Transitory oxidative stress in L929 fibroblasts cultured on poly(epsilon-caprolactone) films

Poly(epsilon-caprolactone) (PCL) is considered as a potential substrate for wide medical applications. In previous studies we carried out the in vitro biocompatibility assessment of PCL films using L929 mouse fibroblasts, obtaining good cell behaviour but a transitory stimulation of mitochondrial activity and cell retraction. Reactive oxygen species (ROS), mainly formed in mitochondria, can impair the function of several cellular components and produce cell oxidative stress by changing the normal red-ox status of the major cell antioxidants as glutathione. The aim of this study was to measure intracellular ROS production and glutathione content of L929 fibroblasts cultured on PCL films. Cell size, internal complexity, cell cycle and lactate dehydrogenase release were also evaluated. The films were treated with NaOH before culture to improve the cell-polymer interaction. PCL induces a transitory but significant oxidative stress in L929 fibroblasts. The treatment of PCL films with NaOH reduces this effect. PCL also induces transitory changes on cell size and complexity. Nevertheless, after 7 days in culture, cells reach control levels for all the studied parameters. Neither cell cycle nor membrane integrity appears affected by this oxidative stress respect to control cells at any culture time. These results underline the cytocompatibility of PCL films and, therefore, its potential utility as a suitable scaffold in tissue engineering.     

Synthesis of partial-stabilized cement (PSC) via sol-gel process

Biomaterials have been widely used to prepare synthetic vascular grafts over the past thirty years, but the inherent thrombogenicity of their surface can lead to graft failure. Endothelial progenitor cells (EPC) are circulating premature cells able to differentiate in either myocardial or endothelial cells (EC). The therapeutic potential of these cells and its easy obtaining technique are important reasons why these cells could be used to improve the performance of vascular grafts. In this study, two different stages of differentiation of EC derived from EPC (EC(1) and EC(2)) were characterized and cultured on poly(epsilon-caprolactone) (PCL) films treated with NaOH (PCL-NaOH). We investigated by immunolabeling the expression of CD31, von Willebrand factor (vWF), and endothelial nitric oxide synthase (eNOS) in these cells during the differentiation process. The proliferation, cell cycle, and mitochondrial function of EC(2) cultured on PCL-NaOH were evaluated at different times. The effect of this biomaterial on the nitric oxide (NO) content was also measured. The mature EC obtained from circulating progenitor cells (EC(2)) showed an appropriate growth and functionality on NaOH-treated films. They conserved their capacity to define vessel-like structures in culture and increased their basal NO production. These results underline the potential usefulness of these EC(2) to get a functional endothelialization of polymers with applications in vascular tissue engineering.     

Biocompatibility markers for the study of interactions between osteoblasts and composite biomaterials

Biphasic calcium phosphate (BCP), a mixture of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), has attracted attention as an excellent bone graft substitute. Mixtures of ceramics with agarose, as natural biodegradable binder, have been recently performed in order to increase the flexibility of the ceramic component and to facilitate the biomaterial preparation. In previous studies we have evaluated the response of both L929 fibroblasts and Saos-2 osteoblasts to hydroxyapatite-betaTCP/agarose disks observing a higher sensitivity of osteoblasts to this biomaterial. In the present study, the use of specific fluorescent probes and antibodies has allowed to evaluate different cell function parameters as biocompatibility markers for the cell/biomaterial interaction of Saos-2 osteoblasts cultured for 7 days on hydroxyapatite-betaTCP/agarose disks. The cell cycle subG(1) fraction, the exposition of phosphatidylserine on the outside surface of the plasma membrane and the analysis of plasma membrane integrity versus cell size, indicate that the interaction with the biomaterial induces a light increase of apoptosis in osteoblasts without producing cell necrosis. The high percentage of viable cells on the biomaterial and the preservation of endothelial nitric oxide synthase (eNOS) expression, eNOS activity and mitochondrial membrane potential (Deltapsi(m)), demonstrate the good biocompatibility of hydroxyapatite-betaTCP/agarose disks and its potential utility for bone substitution and repair.     

Metabolic control of T cell activation and death in SLE

Systemic lupus erythematosus (SLE) is characterized by abnormal T cell activation and death, processes which are crucially dependent on the controlled production of reactive oxygen intermediates (ROI) and of ATP in mitochondria. The mitochondrial transmembrane potential (Deltapsi(m)) has conclusively emerged as a critical checkpoint of ATP synthesis and cell death. Lupus T cells exhibit persistent elevation of Deltapsi(m) or mitochondrial hyperpolarization (MHP) as well as depletion of ATP and glutathione which decrease activation-induced apoptosis and instead predispose T cells for necrosis, thus stimulating inflammation in SLE. NO-induced mitochondrial biogenesis in normal T cells accelerates the rapid phase and reduces the plateau of Ca(2+) influx upon CD3/CD28 co-stimulation, thus mimicking the Ca(2+) signaling profile of lupus T cells. Treatment of SLE patients with rapamycin improves disease activity, normalizes CD3/CD28-induced Ca(2+) fluxing but fails to affect MHP, suggesting that altered Ca(2+) fluxing is downstream or independent of mitochondrial dysfunction. Understanding the molecular basis and consequences of MHP is essential for controlling T cell activation and death signaling in SLE.     

Caspase-8 activation precedes alterations of mitochondrial membrane potential during monocyte apoptosis induced by phagocytosis and killing of Staphylococcus aureus

Human peripheral blood monocytes become apoptotic following phagocytosis and killing of Staphylococcus aureus. Although this type of monocyte apoptosis is known to be initiated by Fas-Fas ligand (FasL) interactions, the downstream signaling pathway has not been determined. In this work the involvement of mitochondria and the kinetics of caspase-8 and caspase-3 activation after phagocytosis of S. aureus were studied. Caspase-8 activity was measured in cell lysates by using the fluorogenic substrate Ac-IETD-AFC. Active caspase-3 levels and mitochondrial membrane potential (Deltapsi(m)) were measured in whole cells by flow cytometry using monoclonal antibodies reacting with activated caspase-3 and chloromethyl-X-rosamine, respectively. The results show that caspase-8 was activated shortly after phagocytosis of bacteria. Caspase-8 activation was followed by progressive disruption of Deltapsi(m), which is associated with the production of reactive oxygen intermediates. The irreversible caspase-8 inhibitor zIETD-FMK prevented the disruption of Deltapsi(m) and the release of cytochrome c from S. aureus-exposed monocytes. Caspase-3 activation occurred following disruption of Deltapsi(m). These results strongly suggest that apoptosis of monocytes that have phagocytosed and killed S. aureus is driven by the Fas-FasL-initiated pathway, which is typical for type II cells.     

Nitric oxide production by endothelial cells derived from blood progenitors cultured on NaOH-treated polycaprolactone films: A biofunctionality study

Poly(ε-caprolactone) (PCL) is a biodegradable polyester whose biocompatibility has been widely demonstrated both in vivo and in vitro. In the last few years, our group has confirmed that NaOH-treated PCL films can serve as a suitable biomaterial for vascular tissue engineering by supporting the culture of primary vascular cells and, more recently, endothelial-like EC₂ cells derived from endothelial progenitor cells (EPC). In the present study, NO production in basal conditions and after stimulation with different agents has been evaluated and related to the reactive oxygen species (ROS) content and the intracellular calcium levels on EC₂ cells cultured on NaOH-treated PCL films. The results obtained demonstrate that EC₂ seeded on NaOH-treated PCL films enhance the basal NO levels and show a faster, more intense response to physiological stimuli such as VEGF, bradykinin and thrombin than vein endothelial cells (ECv). This result could be indicative of a better capacity of EC₂ cells to maintain their endothelial functionality when seeded on polymers. On the other hand, the culture of both EC₂ and ECv cells on NaOH-treated PCL films induces a significant increase in both ROS content and intracellular calcium that is balanced out through the stimulation of NO production in these cells. In conclusion, these results demonstrate the ability of NaOH-treated PCL films to support endothelial cell production of nitric oxide and reinforce the idea of considering the endothelial-like EC₂ cells derived from blood progenitors as an adequate source of endothelial cells to functionalize vascular grafts. Furthermore, NaOH-treated PCL films could be considered as a promising cellular NO production-inducing biomaterial for vascular tissue engineering applications.     

Mitochondrial Dynamics and Motility Inside Living Vascular Endothelial Cells: Role of Bioenergetics

The mitochondrial network is dynamic with conformations that vary between a tubular continuum and a fragmented state. The equilibrium between mitochondrial fusion/fission, as well as the organelle motility, determine network morphology and ultimately mitochondrial/cell function. Network morphology has been linked with the energy state in different cell types. In this study, we examined how bioenergetic factors affect mitochondrial dynamics/motility in cultured vascular endothelial cells (ECs). ECs were transduced with mitochondria-targeted green fluorescent protein (mito-GFP) and exposed to inhibitors of oxidative phosphorylation (OXPHOS) or ATP synthesis. Time-lapse fluorescence videos were acquired and a mathematical program that calculates size and speed of each mitochondrial object at each time frame was developed. Our data showed that inner mitochondrial membrane potential (ΔΨ(m)), ATP produced by glycolysis, and, to a lesser degree, ATP produced by mitochondria are critical for maintaining the mitochondrial network, and different metabolic stresses induce distinct morphological patterns (e.g., mitochondrial depolarization is necessary for "donut" formation). Mitochondrial movement, characterized by Brownian diffusion with occasional bursts in displacement magnitude, was inhibited under the same conditions that resulted in increased fission. Hence, imaging/mathematical analysis shed light on the relationship between bioenergetics and mitochondrial network morphology; the latter may determine EC survival under metabolic stress.     

In vitro biocompatibility assessment of poly(epsilon-caprolactone) films using L929 mouse fibroblasts

Biodegradable and biocompatible materials are the basis for tissue engineering. As an initial step for developing vascular grafts, the in vitro biocompatibility of poly(epsilon-caprolactone) (PCL), recently suggested for several clinical applications, was evaluated in this study using L929 mouse fibroblasts. Different cellular aspects were analyzed in order to know the cell viability during cell culture on PCL films: adhesion, proliferation, morphology, LDH release and mitochondrial function. Since topography and other surface characteristics of materials play an essential part in cell adhesion, PCL membranes with either smooth or rough surface were prepared, characterized and used to carry out cell cultures. During short culture times, PCL produced a significant stimulation of mitochondrial activity evaluated by reduction of the MTT reagent. The results provide evidences of good adhesion, growth, viability, morphology and mitochondrial activity of cells on PCL films. Therefore, it can be concluded that PCL is a suitable and biocompatible material as a scaffold for vascular graft development.     

Capsaicin-induced depolarisation of mitochondria in dorsal root ganglion neurons is enhanced by vanilloid receptors

Capsaicin, a pungent ingredient of hot chilli peppers, triggered Ca(2+) influx in dorsal root ganglion (DRG) neurons, which express specific vanilloid receptors of type 1, with ED(50)<100 nM. An increase in capsaicin concentration to 10 microM inhibited Ca(2+) clearance from the cytosol, but did not affect the amplitude of intracellular Ca(2+) elevation. In DRG neurons, 10 microM capsaicin also produced a significant drop in mitochondrial membrane potential (Deltapsi), as measured with the mitochondria-specific potentiometric fluorescent dye JC-1. Similar loss of mitochondrial potential upon application of capsaicin was observed in non-neuronal primary (human lymphocytes) and transformed (human myeloid leukaemia cell line, HL-60) cells. The EC(50) values for capsaicin-induced mitochondrial depolarisation were 6.9 microM (DRG neurons), 200 microM (human lymphocytes) and 150 microM (HL-60 cells). Removal of extracellular Ca(2+) or an application of the antioxidant trolox attenuated capsaicin-induced dissipation of Deltapsi in DRG neurons, but not in human lymphocytes and HL-60 cells. Rotenone, an inhibitor of complex I of the mitochondrial respiratory chain, and oligomycin, an inhibitor of F(0)F(1)-ATPase, significantly enhanced the mitochondrial depolarisation produced by capsaicin in DRG neurons. In human lymphocytes and HL-60 cells, only oligomycin potentiated the effect of capsaicin. From our results, we suggest that, in DRG neurons and non-neuronal cells, capsaicin dissipates Deltapsi, possibly due to a direct inhibition of complex I of the mitochondrial respiratory chain. The presence of vanilloid receptor-1 in DRG neurons makes their mitochondria 20-30-fold more sensitive to the depolarising effect of capsaicin compared with non-neuronal cells lacking vanilloid receptor-1. The higher sensitivity of DRG neurons to capsaicin may underlie a selective neurotoxicity of capsaicin towards sensory neurons.     

Mitochondria-targeted cytoprotective peptides for ischemia-reperfusion injury

It is now recognized that oxidative injury and mitochondrial dysfunction are responsible for many clinical disorders with unmet needs, including ischemia-reperfusion injury, neurodegeneration, and diabetes. Mitochondrial dysfunction can lead to cell death by apoptosis or necrosis. As mitochondria are the major source of intracellular reactive oxygen species (ROS), and mitochondria are also the primary target for ROS, the ideal drug therapy needs to be targeted to mitochondria. A number of approaches have been used for targeted delivery of therapeutic agents to mitochondria. This review will focus on a novel class of cell-permeable small peptides (Szeto-Schiller peptides) that selectively partition to the inner mitochondrial membrane and possess intrinsic mitoprotective properties. Studies with isolated mitochondrial preparations and cell cultures show that these SS peptides can scavenge ROS, reduce mitochondrial ROS production, and inhibit mitochondrial permeability transition. They are very potent in preventing apoptosis and necrosis induced by oxidative stress or inhibition of the mitochondrial electron transport chain. These peptides have demonstrated excellent efficacy in animal models of ischemia-reperfusion, neurodegeneration, and renal fibrosis, and they are remarkably free of toxicity. The pharmacology of the SS peptides in models of ischemia-reperfusion will be the focus of this review.     

Development of a fibrin composite-coated poly(epsilon-caprolactone) scaffold for potential vascular tissue engineering applications

Poor cell adhesion, cytotoxicity of degradation products and lack of biological signals for cell growth, survival, and tissue generation are the limitations in the use of a biodegradable polymer scaffold for vascular tissue engineering. We have fabricated a hybrid scaffold by integrating physicochemical characteristics of poly(epsilon-caprolactone) (PCL) and biomimetic property of a composite of fibrin, fibronectin, gelatin, growth factors, and proteoglycans to improve EC growth on the scaffold. Solvent cast porous films of poly(epsilon-caprolactone) was prepared using PEG as a porogen. Porosity varied between 5 and 200 microm, and FTIR spectroscopy confirmed structural aspects of PCL. Films kept in PBS for 60 days showed tensile strength and elongation matching native blood vessel. Slow degradation of the scaffold was demonstrated by gravimetric analysis and molecular weight determination. Human umbilical vein endothelial cell (HUVEC) adhesion and proliferation on bare films were minimal. FTIR spectroscopy and environmental scanning electron microscopy (ESEM) of PCL-fibrin hybrid scaffold confirmed the presence of fibrin composite on PCL film. HUVEC was subsequently cultured on hybrid scaffold, and continuous EC lining was observed in 15 and 30 days of culture using ESEM. Results suggest that the new hybrid scaffold can be a suitable candidate for cardiovascular tissue engineering.     

Endothelial Cell–Smooth Muscle Cell Co-Culture in a Perfusion Bioreactor System

Vascular endothelial cells (EC) are exposed to a complex biomechanical environment in vivo and are responsible for relaying important messages to the underlying tissue. EC and smooth muscle cells (SMC) communicate to regulate vascular development and function. In this work, a vascular perfusion bioreactor is used to grow tubular constructs seeded with EC and SMC under pulsatile shear stress in long-term co-culture to study the effects of EC on SMC function. SMC seeded into porous poly(glycolic acid) tubular scaffolds are cultured in the bioreactor for 25 days. Constructs are seeded with EC on day 10 or day 23 creating 2-day (short-term) or 15-day (long-term) EC and SMC co-cultures. Long-term EC–SMC co-culture significantly increases cell proliferation and downregulates collagen and proteoglycan deposition compared to short-term co-culture. After 25 days of culture, 15-day co-culture constructs have a more uniform cell distribution across the construct thickness and SMC express a more contractile phenotype compared to 2-day co-culture constructs. These data demonstrate strong interactions between SMC and EC in the bioreactor under physiologically relevant conditions. Thus, the vascular construct perfusion bioreactor is an important tool to investigate cell–cell and cell–extracellular matrix interactions in vascular cell biology and tissue engineering.     

Mitochondria-targeted redox probes as tools in the study of oxidative damage and ageing

Mitochondrial reactive oxygen species (ROS) and oxidative damage are associated with a range of age-related human pathologies. It is also likely that mitochondrial ROS generation is a factor in stress response and signal transduction pathways. However, current methods for measuring and influencing mitochondrial ROS production in vivo often lack the desired specificity. To help elucidate the potential role of mitochondrial ROS production in ageing, we have developed a range of mitochondria-targeted ROS probes that may be useful in vivo. This was achieved by covalently attaching a lipophilic cation to a ROS-reactive moiety causing its membrane potential-dependent accumulation within mitochondria. Mitochondria-targeted molecules developed so far include antioxidants that detoxify mitochondrial ROS, probes that react with mitochondrial ROS, and reagents that specifically label mitochondrial protein thiols. Here, we outline how the formation and consequences of mitochondrial ROS production can be investigated using these probes.     

氧化应激与2型糖尿病

 胰岛素抵抗、胰岛 细胞功能受损是2型糖尿病的主要病因。高血糖、高血脂导致在代谢过程中，线粒体产生大量活性氧，其可损坏线粒体功能，引起氧化应激反应。氧化应激可以产生以下结果：（1）阻断胰岛素作用通路，导致胰岛素抵抗；（2）降低胰岛素基因表达水平；（3）抑制胰岛素分泌；（4）促进 细胞凋亡等。本文主要针对活性氧的产生、氧化应激诱导胰岛素抵抗和胰岛 细胞功能受损等机制进行了综述。     

Mechanical properties of the extracellular matrix alter expression of smooth muscle protein LPP and its partner palladin; relationship to early atherosclerosis and vascular injury

Lipoma preferred partner (LPP) localizes to focal adhesions/dense bodies, is selectively expressed in smooth muscle cells (SMC) and enhances cell migration. SMCs cultured on denatured collagen or on a rigid substrate, up regulated expression of LPP, its partner palladin, tenascin C (TN-C), phosphorylated focal adhesion kinase (pFAK) and exhibited robust stress fibers. In an endothelial (EC)/SMC hemodynamic flow system, shear stress waveforms mimicking atheroprone flow, applied to the EC layer, significantly decreased expression of SMC LPP and palladin. They were also down regulated with TN-C, in an ApoE murine model of atherosclerosis and with oxidative stress but up regulated in an arterial injury model in response to upstream sequential changes in pFAK, Prx1 and TN-C. In conclusion, expression of LPP and palladin are modulated by a mix of mechanical cues, oxidative stress and substrate composition which translate into their up or down regulation in vessel wall injury and early atherogenesis.     

Insulin-like growth factor-1 protects against prion peptide-induced cell death in neuronal cells via inhibition of Bax translocation

Insulin-like growth factor-1 (IGF-1) is one of the most important components of bovine colostrum. It exhibits antiapoptotic and antioxidative activities. Prion diseases are neurodegenerative disorders caused by cell death through mitochondrial dysfunction and increasing generation of reactive oxygen species (ROS). This study examined the protective effect of IGF-1 on residues 106-126 of the cellular prion protein [PrP (106-126)]-mediated mitochondrial neurotoxicity and oxidative stress. In SH-SY5Y human neuronal cells, treatment with PrP (106-126) decreased the cell viability and IGF-1 pretreatment markedly blocked the PrP?(106-126)-induced neuronal cell death. IGF-1 inhibited PrP?(106-126)-induced intracellular ROS generation and mitochondrial oxidative stress. In addition, IGF-1 blocked the translocation of the Bax protein to the mitochondria induced by PrP (106-126). These results demonstrate that IGF-1 protects neuronal cells against PrP (106-126)-mediated neurotoxicity through an antioxidative effect and blockage of mitochondrial Bax translocation. The results also suggest that regulation of IGF-1 secretion may have a therapeutic potential in the management of mitochondrial dysfunction and oxidative stress-induced neurodegeneration.     

Enhanced smooth muscle cell adhesion and proliferation on protein-modified polycaprolactone-based copolymers

Smooth muscle cells (SMC) were cultured for up to 6 days on copolymer films fabricated from a PCL-PEG-PCL block copolymer or P(epsilon-CL-co-D,L-LA)-PEG-P(epsilon-CL-co-D,L-LA), named P(100/0) and P(70/30), respectively. The films were modified by aminolysis using 1,6-hexanediamine, and fibronectin, fibrinogen, or fibrin layers were subsequently immobilized by physisorption or by covalent coupling using imidoester chemistry. Immobilization of all the tested proteins resulted in significantly enhanced cell adhesion on these polymers. Moreover, we found that covalently immobilized proteins supported significantly greater cell proliferation than physisorbed proteins over 6 days. SMC cultured on P(100/0) films modified by covalently attached fibronectin or fibrin layers proliferated at a rate comparable to that observed on control tissue culture polystyrene. The proposed surface modification schemes were much less efficient in improving cell attachment and proliferation on P(70/30) films. However, prewetting P(70/30) with a phosphate buffer prior to aminolysis significantly improved cell numbers following immobilization of fibronectin. Immunostaining of smooth muscle-specific alpha-actin of SMC grown on protein-modified P(100/0) 8 h and 48 h after cell seeding, confirmed preserved SMC phenotype on all modified surfaces.     

雷帕霉素对小鼠星形胶质细胞体外诱导凋亡的影响

目的:探讨雷帕霉素(rapamycin,Rapa)对小鼠星形胶质细胞体外凋亡的影响。方法:无菌分离并体外培养C57BL/6J幼鼠脑组织星形胶质细胞。通过MTT比色法测定并分析Rapa浓度对幼鼠星形胶质细胞存活的影响;SYTOXGreen荧光染色联合荧光酶标仪检测并分析Rapa对H2O2、ionomycin、deferorxamine等诱导剂作用一定时间内细胞存活的影响;Di OC6(3)染色分析Rapa在H2O2氧化应激损伤条件下对星形胶质细胞线粒体膜电势的影响;分别采用H2DCFDA和Mito SOXTMRed荧光染色联合流式细胞术检测Rapa预适应对星形胶质细胞ROS生成以及线粒体内ROS含量的影响。结果:Rapa能促进H2O2以及ionomycin联合deferorxamine损伤作用下的星形胶质细胞的存活,对线粒体膜电势有保护作用,可降低H2O2损伤作用下星形胶质细胞ROS的产生并可以维持胞内线粒体ROS的含量在较低水平。结论:Rapa能够减少细胞内ROS的生成量并降低胞内线粒体内ROS水平;能够减轻H2O2对细胞线粒体膜的损伤破坏,维护线粒体膜电势的稳定性,进而对氧化应激损伤介导细胞凋亡有一定的抑制作用。     

Oxidative glutamate toxicity involves mitochondrial dysfunction and perturbation of intracellular Ca2+ homeostasis

Glutamate toxicity on PC12 cells is mediated by oxidative stress as a consequence of the inhibition of a cystine uptake system with depletion of GSH. In this study we report that glutamate decreases PC12 cell viability, inhibiting the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). This decrease was prevented by the antioxidants vitamin E, idebenone and L-deprenyl, which were also shown to be effective in reducing the accumulation of reactive oxygen species (ROS) in cells exposed to glutamate, decreasing the fluorescence of 2',7'-dichlorofluorescein (DCF). Incubation of PC12 cells with high glutamate concentrations induced mitochondrial dysfunction, leading to the loss of mitochondrial transmembrane potential, evaluated as a decrease in rhodamine 123 (Rh123) retention by mitochondria, and to the decrease of intracellular ATP levels. The mitochondrial dysfunction, induced by glutamate, can be involved in the observed increase of [Ca2+]i. The elevation of [Ca2+]i occurred after GSH depletion, suggesting that oxidative stress is involved in the disturbances of intracellular calcium homeostasis. In conclusion, our data indicate that glutamate, at concentrations which block cystine uptake in PC12 cells leading to GSH depletion and inducing oxidative stress, increases ROS accumulation and decreases cell survival by a mechanism involving mitochondrial dysfunction and impairment of Ca2+ homeostasis.