A new protocol for functional analysis of adipogenesis using reverse transfection technology and time‐lapse video microscopy

Since the worldwide increase in obesity represents a growing challenge for healthcare systems, research focusing on fat cell metabolism has become a focal point of interest. Here, we describe a small interfering RNA (siRNA)‐technology‐based screening method to study fat cell differentiation in human primary preadipocytes that could be further developed towards an automated middle‐throughput screening procedure. First, we established optimal conditions for the reverse transfection of human primary preadipocytes demonstrating that an efficient reverse transfection of preadipocytes is technically feasible. Aligning the processes of reverse transfection and fat cell differentiation utilizing peroxisome proliferator‐activated receptor γ (PPARγ)‐siRNA, we showed that preadipocyte differentiation was suppressed by knock‐down of PPARγ, the key regulator of fat cell differentiation. The use of fluorescently labelled fatty acids in combination with fluorescence time‐lapse microscopy over a longer period of time enabled us to quantify the PPARγ phenotype. Additionally, our data demonstrate that reverse transfection of human cultured preadipocytes with TIP60 (HIV‐1 Tat‐interacting protein 60)–siRNA lead to a TIP60 knock‐down and subsequently inhibits fat cell differentiation, suggesting a role of this protein in human adipogenesis. In conclusion, we established a protocol that allows for an efficient functional and time‐dependent analysis by quantitative time‐lapse microscopy to identify novel adipogenesis‐associated genes.     

Stable overexpression of DJ‐1 protects H9c2 cells against oxidative stress under a hypoxia condition

It has been well accepted that increased reactive oxygen species (ROS) and the subsequent oxidative stress is one of the major causes of ischemia/reperfusion (I/R) injury. DJ‐1 protein, as a multifunctional intracellular protein, plays an important role in regulating cell survival and antioxidant stress. Here, we wondered whether DJ‐1 overexpression attenuates simulated ischemia/reperfusion (sI/R)‐induced oxidative stress. A rat cDNA encoding DJ‐1 was inserted into a mammalian expression vector. After introduction of this construct into H9c2 myocytes, stable clones were obtained. Western blot analysis of the derived clones showed a 2.6‐fold increase in DJ‐1 protein expressing. Subsequently, the DJ‐1 gene‐transfected and control H9c2 cells were subjected to sI/R, and then cell viability, lactate dehydrogenase, malondialdehyde, intracellular ROS and antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) were measured appropriately. The results showed that stable overexpression of DJ‐1 efficiently attenuated sI/R‐induced viability loss and lactate dehydrogenase leakage. Additionally, stable overexpression of DJ‐1 inhibited sI/R‐induced the elevation of ROS and MDA contents followed by the increase of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) activities and expression. Our data indicate that overexpression of DJ‐1 attenuates ROS generation, enhances the cellular antioxidant capacity and prevents sI/R‐induced oxidative stress, revealing a novel mechanism of cardioprotection. Importantly, DJ‐1 overexpression may be an important part of a protective strategy against ischemia/reperfusion injury. Copyright © 2012 John Wiley & Sons, Ltd.     

Mechanism of membrane pore formation by human gasdermin‐D

Gasdermin‐D (GSDMD), a member of the gasdermin protein family, mediates pyroptosis in human and murine cells. Cleaved by inflammatory caspases, GSDMD inserts its N‐terminal domain (GSDMD^Nterm) into cellular membranes and assembles large oligomeric complexes permeabilizing the membrane. So far, the mechanisms of GSDMD^Nterm insertion, oligomerization, and pore formation are poorly understood. Here, we apply high‐resolution (≤ 2 nm) atomic force microscopy (AFM) to describe how GSDMD^Nterm inserts and assembles in membranes. We observe GSDMD^Nterm inserting into a variety of lipid compositions, among which phosphatidylinositide (PI(4,5)P2) increases and cholesterol reduces insertion. Once inserted, GSDMD^Nterm assembles arc‐, slit‐, and ring‐shaped oligomers, each of which being able to form transmembrane pores. This assembly and pore formation process is independent on whether GSDMD has been cleaved by caspase‐1, caspase‐4, or caspase‐5. Using time‐lapse AFM, we monitor how GSDMD^Nterm assembles into arc‐shaped oligomers that can transform into larger slit‐shaped and finally into stable ring‐shaped oligomers. Our observations translate into a mechanistic model of GSDMD^Nterm transmembrane pore assembly, which is likely shared within the gasdermin protein family.     

N‐acetylcysteine counteracts oxidative stress and prevents hCG‐induced apoptosis in rat Leydig cells through down regulation of caspase‐8 and JNK

We have earlier reported that following persistent stimulation with hCG, oxidative stress‐induced apoptosis in rat Leydig cells was mainly achieved through the extrinsic pathway. In the present study, the role of N‐acetylcysteine (NAC) in counteracting the oxidative stress and the mechanisms of inhibition of apoptosis under such conditions were investigated. NAC (1 mM) intervention with repeated hCG stimulation (50 ng/ml, four times, each with 30 min challenge) prevented the decline in Leydig cell viability and the rise in lipid peroxidation and reactive oxygen species. Simultaneously, the activities of the enzymes glutathione‐S‐transferase, catalase, superoxide dismutase and the intracellular glutathione and antioxidant capacity of the treated cells improved significantly. Apoptotic markers Fas, FasL, and caspase‐8, up‐regulated following repeated hCG exposure, were significantly down‐regulated following NAC co‐incubation. While Bcl‐2 expression was fully restored, Bax and caspase‐9 remained unchanged. NAC treatment induced down‐regulation of upstream JNK/pJNK and down‐stream caspase‐3 in the target cells. Taken together, the above findings indicate that NAC counteracted the oxidative stress in Leydig cells induced as a result of repeated hCG stimulation, and inhibited apoptosis by mainly regulating the extrinsic and JNK pathways of metazoan apoptosis. Mol. Reprod. Dev. 77:900–909, 2010. © 2010 Wiley‐Liss, Inc.     

Characterization of a novel angiogenic model based on stable, fluorescently labelled endothelial cell lines amenable to scale-up for high content screening

Background. Blood vessel formation is important for many physiological and pathological processes and is therefore a critical target for drug development. Inhibiting angiogenesis to starve a tumour or promoting ‘normalization’ of tumour vasculature in order to facilitate delivery of anticancer drugs are both areas of active research. Recapitulation of vessel formation by human cells in vitro allows the investigation of cell—cell and cell—matrix interactions in a controlled environment and is therefore a crucial step in developing HCS (high content screening) and HTS (high throughput screening) assays to search for modulators of blood vessel formation. HUVECs (human umbilical‐vein endothelial cells) exemplify primary cells used in angiogenesis assays. However, primary cells have significant limitations that include phenotypic decay and/or senescence by six to eight passages in culture, making stable integration of fluorescent markers and large‐scale expansion for HTS problematic. To overcome these limitations for HTS, we developed a novel angiogenic model system that employs stable fluorescent endothelial cell lines based on immortalized HMECs (human microvascular endothelial cell). We then evaluated HMEC cultures, both alone and co‐cultured with an EMC (epicardial mesothelial cell) line that contributes vascular smooth muscle cells, to determine the suitability for HTS or HCS. Results. The endothelial and epicardial lines were engineered to express a panel of nuclear‐ and cytoplasm‐localized fluorescent proteins to be mixed and matched to suit particular experimental goals. HMECs retained their angiogenic potential and stably expressed fluorescent proteins for at least 13 passages after transduction. Within 8 h after plating on Matrigel, the cells migrated and coalesced into networks of vessel‐like structures. If co‐cultured with EMCs, the branches formed cylindrical‐shaped structures of HMECs surrounded by EMC derivatives reminiscent of vessels. Network formation measurements revealed responsiveness to media composition and control compounds. Conclusions. HMEC‐based lines retain most of the angiogenic features of primary endothelial cells and yet possess long‐term stability and ease of culture, making them intriguing candidates for large‐scale primary HCS and HTS (of ～10000–1000000 molecules). Furthermore, inclusion of EMCs demonstrates the feasibility of using epicardial‐derived cells, which normally contribute to smooth muscle, to model large vessel formation. In summary, the immortalized fluorescent HMEC and EMC lines and straightforward culture conditions will enable assay development for HCS of angiogenesis.     

DJ‐1 plays an obligatory role in the cardioprotection of delayed hypoxic preconditioning against hypoxia/reoxygenation‐induced oxidative stress through maintaining mitochondrial complex I activity

DJ‐1 was recently reported to mediate the cardioprotection of delayed hypoxic preconditioning (DHP) by suppressing hypoxia/reoxygenation (H/R)‐induced oxidative stress, but its mechanism against H/R‐induced oxidative stress during DHP is not fully elucidated. Here, using the well‐established cellular model of DHP, we again found that DHP significantly improved cell viability and reduced lactate dehydrogenase release with concurrently up‐regulated DJ‐1 protein expression in H9c2 cells subjected to H/R. Importantly, DHP efficiently improved mitochondrial complex I activity following H/R and attenuated H/R‐induced mitochondrial reactive oxygen species (ROS) generation and subsequent oxidative stress, as demonstrated by a much smaller decrease in reduced glutathione/oxidized glutathione ratio and a much smaller increase in intracellular ROS and malondialdehyde contents than that observed for the H/R group. However, the aforementioned effects of DHP were antagonized by DJ‐1 knockdown with short hairpin RNA but mimicked by DJ‐1 overexpression. Intriguingly, pharmacological inhibition of mitochondria complex I with Rotenone attenuated all the protective effects caused by DHP and DJ‐1 overexpression, including maintenance of mitochondria complex I and suppression of mitochondrial ROS generation and subsequent oxidative stress. Taken together, this work revealed that preserving mitochondrial complex I activity and subsequently inhibiting mitochondrial ROS generation could be a novel mechanism by which DJ‐1 mediates the cardioprotection of DHP against H/R‐induced oxidative stress damage.     

The expression of MMP‐1 and MMP‐9 is up‐regulated by smooth muscle cells after their cross‐talk with macrophages in high glucose conditions

Patients with diabetes mellitus have an increased risk of myocardial infarction and coronary artery disease‐related death, exhibiting highly vulnerable plaques. Many studies have highlighted the major role of macrophages (MAC) and smooth muscle cells (SMC) and the essential part of metalloproteases (MMPs) in atherosclerotic plaque vulnerability. We hypothesize that in diabetes, the interplay between MAC and SMC in high glucose conditions may modify the expression of MMPs involved in plaque vulnerability. The SMC‐MAC cross‐talk was achieved using trans‐well chambers, where human SMC were grown at the bottom and human MAC in the upper chamber in normal (NG) or high (HG) glucose concentration. After cross‐talk, the conditioned media and cells were isolated and investigated for the expression of MMPs, MCP‐1 and signalling molecules. We found that upon cross‐talk with MAC in HG, SMC exhibit: (i) augmented expression of MMP‐1 and MMP‐9; (ii) significant increase in the enzymatic activity of MMP‐9; (iii) higher levels of soluble MCP‐1 chemokine which is functionally active and involved in MMPs up‐regulation; (iv) activated PKCα signalling pathway which, together with NF‐kB are responsible for MMP‐1 and MMP‐9 up‐regulation, and (v) impaired function of collagen assembly. Taken together, our data indicate that MCP‐1 released by cell cross‐talk in diabetic conditions binds to CCR2 and triggers MMP‐1 and MMP‐9 over‐expression and activity, features that could explain the high vulnerability of atherosclerotic plaque found at diabetic patients.     

Inhibition of Rho‐kinase protects cerebral barrier from ischaemia‐evoked injury through modulations of endothelial cell oxidative stress and tight junctions

Ischaemic strokes evoke blood–brain barrier (BBB) disruption and oedema formation through a series of mechanisms involving Rho‐kinase activation. Using an animal model of human focal cerebral ischaemia, this study assessed and confirmed the therapeutic potential of Rho‐kinase inhibition during the acute phase of stroke by displaying significantly improved functional outcome and reduced cerebral lesion and oedema volumes in fasudil‐ versus vehicle‐treated animals. Analyses of ipsilateral and contralateral brain samples obtained from mice treated with vehicle or fasudil at the onset of reperfusion plus 4 h post‐ischaemia or 4 h post‐ischaemia alone revealed these benefits to be independent of changes in the activity and expressions of oxidative stress‐ and tight junction‐related parameters. However, closer scrutiny of the same parameters in brain microvascular endothelial cells subjected to oxygen–glucose deprivation ± reperfusion revealed marked increases in prooxidant NADPH oxidase enzyme activity, superoxide anion release and in expressions of antioxidant enzyme catalase and tight junction protein claudin‐5. Cotreatment of cells with Y‐27632 prevented all of these changes and protected in vitro barrier integrity and function. These findings suggest that inhibition of Rho‐kinase after acute ischaemic attacks improves cerebral integrity and function through regulation of endothelial cell oxidative stress and reorganization of intercellular junctions.

     

Fabricating PLGA microparticles with high loads of the small molecule antioxidant N‐acetylcysteine that rescue oligodendrocyte progenitor cells from oxidative stress

Reactive oxygen species (ROS), encompassing all oxygen radical or non‐radical oxidizing agents, play key roles in disease progression. Controlled delivery of antioxidants is therapeutically relevant in such oxidant‐stressed environments. Encapsulating small hydrophilic molecules into hydrophobic polymer microparticles via traditional emulsion methods has long been a challenge due to rapid mass transport of small molecules out of particle pores. We have developed a simple alteration to the existing water‐in‐oil‐in‐water (W/O/W) drug encapsulation method that dramatically improves loading efficiency: doping external water phases with drug to mitigate drug diffusion out of the particle during fabrication. PLGA microparticles with diameters ranging from 0.6 to 0.9 micrometers were fabricated, encapsulating high loads of 0.6–0.9 µm diameter PLGA microparticles were fabricated, encapsulating high loads of the antioxidant N‐acetylcysteine (NAC), and released active, ROS‐scavenging NAC for up to 5 weeks. Encapsulation efficiencies, normalized to the theoretical load of traditional encapsulation without doping, ranged from 96% to 400%, indicating that NAC‐loaded external water phases not only prevented drug loss due to diffusion, but also doped the particles with additional drug. Antioxidant‐doped particles positively affected the metabolism of oligodendrocyte progenitor cells (OPCs) under H₂O₂‐mediated oxidative stress when administered both before (protection) or after (rescue) injury. Antioxidant doped particles improved outcomes of OPCs experiencing multiple doses of H₂O₂ by increasing the intracellular glutathione content and preserving cellular viability relative to the injury control. Furthermore, antioxidant‐doped particles preserve cell number, number of process extensions, cytoskeletal morphology, and nuclear size of H₂O₂‐stressed OPCs relative to the injury control. These NAC‐doped particles have the potential to provide temporally‐controlled antioxidant therapy in neurodegenerative disorders such as multiple sclerosis (MS) that are characterized by continuous oxidative stress.     

TNF‐α promotes survival and migration of MSCs under oxidative stress via NF‐κB pathway to attenuate intimal hyperplasia in vein grafts

The oxidative stress caused by endothelial injury is involved in intimal hyperplasia (IH) in vein grafts. Mesenchymal stem cells (MSCs) can home to injured intima and promote endothelial repair. However, MSC apoptosis is increased accompanied by decreased functional activity under oxidative stress. Thus, we investigate whether tumour necrosis factor‐α (TNF‐α) can promote the survival and activity of MSCs under oxidative stress to reduce IH more effectively, and establish what role the NF‐κB pathway plays in this. In this study, we preconditioned MSCs with TNF‐α (^{TNF‐α‐PC}MSCs) for 24 hrs and measured the activation of the IKK/NF‐κB pathway. EdU and transwell assays were performed to assess proliferation and migration of ^{TNF‐α‐PC}MSCs. Apoptosis and migration of ^{TNF‐α‐PC}MSCs were evaluated in conditions of oxidative stress by analysis of the expression of Bcl‐2 and CXCR4 proteins. ^{TNF‐α‐PC}MSCs were transplanted into a vein graft model, so that cell homing could be tracked, and endothelial apoptosis and IH of vein grafts were measured. The results demonstrated that TNF‐α promotes proliferation and migration of MSCs. Furthermore, survival and migration of ^{TNF‐α‐PC}MSCs under oxidative stress were both enhanced. A greater number of MSCs migrated to the intima of vein grafts after preconditioning with TNF‐α, and the formation of neointima was significantly reduced. These effects could be partially abolished by IKK XII (NF‐κB inhibitor). All these results indicate that preconditioning with TNF‐α can promote survival and migration of MSCs under oxidative stress via the NF‐κB pathway and thus attenuate IH of vein grafts.     

Subcellular dynamics of proteins and metabolites under abiotic stress reveal deferred response of the Arabidopsis thaliana hexokinase‐1 mutant gin2‐1 to high light

Stress responses in plants imply spatio‐temporal changes in enzymes and metabolites, including subcellular compartment‐specific re‐allocation processes triggered by sudden changes in environmental parameters. To investigate interactions of primary metabolism with abiotic stress, the gin2‐1 mutant, defective in the sugar sensor hexokinase 1 (HXK1) was compared with its wildtype Landsberg erecta (Ler) based on time resolved, compartment‐specific metabolome and proteome data obtained over a full diurnal cycle. The high light sensitive gin2‐1 mutant was substantially delayed in subcellular re‐distribution of metabolites upon stress, and this correlated with a massive reduction in proteins belonging to the ATP producing electron transport chain under high light, while fewer changes occurred in the cold. In the wildtype, compounds specifically protecting individual compartments could be identified, e.g., maltose and raffinose in plastids, myo‐inositol in mitochondria, but gin2‐1 failed to recruit these substances to the respective compartments, or responded only slowly to high irradiance. No such delay was obtained in the cold. At the whole cell level, concentrations of the amino acids, glycine and serine, provided strong evidence for an important role of the photorespiratory pathway during stress exposure, and different subcellular allocation of serine may contribute to the slow growth of the gin2‐1 mutant under high irradiance.