primary cultured bone cells to shear flow

We investigated the effect of newborn bovine serum on the intracellular calcium [Ca²⁺]_i response of primary cultured bone cells stimulated by fluid flow. As it has been previously established that these cells exhibit [Ca²⁺]_i responses to fluid flow shear stress in saline media without growth factors or other chemically stimulatory factors, we hypothesized that the addition of serum to the flow medium would enhance the mechanosensitivity of the cells. We examined the effect of a short-term (10–15 min) exposure of the cells to 2 and 10% serum prior to flow stimulation (pretreated) compared to not exposing the cells prior to flow stimulation (unpretreated). The cells were subjected to a well-defined, 90-s flow stimulus with shear stress levels ranging from 0.02 to 3.5 Pa in a laminar flow chamber using a saline medium supplemented with 2 or 10% serum. For pretreatment, the serum concentration was the same from pre-flow to flow exposure. We observed a differential effect in the magnitude of the peak [Ca²⁺]_i response modulated by the concentration of serum in the pre-flow medium. Additionally, ATP-supplemented flow was examined as a comparison to the serum-supplemented flow and exhibited a similar trend in the peak [Ca²⁺]_i flow response that was dependent on ATP concentration and pre-flow exposure conditions. These findings demonstrate that under the conditions of this study, chemical agonist exposure can modulate the [Ca²⁺]_i response in bone cells subjected to fluid flow-induced shear stress.     

人参皂甙Rd预处理可减轻谷氨酸所致的PC12细胞损伤

目的:研究人参皂甙Rd(Ginsenoside Rd)预处理对谷氨酸所致PC12细胞损伤的影响。方法:将体外培养的PC12细胞分为3组,分别为对照组(Control)、谷氨酸损伤组(Glu)和人参皂甙Rd预处理组(Rd)。Control组细胞正常培养;Glu组细胞暴露于含10mM谷氨酸的DMEM培养基中损伤24 h;Rd组细胞经50μM的人参皂甙Rd预处理30 min后,在谷氨酸浓度为10 mM的DMEM培养基中损伤24 h。采用MTT检测细胞活力和乳酸脱氢酶(LDH)检测试剂盒检测LDH释放量;流式细胞仪检测胞内活性氧(ROS)水平;Western blot检测还原型谷胱甘肽蛋白(GSH)表达;专用试剂盒检测细胞内过氧化氢酶(CAT)和超氧化物歧化酶(SOD)含量,相差显微镜观测细胞形态。结果:50μM的人参皂甙Rd预处理30 min,可明显提高谷氨酸诱导的PC12细胞的活力,降低其LDH释放量、胞内ROS含量,并提高胞内GSH蛋白表达,增加CAT、SOD含量并改善细胞形态。结论:人参皂甙Rd预处理可减轻谷氨酸引起的PC12细胞损伤。     

Inhibition of proliferation of contaminating fibroblasts by D-valine in cultures of smooth muscle cells from human myometrium

Replacement of L-valine with D-valine in a standard culture medium can selectively inhibit fibroblast proliferation. The aim of the present study was to investigate whether human myometrial cells cultured with D-valine instead of L-valine can survive and express their characteristics. Cultured cells (95-98%) maintain expression of the intermediate filament desmin, which is the specific marker for mature muscle cells. By transmission electron microscopy, the cells showed the general morphology of smooth muscle cells in culture. Oxytocin in serum-free culture medium at 37 degrees C (5 min) caused a concentration-dependent increase in cellular Na and total Ca, and a decrease in K content as determined by X-ray microanalysis. The percentage of cells cultured with D-valine responding to oxytocin stimulation was larger than that of cells cultured with L-valine, suggesting less contamination of smooth muscle cells by fibroblasts in the presence of D-valine. As shown by measurements with fura-2, D-valine-cultured cells retained the characteristic increase in intracellular free Ca2+ ions after oxytocin stimulation.     

Palmitate increases superoxide production through mitochondrial electron transport chain and NADPH oxidase activity in skeletal muscle cells

The effect of unbound palmitic acid (PA) at plasma physiological concentration range on reactive oxygen species (ROS) production by cultured rat skeletal muscle cells was investigated. The participation of the main sites of ROS production was also examined. Production of ROS was evaluated by cytochrome c reduction and dihydroethidium oxidation assays. PA increased ROS production after 1 h incubation. A xanthine oxidase inhibitor did not change PA-induced ROS production. However, the treatment with a mitochondrial uncoupler and mitochondrial complex III inhibitor decreased superoxide production induced by PA. The importance of mitochondria was also evaluated in 1 h incubated rat soleus and extensor digitorum longus (EDL) muscles. Soleus muscle, which has a greater number of mitochondria than EDL, showed a higher superoxide production induced by PA. These results indicate that mitochondrial electron transport chain is an important contributor for superoxide formation induced by PA in skeletal muscle. Results obtained with etomoxir and bromopalmitate treatment indicate that PA has to be oxidized to raise ROS production. A partial inhibition of superoxide formation induced by PA was observed by treatment with diphenylene iodonium, an inhibitor of NADPH oxidase. The participation of this enzyme complex was confirmed through an increase of p47(phox) phosphorylation after treatment with PA.     

黄连素预处理对6-羟基多巴胺所致PC12细胞损伤的影响

目的:观察黄连素(Berberine,BBR)预处理对6-羟基多巴胺(6-hydroxydopamine,6-OHDA)诱导的PC12细胞的影响,并探讨二型超氧化物歧化酶(Mn-SOD,SOD2)是否介导了BBR的保护作用。方法:将PC12细胞分为5组,分别为正常培养的对照组(Control)、25μM的6-OHDA损伤组、1μM的BBR预处理24 h组(BBR+6-OHDA)、SOD2-siRNA干扰组(SOD2-siRNA+BBR+6-OHDA)和乱序siRNA处理组(SC-siRNA+BBR+6-OHDA),孵育24 h后,采用噻唑蓝法(Methylthiazolyldiphenyl-tetrazolium bromide,MTT)检测细胞活力,试剂盒检测培养基乳酸脱氢酶(Lactic Dehydrogenase,LDH)、细胞内活性氧(Reactive Oxygen Species,ROS)、还原型谷胱甘肽(Glutathione,GSH)和过氧化氢酶(Catalase,CAT)的含量,使用流式细胞仪检测凋亡率,Western blot检测SOD2和凋亡蛋白Cleaved caspase-3的表达。结果:与Control组相比,6-OHDA诱导PC12细胞24 h后,细胞活力显著降低,SOD2表达、GSH和CAT的含量明显减少,培养基上清液LDH活力、细胞凋亡率、Cleaved caspase-3表达和ROS水平显著增加(P<0.05),而BBR预处理可显著恢复6-OHDA诱导的PC12细胞活力、SOD2表达、GSH和CAT水平,并降低细胞凋亡率、凋亡蛋白表达和细胞ROS水平(P<0.05),而SOD2-siRNA显著逆转了BBR预处理产生的上述保护作用(P<0.05),SC-siRNA则未对BBR预处理产生的上述作用造成明显影响(P>0.05)。结论:黄连素预处理可减轻6-OHDA诱导的PC12细胞损伤,而SOD2分子介导了BBR预处理对暴露于6-OHDA的PC12细胞的保护作用。     

DNA damage-induced reactive oxygen species (ROS) stress response in Saccharomyces cerevisiae

Cells are exposed to both endogenous and exogenous sources of reactive oxygen species (ROS). At high levels, ROS can lead to impaired physiological function through cellular damage of DNA, proteins, lipids, and other macromolecules, which can lead to certain human pathologies including cancers, neurodegenerative disorders, and cardiovascular disease, as well as aging. We have employed Saccharomyces cerevisiae as a model system to examine the levels and types of ROS that are produced in response to DNA damage in isogenic strains with different DNA repair capacities. We find that when DNA damage is introduced into cells from exogenous or endogenous sources there is an increase in the amount of intracellular ROS which is not directly related to cell death. We have examined the spectrum of ROS in order to elucidate its role in the cellular response to DNA damage. As an independent verification of the DNA damage-induced ROS response, we show that a major activator of the oxidative stress response, Yap1, relocalizes to the nucleus following exposure to the DNA-alkylating agent methyl methanesulfonate. Our results indicate that the DNA damage-induced increase in intracellular ROS levels is a generalized stress response that is likely to function in various signaling pathways.     

Toll-like receptor 4-mediated signal pathway induced by Porphyromonas gingivalis lipopolysaccharide in human gingival fibroblasts

The lipopolysaccharide (LPS) secreted by Porphyromonas gingivalis is implicated in the initiation and progression of periodontitis. Human gingival fibroblasts (HGFs) are the major constituent of gingival connective tissue. In this study, we examined the expression of Toll-like receptor 4 (TLR4) on HGFs by flow cytometric analysis, and studied the signal transduction induced by LPS stimulation of HGFs by enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation. We show that LPS binds to HGFs, and that HGFs express TLR4 and myeloid differentiation primary response gene 88 (MyD88). P. gingivalis LPS-induced interleukin (IL)-1 production in HGFs was inhibited by anti-TLR4 antibody. P. gingivalis LPS treatment of HGFs activated several intracellular proteins including protein tyrosine kinases, and upregulated the expression of IL-1 receptor-associated kinase (IRAK), nuclear factor-kappaB (NF-kappaB), and activating protein-1 (AP-1), and these events were suppressed by anti-TLR4 monoclonal antibody. Our findings suggest that the binding of P. gingivalis LPS to TLR4 on HGFs activates various second messenger systems.     

The effects of platelet-derived transforming growth factor beta on normal human diploid gingival fibroblasts

Studies of the effects of transforming growth factor (TGF) beta on normal human diploid gingival fibroblasts (HGF) have been carried out to determine possible physiological effects of this growth factor. Responses distinctly different from those characterized using established cell lines were observed. Whether alone, or in combination with EGF (2.5 ng/ml), human platelet-derived TGF-beta (0.1 ng/ml or 1.0 ng/ml) did not induce anchorage-independent growth of HGFs in soft agar assays. However, TGF-beta with EGF acted synergistically in promoting a 1.8-fold increase in anchorage-dependent proliferation of quiescent HGFs. At the same concentrations TGF-beta alone stimulated the incorporation of [35S]methionine into both cellular (cell-layer) and matrix (medium) proteins by as much as 3-fold and 1.7-fold respectively. Densitometric analysis of fluorographs of radiolabeled media proteins separated by SDS-PAGE revealed that the TGF-beta-stimulated protein synthesis was selective. However, synthesis of collagen, the major protein synthesized and secreted by HGFs, was stimulated by TGF-beta to the same extent as the average secreted protein. Protein synthesis and cell proliferation were significantly greater in subconfluent cells compared to confluent and multilayered cells. These effects are likely to reflect physiological activity of platelet-derived TGF-beta which may act to promote the wound healing response.     

Reactive oxygen species as mediators of cell adhesion

The intracellular production of reactive oxygen species (ROS) has a fundamental importance in both cell proliferation and apoptosis induction. Moreover, many experimental and epidemiological evidence indicate that ROS contribute to the initiation and promotion of carcinogenesis, and that drugs or treatments aimed to reduce the tissue content of ROS can be chemopreventive and curative against cancer. Recently, important observations on the role of ROS as physiological regulators of intracellular signaling cascades activated by growth factors through their tyrosine-kinase receptors have shed new light on the possible mechanisms that can sustain the promoting activity of ROS. The downstream effect of ROS production is the reversible oxidation of proteins. Redox sensitive proteins include protein tyrosine phosphatases (PTPs) as the active-site cysteine is the target of specific oxidation, and this modification can be reversed by intracellular reducing agents. The reversible oxidation of PTPs family member was demonstrated firstly for PTP1B during EGF signaling and then for LMW-PTP and SHP-2 during PDGF stimulation. The inhibition exerted by ROS on tyrosine-phosphatases helps the propagation of RTK signals mediated by protein tyrosine phosphorylation, generally associated with the proliferative stimulus. Our new data are consistent with a model in which ROS take a role in integrin signaling, and in which synergistic activation of Rac-1 by growth factors and adhesion molecules translates in a critical increase of intracellular oxidants up to a threshold level where inhibition of the tyrosine phosphatase LMW-PTP takes place. In seeking for potential molecular mechanisms for oxidative signaling by integrins, we found that transient oxidation/inactivation of LMW-PTP, a known negative regulator of RTK signaling, occurred during fibroblast adhesion to matrix, with a kinetic which paralleled the generation of ROS. Moreover, overexpression of LMW-PTP in NIH-3T3 fibroblasts delayed cell attachment to the substrate. Finally, constitutively high levels of intracellular ROS, as are observed in cells expressing active Rac, would attenuate anchorage dependence for growth, by substituting for integrin signaling in non adherent cells.     

Short-term exposure of high glucose concentration induces generation of reactive oxygen species in endothelial cells: implication for the oxidative stress associated with postprandial hyperglycemia

Recent studies demonstrating a close relationship between postprandial hyperglycemia and the incidence of atherosclerotic cardiovascular disease prompted us to investigate the generation and source of reactive oxygen species (ROS) in endothelial cells stimulated by short-term exposure to a high glucose concentration. In addition, we investigated the effect of insulin on ROS production induced by high glucose concentration. Cultured bovine aortic endothelial cells demonstrated a significant increase in intracellular ROS generation after a 3-h exposure to 25 mM glucose (131.4% versus 5 mM glucose). This increased generation of ROS was suppressed by an inhibitor of NAD(P)H oxidase. Intracellular ROS production in cells exposed to 3 h of high glucose concentration was increased significantly by the presence of a physiological concentration of insulin. However, after a 1-h exposure to high glucose levels, ROS generation in cells incubated with insulin was only about 80% of that measured in cells incubated without insulin. The generation of intracellular nitric oxide (NO) resulting from an acute insulin effect may account for this difference. In conclusion, acute hyperglycemia itself may possibly cause endothelial oxidative stress in patients with postprandial hyperglycemia. Endothelial oxidative stress may be determined by the interaction between NO and superoxide generation.     

Phosphatidic acid induces leaf cell death in Arabidopsis by activating the Rho-related small G protein GTPase-mediated pathway of reactive oxygen species generation

Phosphatidic acid (PA) level increases during various stress conditions. However, the physiological roles of this lipid in stress response remain largely unknown. In this study, we report that PA induced leaf cell death and elevated the levels of reactive oxygen species (ROS) in the whole leaf and single cells. To further elucidate the mechanism of PA-induced cell death, we then examined whether Rho-related small G protein (ROP) 2, which enhanced ROS production in an in vitro assay, is involved in PA-induced ROS production and cell death. In response to PA, transgenic leaves of Arabidopsis expressing a constitutively active rop2 mutant exhibited earlier cell death and higher levels of ROS than wild type (WT), whereas those expressing a dominant-negative rop2 mutant exhibited later cell death and lower ROS. However, in the absence of exogenous PA, no spontaneous cell death or elevated ROS was observed in constitutively active rop2 plants, suggesting that the activation of ROP GTPase alone is insufficient to activate the ROP-mediated ROS generation pathway. These results suggest that PA modulates an additional factor required for the active ROP-mediated ROS generation pathway. Therefore, PA may be an important regulator of ROP-regulated ROS generation and the cell death process during various stress and defense responses of plants.     

Manganese superoxide dismutase activity regulates transitions between quiescent and proliferative growth

In recent years, the intracellular reactive oxygen species (ROS) levels have gained increasing attention as a critical regulator of cellular proliferation. We investigated the hypothesis that manganese superoxide dismutase (MnSOD) activity regulates proliferative and quiescent growth by modulating cellular ROS levels. Decreasing MnSOD activity favored proliferation in mouse embryonic fibroblasts (MEF), while increasing MnSOD activity facilitated proliferating cells' transitions into quiescence. MnSOD +/- and -/- MEFs demonstrated increased superoxide steady-state levels; these fibroblasts failed to exit from the proliferative cycle, and showed increasing cyclin D1 and cyclin B1 protein levels. MnSOD +/- MEFs exhibited an increase in the percentage of G(2) cells compared to MnSOD +/+ MEFs. Overexpression of MnSOD in MnSOD +/- MEFs suppressed superoxide levels and G(2) accumulation, decreased cyclin B1 protein levels, and facilitated cells' transit into quiescence. While ROS are known to regulate differentiation and cell death pathways, both of which are irreversible processes, our results show MnSOD activity and, therefore, mitochondria-derived ROS levels regulate cellular proliferation and quiescence, which are reversible processes essential to prevent aberrant proliferation and subsequent exhaustion of normal cell proliferative capacity. These results support the hypothesis that MnSOD activity regulates a mitochondrial 'ROS-switch' favoring a superoxide-signaling regulating proliferation and a hydrogen peroxide-signaling supporting quiescence.     

Cation transport in murine L fibroblasts cultured long term in a serum-free medium

Cation fluxes and intracellular content in mouse fibroblasts L, growing for more than three years in the Dulbecco modified Eagle's serum-free medium (clone L625sf) were measured as a function of culture density. The cells show no density-dependent inhibition of growth and in continuously growing cultures of L625sf cells internal potassium, and rubidium influx was found to remain high within a wide range of densities (5.10(4)-20.10(4) cells/cm2). A close correlation was revealed between the potassium transport and the proliferative state of L625sf cultures: the addition of 5% calf serum to logarithmically growing cultures leads to the increase in culture growth rate as well as to the increase in ouabain-inhibited rubidium influx and intracellular potassium content; a delay in culture growth rate due to medium depletion is accompanied by decreasing both the rubidium influx and the intracellular potassium content. It is concluded that L625sf cells being capable of multiplicating in serum-free medium remain sensitive to growth factors of serum and may be used for study of growth factor induction of cell proliferation and for identification of autocrine factors of cell growth.