Statin drugs are one of the most commonly prescribed pharmaceuticals by physicians. By blocking the rate-limiting step in the cholesterol biosynthesis pathway, statins inhibit cholesterol synthesis, which benefits patient health. However, since many other important cellular processes are regulated within this pathway, they may also be influenced by statin therapy. These pleiotropic effects of statins have not been fully investigated, but are believed to positively influence endothelial cells (ECs), which line the vasculature in a confluent monolayer. Few studies have considered the effect of blood flow on ECs and how this may augment EC response to statins.2 using a three-dimensional tissue culture model to provide a realistic hemodynamic environment. ECs are analyzed for morphology using light microscopy as well as cytoskeletal structure and alignment using confocal microscopy. Statistical analysis is performed on the results using both the one-way analysis of variance with Bonferroni post-tests and the two-tailed t test.In this study, the effect of statin treatment on ECs is investigated for cells stimulated with tumor necrosis factor alpha (TNF-α), an inflammatory cytokine that promotes an atheroprone endothelium. Additionally, ECs are exposed to a physiologically relevant wall shear stress (WSS) of 12.5 dynes/cmWe have shown that statin treatment caused cells to adapt to a rounded, atheroprone morphology, with a significantly higher shape index. Oppositely, TNF-α stimulation caused cells to elongate to an atheroprotective morphology, with a significantly lower shape index. WSS and TNF-α were unable to reverse any statin-induced cell rounding or F-actin disruption.Further work is therefore needed to determine why statin drugs cause cells to have an atheroprone morphology, but an atheroprotective genotype, and why TNF-α stimulation causes an atheroprotective morphology, but an atheroprone genotype. Despite the morphological changes due to statins or stimulation, ECs still respond to WSS. Understanding how statins influence ECs will allow for more targeted treatments for hypercholestemia and potentially other diseases. Despite improved treatment options available, cardiovascular diseases, such as atherosclerosis, remain a leading cause of death in North America . AtherosClinically, statin drugs [inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase] have been successful in reducing patient cholesterol levels –11, reduECs are a dynamic interface between the blood and vascular tissue and play a pivotal role in disease formation. Dysfunction of the ECs in response to biochemical and hemodynamic stresses can create an inflammatory phenotype associated with atherosclerosis , 19. EarEC morphology is believed to be a key indicator of cellular health , 26, 27.As atherosclerosis is an inflammatory disease, we have continued our studies by looking at the response of an inflamed endothelium to statin therapy. Tumor necrosis factor alpha (TNF-α) is implicated in various inflammatory diseases and is commonly used to simulate an inflamed, atheroprone EC phenotype , 36. TNFThe methods used here are based upon methods previously described elsewhere –25, but ® 184 silicone elastomer (Dow Corning) around a 2 mm diameter stainless steel rod  in a Plexiglas mold for 24 h at 45°C. To sterilize for cell culture, models were boiled in reverse osmosis water for 30 min. A suitable surface for cell growth was created by coating the inner lumen of the model with 40 μg/mL fibronectin . The models were sealed using PVDF filters  and attached to a LabQuake rotor (Barnstead/Thermolyne) and left to rotate overnight within an incubator set at 37°C and 5% CO2 (Thermo Scientific).Flexible, transparent tissue culture models were prepared by curing Sylgard6 cells/mL, and left on the rotor for 24 h to create a confluent monolayer of cells within the tissue culture models.Human abdominal aortic endothelial cells (HAAECs) from a 20-year-old male  were obtained from the supplier and expanded as per supplier protocol. For individual experiments, 1 mL of frozen HAAECs was thawed and cultured within a T175 flask  coated with gelatin  using basal PromoCell media with SupplementMix  containing 0.02 mL/mL fetal calf serum, 0.004 mL/mL endothelial cell growth supplement, 0.1 ng/mL human recombinant epidermal growth factor, 1 ng/mL human recombinant basic fibroblast growth factor, 90 μg/mL heparin, and 1 μg/mL hydrocortisone, with 10% fetal bovine serum  and 1% penicillin–streptomycin . The media was changed every 48 h and HAAECs were split into 4 T175 flasks after 4 days when they became confluent. HAAECs were seeded into the fibronectin-coated models at passage 5, with a concentration of 1.25 × 10Simvastatin , mevalonate , and TNF-α  were obtained from the suppliers and stock solutions of 0.5 mM simvastatin, 50 mM of mevalonate, and 0.002 mg/mL TNF-α were prepared. These stock solutions were added to either the perfusion media, or within the lumen of static models, to obtain the final working concentrations of 10 μM simvastatin, 200 μM mevalonate, and 10 ng/mL TNF-α. MTT assays were used to ensure no significant cell death due to simvastatin, mevalonate, or TNF-α treatment (unpublished data). These particular concentrations were chosen to elicit a reproducible response in vitro , althougA flow diagram of the experimental time line is shown in Figure 2, a hemodynamically relevant level of WSS [All perfused models were sheared at 12.5 dynes/cml of WSS . At t = ® (Mathworks) code to analyze the shape index (SI), a metric that ranges from 0 to 1 [Models were analyzed for both EC morphology and F-actin cytoskeleton structure. For morphology analysis, the models were cut into 1 cm sections and then cut in half through the lumen. The bottom half of the model was analyzed by staining the EC nucleus with a 4% solution of crystal violet  in 1× PBS for 9 min and viewed under a light microscope  with digital camera . A single image was taken of each model piece, with ten representative ECs analyzed from each image. Images were analyzed using Photoshop (Adobe) and a MATLABm 0 to 1 –42, descm 0 to 1 , 43.® 647 , at a 1:40 dilution in 1× PBS with 1% bovine serum albumin  at room temperature. ECs were rinsed and the model was further cut so that only the bottom of the channel remained. The model piece was mounted within a cover slip bottomed petri dish  using a 0.2% Dabco  solution in glycerol. A laser scanning confocal microscope  with Zen (2008) software (Zeiss) was used to image the ECs with a 10×/0.3 DICI or 20×/0.5 DICII objective lens. Image analysis was completed with Photoshop (Adobe) and ImageJ .To analyze the F-actin cytoskeleton, 1 cm model sections were cut and the ECs were blocked and permeabilized with 0.1% Triton™ X-100  at room temperature for 5 min. ECs were washed and then dyed with phalloidin-conjugated Alexa FluorEach experiment was performed independently in triplicate. Prism™ 5 (GraphPad) was used to analyze the data. One-way analysis of variance (one-way ANOVA) with Bonferroni post-tests or unpaired two-tailed t tests were used. A P value of less than 0.05 was considered to be statistically significant.Stimulation with 10 ng/mL TNF-α for 24 h caused a significant decrease in the SI when compared to the non-stimulated, static control, Figure 2, there was a significant decrease in SI when compared to the non-stimulated, perfused control, Figure When ECs were stimulated with 10 ng/mL TNF-α at a WSS of 12.5 dynes/cmA significant increase in the SI was observed when TNF-α stimulated ECs were treated with 10 μM simvastatin under static conditions when compared to the TNF-α stimulated, static control cells, Figure 2 of WSS and treated with 10 μM simvastatin had a significantly higher SI than the TNF-α stimulated, perfused, control cells, Figure Stimulated ECs exposed to 12.5 dynes/cmWhen non-stimulated ECs exposed to flow and TNF-α stimulated ECs left under static conditions were compared, there was no significant difference observed, Figure 2 of WSS when compared to the static control, Figure There is no significant difference in EC SI when the TNF-α stimulated ECs were treated with 10 μM simvastatin and exposed to 12.5 dynes/cmWhen TNF-α stimulated ECs were treated with 10 μM simvastatin, the F-actin cytoskeleton became fragmented and disorganized. This was observed for both static and flow conditions, Figure When TNF-α stimulated ECs were treated with both 10 μM simvastatin and 200 μM mevalonate there was no significant difference from the TNF-α stimulated control cells, under both static and perfused conditions, Figure When TNF-α stimulated ECs were treated with both 10 μM simvastatin and 200 μM mevalonate, the statin-induced F-actin cytoskeleton fragmentation did not occur. This was observed for both static and flow conditions, Figure 2 of steady WSS, statin treatment, and TNF-α stimulation has on HAAEC morphology within three-dimensional tissue culture models. As in accordance with other published literature [2 when compared to the static control. We observed this trend for all of the conditions considered: with and without statin treatment, with or without mevalonate treatment, and with or without TNF-α stimulation.In this work, we investigated the effect of simvastatin on an inflamed endothelium. We show that ECs treated with simvastatin or stimulated with TNF-α are still able to sense and respond to WSS. We quantified the influence that 12.5 dynes/cmterature , 42, 44,terature  of 12.5 It is well established that in the presence of WSS, ECs will elongate and align in the direction of flow , 42, 44.We have also observed that EC stimulation with TNF-α causes a significant elongation (reduction in SI) visually and quantitatively under both static and flow conditions, Figures 2 of steady WSS and TNF-α stimulated ECs exposed to 12.5 dynes/cm2 of steady WSS, Figure The observed elongation is likely related to the EC cytoskeleton. The EC cytoskeleton is believed to mechanotransduce physical stimuli through the cell to biochemical changes , 47. ResIt is believed that the activation of RhoA, by either steady WSS or stimulation with TNF-α, causes the F-actin cytoskeleton to become elongated and stretches out the cell . StudiesIn this work, we have observed a decrease in EC SI with TNF-α stimulation, but an increase in EC SI with statin treatment, even with stimulation and WSS, Figure In our work, we use mevalonate to ensure that the effects that we are observing due to statin treatment are regulated within the cholesterol biosynthesis pathway . Indeed,We have shown that both statin therapy and TNF-α stimulation induce changes in the cell, but that ECs still respond to WSS despite the statin-induced fragmentation. These results suggest that morphology and the state of the F-actin cytoskeleton may not be a true indicator of cellular health, and alternative methods of analysis should be considered. It also shows that EC mechanotransduction occurs despite changes in the F-actin cytoskeleton. Future work will focus on making a quantitative link between statin drugs and RhoA regulation, with the hopes of designing more specific and improved statin drugs.In this work, we studied how an inflamed (TNF-α stimulated) endothelium responds to steady WSS when treated with 10 μM of simvastatin. Statin treatment or TNF-α stimulation did not hinder the ability for ECs to sense and respond to WSS despite significant alterations in the F-actin cytoskeletal structure. Although statin drugs are thought to be beneficial to ECs, they caused the cells to adapt a rounded morphology and fragmented F-actin cytoskeleton, usually considered unhealthy. Conversely, TNF-α stimulation caused the cells to become more elongated, which is usually indicative of a healthy, atheroprotective EC. Neither WSS nor TNF-α stimulation was able to abrogate the statin-induced rounding of cells, or the F-actin cytoskeleton rearrangement due to statin treatment. ECs were still able to respond to WSS by elongating despite changes in the F-actin cytoskeleton structure. This work suggests that an alternate method of determining cellular health is necessary, as cellular morphology does not always correlate with cellular phenotype.


Tumour necrosis factor (TNF)-α has been considered to induce ischaemia-reperfusion injury (IRI) of liver which is characterized by energy dysmetabolism. Peroxisome proliferator–activated receptor-γ co-activator (PGC)-1α and mitofusion2 (Mfn2) are reported to be involved in the regulation of mitochondrial function. However, whether PGC-1α and Mfn2 form a pathway that mediates liver IRI, and if so, what the underlying involvement is in that pathway remain unclear. In this study, L02 cells administered recombinant human TNF-α had increased TNF-α levels and resulted in down-regulation of PGC-1α and Mfn2 in a rat liver IRI model. This was associated with hepatic mitochondrial swelling, decreased adenosine triphosphate (ATP) production, and increased levels of reactive oxygen species (ROS) and alanine aminotransferase (ALT) activity as well as cell apoptosis. Inhibition of TNF-α by neutralizing antibody reversed PGC-1α and Mfn2 expression, and decreased hepatic injury and cell apoptosis both in cell culture and in animals. Treatment by rosiglitazone sustained PGC-1α and Mfn2 expression both in IR livers, and L02 cells treated with TNF-α as indicated by increased hepatic mitochondrial integrity and ATP production, reduced ROS and ALT activity as well as decreased cell apoptosis. Overexpression of Mfn2 by lentiviral-Mfn2 transfection decreased hepatic injury in IR livers and L02 cells treated with TNF-α. However, there was no up-regulation of PGC-1α. These findings suggest that PGC-1α and Mfn2 constitute a regulatory pathway, and play a critical role in TNF-α-induced hepatic IRI. Inhibition of the TNF-α or PGC-1α/Mfn2 pathways may represent novel therapeutic interventions for hepatic IRI. Ischaemia-reperfusion injury (IRI) is the most important cause of liver dysfunction and failure after restoration of liver ischaemia, as occurs during liver transplantation , surgicaTumour necrosis factor (TNF)-α is a key inflammatory cytokine released by infiltrated leucocytes during IRI ,5. InhibEnergy metabolism in mammalian cells is dependent on continuous mitochondrial activity such as mitochondrial fusion, fission and mitochondrial remodelling . MitofusThe aim of the study was to determine the role of the TNF-α/PGC-1α/Mfn2 pathway in the pathogenesis of liver IRI in a rat model of IRI.Expanded materials and methods are available in the Inbred male Sprahue-Dawley rats (200–250 g) and BALB/c mice (6 and 8 weeks old) were purchased from the Center of Experimental Animals . Animal handling procedures were conducted in compliance with guidelines for the Care and Use of Laboratory Animals published by NIH, and all animal experimental protocols were approved by the Animal Care and Use Committee of Tongji Medical College. Human liver specimens were obtained from patients with partial liver resection in Union Hospital, Tongji Medical College of Huazhong University of Science and Technology. The experiments were carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association and the protocols approved by the Institutional Research Review Board at Union Hospital, Tongji Medical College of Huazhong University of Science and Technology with informed consent.Recombinant lentiviral vectors carrying a specific albumin promoter and an Mfn2 (Ltv-Mfn2) gene downstream fused to enhanced green fluorescence protein (EGFP) were generated as described previously –19.L02 cell line was purchased from China Center for Type Culture Collection .For drug treatment, L02 cells were first incubated by recombinant human TNF-α  with or without neutralizing anti-TNF-α  for 24 hrs. Additional recombinant human TNF-α  was administered to L02 cells after pre-treatment with rosiglitazone  for 24 hrs or transfection with Ltv-human-Mfn2 for 72 hrs.For transfection treatment, L02 cells were transfected with Ltv-human-vector or Ltv-human-Mfn2 in the presence of polybrene (5 μg/ml). Transfection efficiency was monitored by fluorescence microscopy, quantitative PCR and Western Blotting after 48 hrs after transfection . Experimet al. [Rat model of partial warm hepatic IRI was produced as described by Ji H et al. .7 TU/ml) in the presence of polybrene  administered by intravenous injection 1 week before the IR operation. Transfection efficiency was monitored by fluorescence microscopy and quantitative PCR  by intraperitoneal injection 2 weeks before the IR operation, and 10 μg of rat neutralizing anti-TNF-α  was infused as soon as blood reperfusion was established. For transfection treatment, rats were exposed to 1 ml of Opti-MEM I medium, containing Ltv-rat-Mfn2 or Ltv-rat-vector .6 and 20 mg liver sample per group were lysed in lysis buffer from an ATP Assay Kit . They were centrifuged at 12,000 g for 10 min., and the supernatants collected. ATP production was then analysed according to the manufacturer's instructions.L02 cells, 1 × 106 cells, and plasma samples were collected and centrifuged at 1000 g for 10 min. ALT activity was then detected in the supernatants by a colorimetric assay according to the manufacturer's instructions.Supernatants of L02 cells and plasma samples were analysed for ALT activity by using an ALT Assay Kit . Briefly, supernatants of 1 × 106 cells, and 1 g liver samples were collected, rinsed with cleaning liquid from the kit, and then centrifuged at 300 × g for 5 min. ROS content of the precipitations was then measured by fluorescence spectrophotometry according to the manufacturer's instructions.L02 cells and liver samples were analysed for ROS content by using a Reactive Oxygen Species Assay Kit . Briefly, 1 × 10Each plasma sample was analysed for TNF-α by ELISA using an ELISA kit  according to the manufacturer's instructions.L02 cell apoptosis was quantitatively assessed by flow cytometry  after labelling by propidium  and allophycocyanin-labelled Annexin V .Apoptosis in liver samples was detected by using an In Situ Cell Death Detection Kit  according to the manufacturer's instructions.RNA from L02 cells and liver samples per group was isolated by TRIzol™ Reagent  according to the manufacturer's protocol. RNA levels were measured by reverse transcription–polymerase chain reaction by using iQ SYBR Green Supermix in an iCycler Real-Time PCR Detection System .Western Blotting was used to measure the levels of TNF-α, PGC-1α and Mfn2 in each group of L02 cells, and liver samples. Optical density of the bands was quantified by using NIH Image J software.anova followed by Student–Newman–Keuls test where appropriate, was used to evaluate the statistical significance. Values of P < 0.05 were considered statistically significant. Experiments were performed in triplicate.All data were presented as mean ± SEM. Six rodents per group were used in the experiments. After demonstration of homogeneity of variance with the Bartlett test, one-way versus 14.17 ± 1.28 pmol) . Apoptosis in these cells as determined by PI and Annexin V labelling showed that there was more apoptosis in human TNF-α-treated versus control cells (24.5% versus 2.0%), reflecting hepatocyte injury , whereas neutralizing anti-TNF-α decreased it (43.04 ± 5.68 RLU) in L02 cells . ALT activity in plasma from rat with liver IR was also significantly increased  with decreased ROS production  (Fig. In vitro, TNF-α induces mitochondrial injury, ATP production deficiency and increase in ROS that leads to hepatocyte injury and apoptosis by sequentially activating the PGC-1α/Mfn2 pathway. In vivo, rats which underwent IR operations had increased TNF-α and the down-regulation of the PGC-1α/Mfn2 pathway that results in hepatocyte injury, as was observed in the cell culture experiments. Furthermore, either inhibition of TNF-α or activation of PGC-1α/Mfn2 pathway by pharmacologic inhibitors or gene transfection decreased IRI in hepatocytes. These findings suggest that liver IRI induced by TNF-α is dependent on the PGC-1α/Mfn2 pathway.IRI increases the risk of death by causing liver dysfunction in patients with liver transplantation , surgicaTumour necrosis factor-α is produced by a variety of inflammatory cells including T cells, B cells, NK cells and macrophages during various pathological and pathophysiological processes, such as tumour development –24, inflTumour necrosis factor-α has been implicated in the progression of IRI in variety organs, and inhibition of TNF-α has been shown to protect organs from IRI. These organs include heart , brain , kidney etc. [Mitochondrial swelling, ATP production deficiency and excess ROS represent characteristics of IRI ,32. Mfn2etc. . Loss oretc. ,34. In tetc. ,14. ThesPGC-1α has been documented to regulate activation of the MEK1/2 and NF-κB signalling pathway in muscle cells . HoweverOther studies have reported that the PGC-1α/Mfn2 regulatory pathway is functional in diseases of energy dysmetabolism, such as type 2 diabetes which support our finding that PGC-1α/Mfn2 plays a key role in the regulation of hepatic mitochondrial function whose abnormality is involved in the pathogenesis of hepatic IRI, .In summary, the findings reported here indicate that the PGC-1α/Mfn2 pathway plays a crucial role in the TNF-α-mediated hepatic IRI. Targeted manipulation of TNF-α and PGC-1α/Mfn2 pathway might protect hepatocytes from IRI.


Retinal inflammatory diseases induced by cytokines, such as tumor necrosis factor-α (TNF-α) are associated with an up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the retinal pigment epithelial cells (RPECs). Retinal pigment epithelium (RPE) is a monolayer of epithelial cells that forms the outer blood-retinal barrier in the posterior segment of the eye, and is also implicated in the pathology of, such as neovascularization in age-related macular degeneration (AMD). However, the detailed mechanisms of TNF-α-induced ICAM-1 expression are largely unclear in human RPECs. We demonstrated that in RPECs, TNF-α could induce ICAM-1 protein and mRNA expression and promoter activity, and monocyte adhesion. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of PKCs (Ro318220), PKCδ (Rottlerin), MEK1/2 (U0126), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of TNFR1, TRAF2, JNK2, p42, or c-Jun. We showed that TNF-α could stimulate the TNFR1 and TRAF2 complex formation. TNF-α-stimulated JNK1/2 was also reduced by Rottlerin or SP600125. However, Rottlerin had no effect on TNF-α-induced p42/p44 MAPK phosphorylation. We observed that TNF-α induced c-Jun phosphorylation which was inhibited by Rottlerin or SP600125. On the other hand, TNF-α-stimulated ICAM-1 promoter activity was prominently lost in RPECs transfected with the point-mutated AP-1 ICAM-1 promoter plasmid. These results suggest that TNF-α-induced ICAM-1 expression and monocyte adhesion is mediated through a TNFR1/TRAF2/PKCδ/JNK1/2/c-Jun pathway in RPECs. These findings concerning TNF-α-induced ICAM-1 expression in RPECs imply that TNF-α might play an important role in ocular inflammation and diseases. Age-related macular degeneration (AMD) is the major cause of blindness. The pathogenesis of AMD involves demise of the retinal pigment epithelium (RPE) followed by death of photoreceptors . The RPETumor necrosis factor-α (TNF-α) is a potent proinflammatory cytokine implicated in tissue damages . The imp2+-binding domains; the novel PKCs  have DAG- but not Ca2+-binding domains; the atypical PKCs  have neither Ca2+- nor DAG-binding domains [Cellular responses to TNF-α are initiated by the type 1 TNFR (TNFR1) and TNFR2 . Most TN domains . Moreove domains , PKCδ [9 domains  activati domains . Moreove domains . Huang e domains . These rIt has been well established that inflammatory responses following exposure to extracellular stimuli are highly dependent on activation of AP-1 or NF-κB, which plays an important role in the expression of several target genes, such as ICAM-1 . AP-1 isIn addressing these questions, experiments were undertaken to investigate the mechanisms underlying TNF-α-induced ICAM-1 expression mediated through PKCδ/JNK1/2/c-Jun activation in RPECs. These findings suggest that in RPECs, TNF-α-induced ICAM-1 expression was, at least in part, mediated through a TNFR1/PKCδ/JNK1/2-dependent c-Jun pathway. These results provide new insights into the mechanisms by which TNF-α induced expression of ICAM-1 on RPECs and thus exaggerated the inflammatory responses.Anti-β-actin, anti-ICAM-1, anti-p42, anti-JNK2, anti-TNFR1, anti-c-Jun, and anti-TRAF2 antibodies were from Santa Cruz . Anti-phospho-PKCδ, anti-phospho-p42/p44 MAPK, anti-phospho-c-Jun, and anti-phospho-JNK1/2 antibodies were from Cell Signaling . Ro318220, Rottlerin, Gö6976, U0126, SB202190, SP600125, and Tanshinone IIA were from Biomol . Anti-TNFR neutralizing antibody, anti-ICAM-1 neutralizing antibody, and TNF-α were from R&D Systems . All other reagents were from Sigma .2 atmosphere. When reached confluence, the cells were subcultured to (1 ml/well) 12-well culture plates and (10 ml/dish) 10-cm culture dishes for the measurement of kinases phosphorylation, protein expression, and mRNA accumulation. In these experiments, TNF-α was added to the serum-free medium and incubated for the indicated time intervals. When the inhibitors were used, they were added 1 h before TNF-α treatment. The concentrations of these inhibitors, vehicle DMSO or TNF-α used alone had no toxic effect and change in the cell viability on RPECs, excluded by LDH release test or XTT assay (data not shown).Human RPECs were from ScienCell Research Lab  and cultured in DMEM/F-12 supplemented with 10% FBS and antibiotics  at 37°C in a humidified 5% COHuman siRNAs of scrambled, TNFR1 (HSS110866), TRAF2 (HSS186411), p42 (HSS108535), JNK2 (HSS108550), and Jun (HSS105641) were from Invitrogen . Human RPECs (passages 4 and 5) were cultured onto 12-well plates. At 70–80% confluence, transient transfection of siRNAs was carried out using Metafectene transfection reagent. Briefly, siRNA (100 nM) was formulated with Metafectene transfection reagent according to the manufacturer’s instruction. The transfection complex was diluted into 400 μl of DMEM/F-12 medium and added directly to the cells. The medium was replaced with serum-free DMEM/F-12 after 24 h. Cells were analyzed at 72 h after transfection by Western blot analysis. The transfection efficiency (approximate 60%) was determined by transfection with EGFP.g at 4°C for 1 h to yield the whole cell extract, as previously described [Growth-arrested cells were incubated with TNF-α at 37°C for the indicated time intervals. The cells were washed, scraped, collected, and centrifuged at 45000 × escribed . SamplesTotal RNA was extracted using TRIzol reagent. mRNA was reverse-transcribed into cDNA and analyzed by real-time PCR using SYBR Green PCR reagents  and primers specific for ICAM-1 and GAPDH mRNAs. The levels of ICAM-1 mRNA expression were determined by normalizing to that of GAPDH expression. The primers used were as follows: 5′-GGCCTCAGTCAGTGTGA-3′ (sense) and 5′-AACCCCATTCAGCGTCA-3′ (anti-sense) for ICAM-1; 5′-GCCAAAAGGGTCATCATCTC-3′ (sense) and 5′-GGCCATCCACAGTCTTCT-3′ (anti-sense) for GAPDH.The human ICAM-1 (pIC-339) firefly luciferase was kindly provided by Dr. P. T. van der Saag . Additionally, the introduction of a mismatched primer mutation into the AP-1 to generate pGL3-ICAM-1 ΔAP-1 was performed, using the following (forward) primer: ΔAP-1: 5′-TTTTTCAAGCTTAGCCTGGCCG-3′. All plasmids were prepared by using QIAGEN plasmid DNA preparation kits. ICAM-1-luc activity was determined as previously described  using a 6 cells/ml) at 37°C for 1 h. Non-adherent THP-1 cells were removed and plates were gently washed twice with PBS. The numbers of adherent THP-1 cells were determined by counting four fields per 200X high-power field well using a fluorescence microscope . Experiments were performed in triplicate and repeated at least three times.RPECs were grown to confluence in 6-well plates, incubated with TNF- for 6 h, and then adhesion assays were performed. Briefly, THP-1 cells (human acute monocytic leukemia cell line) were labeled with a fluorescent dye, 10 μM BCECF/AM, at 37°C for 1 h in RPMI-1640 medium  and subsequently washed by centrifugation. Confluent RPECs in 6-well plates were incubated with THP-1 cells . Quantitative data were expressed as the mean±S.E.M. and analyzed by one-way ANOVA followed with Tukey’s post-hoc test. TNF-α has been shown to induce ICAM-1 expression in various cell types ,12. ThusThe effects of TNF- are predominantly mediated through TNFR1, which contains a death domain that fosters protein-protein interactions, particularly with other death-domain proteins . To inveSome studies have indicated that the expression of ICAM-1 is mediated through the activation of PKCs ,10. ThusMAPKs integrate signals from numerous receptors and translate these signals into cellular functions. MAPKs are critical for metabolism, migration, production of pro-inflammatory mediators, survival and differentiation. Several reports have suggested that activation of MAPKs leads to the expression of inflammatory genes, including ICAM-1 ,12. In tIt has been well established that inflammatory responses following exposure to extracellular stimuli are highly dependent on activation of AP-1, which plays an important role in the expression of several target genes, such as ICAM-1 . Among AAMD is a blinding disease, affecting millions of elderly individuals. In the most severe manifestation of AMD, blood vessels grow from the choroid into the subretinal pigment epithelial (subRPE) and subretinal space, leading to severe visual loss that can be sudden and permanent . RetinalICAM-1 CD54) is an endothelial- and leukocyte-associated transmembrane protein long known for its importance in stabilizing cell-cell interactions and facilitating leukocyte endothelial transmigration. ICAM-1 plays a role in pathological processes including inflammation, arthritis, cardiovascular diseases, and eye diseases. Accumulating evidence demonstrates that TNF-α may activate downstream protein kinases leading to the expression of inflammatory proteins ,4. All k is an enPKC represents a family of more than 11 phospholipid-dependent Ser/Thr kinases that are involved in a variety of pathways that regulate cell growth, death, and stress responsiveness . PKC isoThe MAPKs family consists of three major members: ERK1/2, p38 MAPK, and JNK1/2. MAPKs are important intracellular signalings and play critical roles in inflammatory responses. Previous study has indicated that TNF-α induced ICAM-1 expression and monocyte adhesion via MAPKs in A549 cells . RecentlIt has been well established that inflammatory responses following exposure to extracellular stimuli are highly dependent on activation of AP-1 or NF-kB, which plays an important role in the expression of several target genes . AP-1 isBase on the observation from literatures and our findings,


Neorickettsia . Some Neorickettsia are able to invade cells of the digenean's vertebrate host and are known to cause diseases of wildlife and humans. In this study we report the results of screening 771 digenean samples for Neorickettsia collected from various vertebrates in terrestrial, freshwater, brackish, and marine habitats in the United States, China and Australia. Neorickettsia were detected using a newly designed real-time PCR protocol targeting a 152 bp fragment of the heat shock protein coding gene, GroEL, and verified with nested PCR and sequencing of a 1371 bp long region of 16S rRNA. Eight isolates of Neorickettsia have been obtained. Sequence comparison and phylogenetic analysis demonstrated that 7 of these isolates, provisionally named Neorickettsia sp. 1–7  represent new genotypes and one (obtained from Metagonimoides oregonensis) was identical to a published sequence of Neorickettsia known as SF agent. All digenean species reported in this study represent new host records. Three of the 6 digenean families  are also reported for the first time as hosts of Neorickettsia. We have detected Neorickettsia in digeneans from China and Australia for the first time based on PCR and sequencing evidence. Our findings suggest that further surveys from broader geographic regions and wider selection of digenean taxa are likely to reveal new Neorickettsia lineages as well as new digenean host associations.Digeneans are endoparasitic flatworms with complex life cycles including one or two intermediate hosts  and a vertebrate definitive host. Digeneans may harbor intracellular endosymbiotic bacteria belonging to the genus  Neorickettsia (family Anaplasmataceae) is a genus containing obligate intracellular endosymbionts of digeneans . Although relatively small, this genus has received significant attention in the recent years, with a rapid increase in the number of known species level lineages in this group Neorickettsia are vertically transmitted through all stages of complex digenean life cycles. Additionally, they are capable of being horizontally transmitted to the vertebrate hosts of the digenean, both human and wildlife, where they can cause disease Neorickettsia sennetsu), or even fatal, e.g., salmon dog poisoning (Neorickettsia helminthoeca), and Potomac horse fever (Neorickettsia risticii) Neorickettsia have now been reported from several countries and all continents including Antarctica Neorickettsia in vertebrate hosts, mainly horses. It should be noted that positive immunological tests of horses could be a result of either actual infection or a previous vaccination against Neorickettsia risticii (causative agent of Potomac horse fever). Therefore, immunological results alone without PCR-based confirmation need to be considered with caution Neorickettsia has been limited, which prevents understanding of the actual diversity of these bacteria and potential sources of infection of vertebrate animals including humans. Only 21–22 digenean species, in most cases identified only to genus or family level, have been previously confirmed as hosts of NeorickettsiaNeorickettsia detected in vertebrate host tissues . As posited by Vaughan et al. Neorickettsia will reveal additional host associations of these bacteria and new pathways of their circulation in nature. This has already been demonstrated in a recent study by Tkach et al. Neorickettsia in 7 species of digeneans belonging to 7 different families.Neorickettsia that are likely to represent additional species based on levels of 16S rRNA sequence divergence Neorickettsia, 6 have been found in North America. Circulation of digenean hosts of known Neorickettsia occurs in either freshwater , freshwater/terrestrial , or fully terrestrial (N. risticii) environments Neorickettsia from digeneans having fully marine life cycles. Presence of Neorickettsia in tissues of marine fishes in Antarctica and the Gulf of Mexico Currently, there are 3 named species and 10 not formally named genotypes of Neorickettsia in previously understudied or unstudied regions. To accomplish this goal, we screened for Neorickettsia numerous DNA extracts from an extensive collection of adult digeneans collected from various vertebrates as well as snail hosts in the United States , Australia, and China. As a result, we have found 7 new genetic lineages of Neorickettsia some of which may potentially represent new species. All of our records represent new digenean host associations. Our findings also expand the range of circulation pathways known for Neorickettsia. We have for the first time detected Neorickettsia from Australia and China using PCR-based detection and DNA sequencing. We have also conducted a molecular phylogenetic analysis in order to estimate interrelationships among the newly discovered genotypes with previously known named species and not yet named lineages of Neorickettsia.In this study, we used real-time PCR-based detection methods to study the diversity of Digeneans were collected from marine and freshwater fishes, amphibians, reptiles, birds, mammals, and invertebrates from 2009 through 2012 from multiple localities in China, Australia, Argentina, Costa Rica, and several states in the USA . An IACUC protocol 10100105 was issued by the University of Southern Mississippi for collecting and humane euthanasia of wild animals in Mississippi, Florida, and Louisiana. Birds were collected by shotgun in accordance to the state and federal permits, raccoons were live trapped and euthanized in the field by firearm  in accordance to issued permits, and amphibians and reptiles were collected by hand and humanly euthanized by immersion in a solution of chlorobutanol (chloretone) in water in accordance with the issued permits and IACUC protocol. IACUC protocols were not required for invertebrate collecting in Oregon, North Dakota, and Minnesota or for fish. Fish were purchased dead from food markets in Argentina, Costa Rica, and China. In Australia, fish were caught by cast net by one of the authors (EP) from around Townsville (Queensland), Darwin (Northern Territory) and Broome . Fish were placed on ice after capture, and if necessary were sacrificed with the use of a 250 mg/l bath of Tricaine methane sulfonate (MS-222) in accordance with the issued permits.Neorickettsia screening was opportunistic, but as inclusive as possible. Required scientific collecting permits were obtained in all cases. The Florida Fish and Wildlife Conservation Commission issued the permits LSSC-11-00074 and FNW-13-05 that allowed for the collection of invertebrates, amphibians, reptiles, and mammals as well as freshwater fishes in Florida. The Mississippi Department of Wildlife Fisheries and Parks issued a permit 0123131 for the collection of invertebrates, amphibians, reptiles, mammals, and freshwater and marine fishes in Mississippi, and the Louisiana Wildlife and Fisheries issued a permit LNHP-13-017 also for the collection of the above mentioned animals in Louisiana. Additionally, a federal permit (MB681207-0) was issued by the US Fish and wildlife service for collection of reptiles, mammals, birds and freshwater fish in Mississippi and Louisiana. Three separate permits were issued in Australia for the collection of fish; Western Australia ; Northern Territory ; and Queensland (Department of Primary Industries and Fisheries 133621). Only abundant commercial fish species were purchased from local markets and fishermen in China, Argentina, and Costa Rica, and therefore, permits were not required. Additionally, aquatic snails were collected from North Dakota, Minnesota, and Oregon. Snails were identified using Burch's “North American Freshwater snails”Neorickettsia a negative result does not necessarily mean that a certain species of digenean cannot be a host for Neorickettsia. As discussed by Tkach et al. Neorickettsia endosymbionts and therefore, different individuals of the same digenean species may or may not be infected.All digenean samples were collected over a period of 7 years as parts of several independent projects dealing with parasite biodiversity and systematics. Thus, the source of DNA available for Live worms were rinsed in saline, examined briefly, killed with hot water, and fixed in 70% ethanol that allowed for both morphological examination and molecular study. When numerous specimens were found, some were fixed in 95% ethanol for molecular work. Fixed worms were stained in aqueous alum carmine, Mayer's hematoxylin, or Van Cleave's hematoxylin; dehydrated in a graded ethanol series; cleared in clove oil (carmine and Van Cleave's) or methyl salicylate (Mayer's); and mounted permanently in Damar Gum for morphological identification.Genomic DNA was extracted from individual adult worms and pooled larval stages either using the Qiagen DNAeasy tissue kit  following the manufacturer's instructions or the guanidine thiocyanate method according to Tkach and Pawlowski Neorickettsia using a real-time PCR protocol designed and extensively tested by one of the authors (SEG). Five microliters of each DNA extract were used. The real-time PCR amplified a 152-bp portion of the 3′ end of the heat shock protein coding gene, GroEL. The primer pair (designed by SEG) used is listed in DNA extracts were first tested for the presence of N. sennetsu used as a positive control was graciously provided by Dr. Sabine Dittrich (Lao Oxford Mahosot Wellcome Trust Research Unit). Pure water was used for negative controls in both real-time and nested PCRs.The real-time PCR reactions were run on a Bio-Rad CFX96 Touch real-time PCR detection system  using iTaq universal sybr green supermix  according to the manufacturer's instructions. A two-step program was used with a denaturation temperature of 95°C for 3 seconds, followed by an annealing and extension temperature of 64°C for 25 seconds and 36 cycles. In addition, a melt curve was run starting from 72°C and increasing at 0.2°C increments every 5 seconds until reaching 87°C. The nested PCR reactions were run on EP Gradient thermocycler  using Quick load OneTaq mastermix  according to the manufacturer's instructions. Annealing temperature of 56°C and 40 cycles were used in both first and nested PCRs. DNA of Neorickettsia positive cercariae or metacercariae were identified to the lowest possible taxonomic level using a partial sequence of the nuclear large ribosomal subunit gene (28S). Digenean DNA was amplified from the extracted DNA by PCR using the primer pair listed in Adult digeneans were identified based on their morphology using stained whole mounts. Identification to generic level was done using the three-volume “Keys to the Trematoda” Neorickettsia and digeneans were purified using the Zymo DNA Clean & Concentrator -5 or ExoSap PCR clean-up enzymatic kit from Affimetrix  according to the manufacturer's instructions. The PCR products were cycle-sequenced using ABI BigDye chemistry, ethanol precipitated, and run on an ABI Prism 3100 automated capillary sequencer. Contiguous sequences of Neorickettsia and digeneans were assembled using Sequencher ver. 4.2  and submitted to GenBank under accession numbers KF661342-KF661351 and KF878083- KF878084.PCR amplicons of both Newly obtained sequences  and sequPhylogenetic analysis was carried out using Bayesian inference (BI) as implemented in the MrBayes program, version 2.01 Neorickettsia. A list of digenean families screened and the number of extracts corresponding to each family are provided in Neorickettsia in 8 digenean species belonging to 7 different families ; Cedar Key, Florida ; Williford Springs, Florida ; Daya Bay, Guangdong, China (22°43′N 114°32′E); Eli Creek, Queensland, Australia . We have detected Neorickettsia from digeneans for the first time from the Australian continent and China as well as from Oregon and Florida. One of the forms discovered in our study is identical to the SF agent  as an outgroup. The alignment included a total of 1,258 sites, of which 1,249 could be aligned unambiguously. Positions that could not be aligned unambiguously were excluded from the analysis. Bayesian analysis produced a tree where all Neorickettsia sequences clearly fall into a well-defined clade, with a 100% support  and Neorickettsia sp. 2 (from Saccocoeliodes beauforti from Florida). All internal sub-clades of clade II are well resolved and 100% supported. Five lineages of this group are from North America while one is from Southeast Asia.The “d clades  and 2. CNeorickettsia species, including all three currently recognized named species of these bacteria, namely N. helminthoeca, N. risticii, and N.sennetsu  in the USA. All 8 digenean species detected as hosts of Neorickettsia in our study  in Antarctica Mugil cephalus in the Gulf of Mexico Neorickettsia  from three digenean species with completely marine life cycles, namely Saccocoeliodes beauforti, Saccocoelioides lizae, and Bacciger sprenti. This suggests that Neorickettsia can be encountered in almost every type of environment suitable for digenean life cycles.Our study has revealed a new circulation pathway of Neorickettsia genotypes obtained from Metagonimoides oregonensis in Florida fully matched the sequence of SF agent. SF agent was initially found in metacercariae of the heterophyid Stellantchasmus falcatusS. falcatus and M. oregonensis belong to the family Heterophyidae, therefore, there is a probability that SF agent could be found in other members of this large digenean family, members of which are parasitic primarily in fish-eating birds and mammals including humans. SF agent causes mild clinical symptoms in experimentally infected mice and dogs, but not monkeys or humans One of our Channa striata most likely represents a separate genus of Anaplasmataceae. On the other hand, the Anaplasmataceae sp. from Mastacembelus armatus considered by Seng et al. Neorickettsia clade” Saccocoelioides lizae has only been found off the cost of South-eastern China Neorickettsia sp. 2 may be found in other areas along the coast of the southeastern United States and that Neorickettsia sp. 4 may be found in other costal countries in Southeast Asia.is study , is a glDeropegus aspina is only known from the Pacific Northwest of the United States in salmonid fishes, including Salmo clarki, S. gairdneri, Oncorhynchis kisutch, O. tshawytscha, and a frog, Rana boyli, Neorickettsia sp. 7 is limited in its distribution to the Pacific coast of the USA and Canada.Bacciger sprenti (family Faustulidae) carrying Neorickettsia in our study was obtained from fish Selenotoca multifasciata  and not in 11 individuals of Mugil cephalus or 49 individuals of Mugil georgii. This led Cribb et al. Bacciger sprenti has only been reported from marine fishes in Australian coastal waters, therefore, we can assume Neorickettsia sp. 5 is a genotype unique to Australia.fasciata . This diNeorickettsia is very geographically uneven and is clearly associated with areas where most studies have taken place. The majority of genotypes were found in the USA and several countries in southeastern and eastern Asia Neorickettsia in Australia, there is no well-documented information supported by PCR/sequence data from Africa, Europe, most of South America, most of Asia, and nearly all island countries Neorickettsia and rather reflects insufficient knowledge due to the lack of broad screening efforts. With more than 18,000 described species of digeneans Neorickettsia to be found and characterized.Currently known distribution of


Lepomis macrochirus) escape responses to examine this potential trade-off, determining the full repertoire of escape behaviour for individual bluegill sunfish and linking this to performance as indicated by escape velocity and acceleration. Fish escapes involve an initial C-bend of the body axis, followed by variable steering movements. These generate thrust and establish the escape direction. Directional changes during the initial C-bend were less variable than the final escape angle, and the most frequent directions were associated with high escape velocity. Significant inter-individual differences in escape angles magnified the overall variation, maintaining unpredictability from a predator perspective. Steering in the latter stages of the escape to establish the final escape trajectory also affected performance, with turns away from the stimulus associated with reduced velocity. This suggests that modulation of escape behaviour by steering may also have an associated performance cost. This has important implications for understanding the scope and control of intra- and inter-individual variation in escape behaviour and the associated costs and benefits.Successful predator evasion is essential to the fitness of many animals. Variation in escape behaviour may be adaptive as it reduces predictability, enhancing escape success. High escape velocities and accelerations also increase escape success, but biomechanical factors likely constrain the behavioural range over which performance can be maximized. There may therefore be a trade-off between variation and performance during escape responses. We have used bluegill sunfish ( Effective predator evasion is a vital component of fitness for many animals . Given tEscape performance is dictated by a suite of interacting physical factors. For example, during fish escape responses rapid muscle contraction and bending of the body axis transfers momentum to the surrounding water, potentially generating high escape velocities . In compDespite the potential physical constraints, fish escape responses appear to be quite variable . This maThe goals of the current study were to quantify the extent of individual variability in fish escape behaviour, place this in context with the overall scope for behavioural variability across individuals, and determine the extent to which behavioural variation and flexibility were constrained by a trade-off between variability and mechanical performance. Data to indicate the overall scope for intra- and inter-individual variation in escape behaviour and allow determination of behavioural repeatability for fish escapes are scarce , and basLepomis macrochirus), encompassing the intra- and inter-individual variation in escape angle, and linked this to escape performance, specifically velocity and acceleration, variables associated with both escape success (In the present study we have quantified the repertoire of escape behaviours for bluegill sunfish ( success  and effe success . High-spt(14) = 2.49, p<0.05). For the composite distribution of final angles the circular variance of 0.30 was also significantly greater than in individual distributions  = 2.71, p<0.05, Inter-individual variation in behaviour  magnifieEscape performance was associated with the frequency with which stage 1 escape angles were used by an individual . Peak COCircular variance was significantly greater for final compared to stage 1 distributions  = 5.98, p<0.05; Fish most frequently use escape angles that are associated with high performance. This suggests that a high level of variation in escape behaviour is associated with a performance cost ; Table 2Pterophyllum eimekei) where ‘single bend’ escapes that lack a stage 2 counter-turn were associated with lower peak velocities than ‘double bend’ escapes where stage 2 was present  indicates the proportion of phenotypic variability attributable to differences between individuals. It also sets the upper limit for the heritability of a given trait  with an absence of consistently poor performers. As these were wild-caught fish, this may reflect narrowing of the scope for inter-individual variation through removal of low performance phenotypes  were collected from Lake Waban, MA, USA using baited hooks in August and September 2011. Fish were maintained in pairs in divided 20 gallon aquaria at 21°C, and fed on earthworms ad libitum. Kinematic data indicating changes in fish heading through the escape response were obtained from 15 fish  with sufficient numbers of observations per individual to establish the frequency distribution of escape angles. Analyses of velocities and accelerations were carried out for 7 of these fish . Fish were collected under license from the Massachusetts Department of Fish and Game, and all procedures were approved by the Institutional Animal Care and Use Committee at Wellesley College.Bluegill sunfish (Video recordings were obtained in a 45×90 cm tank with 15 cm water depth . Escapesθ) was calculated as:xd and yd are the distances between the COM and snout in the X and Y directions.Video sequences were downloaded to a personal computer using AOS Digital Imaging software . The COM of bluegill sunfish is located approximately 40% of total body length from the snout when the fish is in a straight position , althougEscape responses are typically divided into two kinematic phases . Phase 1If the development of high velocities and accelerations is restricted to a relatively narrow range of body movements by hydrodynamic factors and the axial mechanics of the body, then within a frequency distribution of kinematic variables the most frequently adopted patterns of movement may be associated with high performance. Conversely, infrequently observed behaviours at the margins of the distribution may be associated with low performance. We used regression analyses to determine whether there was a relationship between escape performance  and the frequency distributions of stage 1 and escape angles for each individual.2(n) bins, where n was the number of observations , and have a relatively narrow range, the data do not have a circular distribution and are suitable for analysis with linear statistical models. ANOVA with a fish identifier as a random factor and turn direction (left vs. right) as a fixed factor was used to test for inter-individual differences in steering angle. PPMC was used to determine the strength and sign of relationships between performance  and steering angle. A two tailed t-test was also applied within a linear regression analysis to determine whether the slope of each performance-probability relationship was detectably different from zero. Data were tested for normality using a Kolmogorov–Smirnov test (p<0.05) and Levene's equality of error variances test (p<0.05). All data were log-transformed to achieve normality. Negative values were adjusted by absolute value transformation before log transformation. Untransformed data are presented in figures.Stage 1 and escape angle data were analysed for uniformity and left–right symmetry using a circular statistics package . Rayleigh's test established that angle distributions were non-uniform for both left and right turns in all fish . Circular variance, equivalent to a coefficient of variation for non-directional data, was used as a relative indicator of the dispersion of the distributions, with 0 indicating concentration at a single direction. Frequency distributions for left and right turns for each fish were compared using a Mardia-Watson-Wheeler test . This isr, was calculated as follows:s2 is the within-group variance (s2 is the variance in behaviour exhibited by individuals, and r therefore indicates the proportion of the total phenotypic variance that is attributable to the between-individual variance. The variance components were calculated from the mean squares derived from one-way ANOVA with an identifier for each individual as a random factor as follows:AMS and WMS are the across and within groups mean squares and n0 is related to the number of observations obtained per individual as follows:a is the number of individuals and ni is the sample size of the ith group. ANOVA is generally not suitable for analysis of directional data as the frequency distribution may be ‘wrapped’ around a circle due to the equivalency of 0° and 360°. However, given that the starting angle was defined as 0° and that no angular changes exceeded 360°, mean squares values obtained by ANOVA give a reasonable estimate of repeatability in this case. The F-statistic and p-value obtained by ANOVA indicate whether the repeatability is significantly greater than zero (The behavioural repeatability, variance . In thishan zero . To accohan zero . Linear


In pathological conditions, microglia release large amounts of TNF-α; this de novo production of TNF-α is an important component of the so-called neuroinflammatory response that is associated with several neurological disorders. In addition, TNF-α can potentiate glutamate-mediated cytotoxicity by two complementary mechanisms: indirectly, by inhibiting glutamate transport on astrocytes, and directly, by rapidly triggering the surface expression of Ca+2 permeable-AMPA receptors and NMDA receptors, while decreasing inhibitory GABAA receptors on neurons. Thus, the net effect of TNF-α is to alter the balance of excitation and inhibition resulting in a higher synaptic excitatory/inhibitory ratio. This review summarizes the current knowledge of the cellular and molecular mechanisms by which TNF-α links the neuroinflammatory and excitotoxic processes that occur in several neurodegenerative diseases, but with a special emphasis on amyotrophic lateral sclerosis (ALS). As microglial activation and upregulation of TNF-α expression is a common feature of several CNS diseases, as well as chronic opioid exposure and neuropathic pain, modulating TNF-α signaling may represent a valuable target for intervention.Tumor necrosis factor alpha (TNF- The cleavage of the extracellular domain of tmTNF-α by the matrix metalloprotease TNF-α-converting enzyme (TACE) releases a soluble TNF-α (sTNF-α) homotrimer. Remarkably, both tmTNF-α and sTNF-α are biologically active and their signal transduction involves binding to two distinct surface receptors, TNF-α receptor 1 (TNFR1 or p55TNFR) and TNF-α receptor 2 (TNFR2 or p75TNFR), which are different in their expression pattern, downstream signal-transduction cascades, and binding affinity for TNF-α [α, and those related to cell survival and proliferation involved TNFR2 activation, now it is becoming clear that TNFR2 can also induce cell death [α trimer to the extracellular domain of TNFR1 induces receptor trimerization and recruitment of the adaptor protein TNF receptor-associated death domain (TRADD), which in turn recruits additional adaptor proteins: receptor-interacting protein (RIP), TNF receptor-associated factor 2 (TRAF2), and Fas-associated death domain (FADD). This latter protein mediates recruitment and activation of caspases 8 and 10 that initiate a protease cascade that leads to apoptosis [κB), the extracellular signal-regulated kinase (ERK), the c-Jun N-terminal kinase (JNK), the p38 mitogen-activated protein kinase (p38 MAPK), the acidic sphingomyelinase (A-SMase), and the neutral sphingomyelinase (N-SMase) pathways. These pathways regulate the expression of several genes, and some of them, particularly those regulated by the NF-κB pathway, have antiapoptotic effects. With the exception of the SMase pathways, the above signal transduction pathways can also be induced through TNFR2 signaling because TRAF2  can directly associate to the intracellular domain of the TNFR2 receptor .Neuroinflammation in the CNS refers to the collective response of microglia, and to a lesser extent of astrocytes and oligodendrocytes, against diverse insults  designed to remove or inactivate the noxious agents and to inhibit and reverse their detrimental effects. The glial response can be considered as an innate immune mechanism, whereas the participation in the neuroinflammatory process of lymphocytes (mainly T cells) carrying binding sites for specific antigens is an acquired immune mechanism . In neurα [γ) is a potent inducer of TNF-α gene expression in microglia [κB, and ERK1/2 [α expression. In our laboratory, we demonstrated that the sole inhibition of the mitogen-activated protein kinase and ERK kinase (MEK)/ERK signaling pathway with U0126 or apigenin was enough to inhibit the LPS or the IFN-γ-stimulated TNF-α expression in the BV-2 microglial cell line [γ is produced by T cells but not in significant amounts by any CNS resident cells, including microglia [γ [+ and CD8+ T cells were found to invade the brain in postmortem human specimens and in a mouse model of PD [+ and CD8+ T cells can have detrimental [γ which, via the MEK/ERK signaling pathway, induces in microglia an increased de novo TNF-α production and release  were neurotoxic when combined [α potentiated glutamate neurotoxicity and that this effect could be blocked by competitive (2-APV) and noncompetitive (MK-801) NMDA receptor antagonists [α or kainate (KA) alone into the thoracic gray matter resulted in almost no tissue damage; however, the combination of these substances at the same doses produced a large area of tissue necrosis and neuronal cell death, an effect that could be reverted by the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline 2,3--dione (CNQX) [The first reports demonstrating that TNF-combined . Similaragonists . Later, e (CNQX) .α induced neuronal death was investigated. In mouse primary microglia it was demonstrated that TNF-α, through the TNFR1 pathway, induces excitotoxicity by promoting microglial glutamate release from hemichannels of gap junctions in an autocrine manner [α release, and when this microglial-conditioned medium was added to cerebellar granule neurons in culture resulted in caspase-3 activation and apoptotic cell death. The authors also identified microglial-released Fas ligand as an essential cofactor for the TNF-α-induced neurotoxicity [α derived from KA-activated microglia also resulted in apoptotic neuronal cell death [α promotes further microglial TNF-α production and release through TNFR1 signaling [α induces glutamate release that acts on microglial mGluR2 to induce more TNF-α production  inhibits glutamate uptake by inducing a downregulation of EAAT2/GLT-1 mRNA [α induced binding of NF-κB to the EAAT2 promoter [κB in regulating GLT-1 expression was further confirmed in our laboratory. We used spinal cord organotypic cultures to create a model of chronic glutamate excitotoxicity in which glutamate transporters were inhibited by threohydroxyaspartate (THA) to induce motoneuron death. Exposure to THA induced microglial activation and TNF-α release. In the presence of exogenous TNF-α (20 ng/mL), THA-induced excitotoxic motoneuron death was potentiated. Coexposure to TNF-α and THA also resulted in downregulation of GLT-1 and in increased extracellular glutamate levels. The downregulation of GLT-1, as well as the excitotoxic motoneuron death, could be prevented by NF-κB inhibition [In astroglia, the interaction of TNF-ocytosis , 78. Thee system , 80; howiewed by )  was applied for a short time period (30 minutes) in hippocampal-entorhinal complex slice cultures, it reduced glutamate transport without affecting GLT-1 or GLAST expression [α are not clear. On the one hand, TNF-α is a clear inducer of oxidative stress in the CNS [α, as explained before, can activate caspases, including caspase-3, which can also be activated by oxidative stress [When TNF-pression . The mec the CNS , 88, 89, the CNS , 91, thu the CNS . On the e stress . In thise stress .α have been described  are ligand-gated channels that mediate fast excitatory synaptic transmission in the vertebrate CNS. These receptors are tetramers assembled from glutamate receptor (GluR) 1, 2, 3, and 4  subunits around an aqueous pore in the membrane , 101. Thermeable , 106. Innditions ; see alsnditions .α has an important role in the regulation of AMPARs trafficking being a critical component of the homeostatic regulatory system controlling synaptic plasticity [α increased their expression of the GluR1 subunit, resulting in an increased susceptibility to KA-induced necrosis. The effect of TNF-α implicated both the A-SMase and the NF-κB signaling pathways [α, within 15 min, increased the surface expression of GluR1-containing AMPARs, and these changes were accompanied by dramatic changes in AMPAR-mediated excitatory postsynaptic currents [α on AMPA-induced excitotoxicity. In mouse hippocampal slice cultures it was reported that pretreatment (24 h) of cultures with 10 ng/mL TNF-α potentiated AMPA-induced neuronal death; however, decreasing the concentration of TNF-α to 1 ng/mL resulted in neuroprotection. The authors demonstrated that the “high-dose” toxic effect was mediated by TNFR1 whereas the “low-dose” protective effect implied the TNFR2 [α of AMPA toxicity was further confirmed in a work performed also in hippocampal cells and demonstrating that, by activating neuronal TNFR1, TNF-α increased the surface AMPARs, but remarkably, TNF-α preferentially increased the synaptic expression of GluR2-lacking (Ca2+ permeable) AMPARs. This effect was mediated through a phosphatidylinositol 3-kinase- (PI3-K-) dependent process [α of KA-induced neuronal death was confirmed later by the same group; they demonstrated that the specific PI3-K inhibitor LY294002 reverted the TNF-α effect on hippocampal neurons. Moreover, and in agreement with the “GluR2 hypothesis,” the potentiating effect was also reverted by the synthetic joro spider toxin analog NASPM, which selectively blocks Ca2+ permeable-AMPARs [α triggers a rapid induction of Ca2+ permeable-AMPARs in hippocampal pyramidal neurons; the effect was rapid (15 min) and since TNF-α exposure did not alter mRNA levels for either GluR1 or GluR2 subunit, it was proposed that TNF-α acts at posttranscriptional level to induce rapid increases in surface subunit expression [TNF-asticity . In 2002pathways . Similarhe TNFR2 . The rol process . The role-AMPARs . These rpression . in vivo nanoinjections of TNF-α into the ventral horns resulted in increased GluR1 and decreased GluR2 at both extrasynaptic and synaptic plasma membrane sites. The effect was seen in the neuropil 60 min after TNF-α nanoinjection and could also be detected in the somata of large spinal motoneurons [α [α induced GluR2-lacking AMPARs trafficking to the membrane is likely to contribute to postinjury excitotoxicity in spinal cord motoneurons. However, another study has reported TNF-α to reduce AMPAR-mediated calcium entry in cultured motoneurons by increasing cell surface expression of the GluR2 subunit [The pathological relevance of the above findings was first described in a model of cervical spinal cord contusion injury. In this model, increased synaptic AMPAR numbers were found at synapses ipsilateral to the lesion at 90 min and 3 h after injury. Interestingly,oneurons . In a suurons [α . Togethe subunit .2+ permeable-AMPARs under basal conditions, and it has been proposed that this circumstance would render them more susceptible to neurodegeneration in ALS [α [α has been shown to potentiate AMPAR-mediated excitotoxicity on lumbar spinal cord motoneurons both by decreasing GLT-1 expression [2+ permeable-AMPARs via a PI3-K and protein kinase A- (PKA-) dependent mechanism [ in vitro and in vivo, that VEGF increases the expression of GluR2 subunit of AMPARs of spinal cord motoneurons, thus minimizing their vulnerability to AMPA-mediated excitotoxicity [Adult spinal cord motoneurons possess significant numbers of Can in ALS –120. Actn in ALS , 122; thn ALS [α , 123. Inpression , and alsechanism . Interesechanism ), and ittoxicity .α on N-methyl-D-aspartate receptors (NMDARs) trafficking are less studied; however, the results obtained are similar to those on AMPARs. Thus, in hippocampal neurons TNF-α induced a rapid increase in the surface expression of the NR1 subunit of NMDARs and also, via N-SMase2, promoted a specific clustering of phosphorylated NR1 subunits into lipid rafts [α has also been shown to potentiate NMDAR-mediated excitotoxicity in cortical neurons [The effects of TNF-id rafts . Similar neurons .α also regulates inhibitory synapse function. An in vivo study in the rat spinal cord indicated that TNF-α increased within 60 min synaptic and total gamma-aminobutyric acid A receptors (GABAARs) in the neuropil and in the plasma membrane of motoneurons. However, the effect of TNF-α on GABAAR trafficking was complex, displaying a nonlinear dose-dependent relationship [ARs have been implicated in maladaptive spinal plasticity in a model of instrumental learning [α is necessary and sufficient for generating lasting inhibition of spinal learning and that the effect of this cytokine also involves Ca2+ permeable-AMPARs, since it was reverted by a GluR2-lacking AMPA receptor antagonist [ in vitro study in mature rat and mouse hippocampal neurons in culture demonstrated that acute (45 min) application of TNF-α induced a rapid and persistent decrease of inhibitory synaptic strength as well as a downregulation of cell-surface levels of GABAARs. The trafficking of these receptors in response to TNF-α was mediated through the activation of neuronally expressed TNFR1 and required the activation of PI3-K, p38 MAPK, protein phosphatase 1, and dynamin GTPase [TNF-tionship . The autlearning . Interestagonist . More ren GTPase .α potentiates excitotoxicity by rapidly increasing excitatory synaptic strength through increased AMPA and NMDA receptors surface expression and also that neurons respond to elevated levels of the cytokine weakening their inhibitory synaptic strength through a decreased presence of GABAARs in the plasma membrane. Thus, the net effect of TNF-α is to alter the balance of excitation and inhibition resulting in a higher synaptic excitatory/inhibitory ratio [α in cerebrospinal fluid has been described [Together, the findings presented here indicate that TNF-ry ratio   and microglia and neurons .It is now widely accepted that most developmental, lesional, and degenerative nervous system disorders involve common interconnected neurotoxic mechanisms. α, associated with neuroinflammation and excitotoxicity, have been reported, but also describes a broader situation in which activated microglia releases significant amounts of TNF-α. This is the case of opioid tolerance and neuropathic pain, two situations that are modulated by TNF-α [α mRNA expression in the rat spinal cord [2+ permeable-AMPA and NMDA receptors [α antagonist [α mediated increased insertion of Ca2+ permeable-AMPARs in spinal cord neurons plays a major role in inflammatory pain and may represent a path by which glia contribute to neuronal sensitization and pathological pain [It is noteworthy that the scheme shown in by TNF-α , 137. Chnal cord ; this efeceptors . All thetagonist , 139. Sical pain .α is a key mediator in the pathological mechanisms of a large number of neurological disorders including ischemia, AD, PD, MS, and ALS [α action seems to be an attractive disease-modifying strategy. The different strategies employed for TNF-α inhibition have been reviewed elsewhere [α and tmTNF-α, the antibiotic minocycline that decreases TNF-α synthesis, the immunomodulatory drug thalidomide and its derivatives that enhance the degradation of TNF-α mRNA [α production. Clinical trials examining the effects of TNF-α inhibition have been conducted on patients with MS, AD, and ALS. Although promising effects were obtained in AD patients with substantial cognitive and behavioral improvements [α gene knockout did not affect life span or the extent of motoneuron loss in the superoxide dismutase 1 (SOD1) transgenic mice model of ALS, thus suggesting that TNF-α alone is not a key factor in motoneuron degeneration [As TNF- and ALS  and alsolsewhere  and inclF-α mRNA , and TACovements , 142, thovements , 144. Moneration .α has both neuroprotective and neurotoxic effects related to the different signaling pathways activated by their receptors [α receptors were more susceptible to ischemia and excitotoxic injury [β and TNF-α) have redundant functions in vivo; thus, in the TNF-α knockout mice an increase in the transcripts encoding for IL-1β was detected [α often works in concert with other cytokines  to promote neuronal death [α and/or its potentiation of excitotoxicity will be essential in the management of chronic and acute neurodegenerative diseases.The above findings can be explained first because TNF-eceptors . In thisc injury , 148. Sedetected ; and, thal death , 150. Neα plays a physiological role in controlling synaptic transmission and plasticity in the healthy CNS by modulating ionotropic glutamate receptors trafficking. However, excessive TNF-α levels, as a result of different types of injury, have an inhibitory effect on glutamate transporters, resulting in increased glutamate concentration in the CNS parenchyma. In this context, even slight increases in TNF-α induced Ca2+ permeable-AMPA and/or NMDA receptors trafficking become toxic for neurons. As microglial activation and upregulation of TNF-α expression is a common feature of several CNS diseases, as well as chronic opioid exposure and neuropathic pain, modulating TNF-α signaling may represent a valuable target for intervention.TNF-


Background: Rats subjected to bile duct ligation (BDL) exhibit increased systemic oxidative stress and brain dysfunction characteristic of hepatic encephalopathy (HE), including fatigue, neurotransmitter alterations, cognitive and motor impairment, and brain inflammation. The levels of tumor necrosis factor-alpha (TNF-α) and asymmetric dimethylarginine (ADMA) are both increased in plasma and brain in encephalopathy induced by chronic liver failure. This study first determined the temporal profiles of TNF-α and ADMA in the plasma, brain cortex, and hippocampus in young BDL rats. Next, we examined whether etanercept was beneficial in preventing brain damage.Methods: Young rats underwent sham ligation or BDL at day 17 ± 1 for 4 weeks. Treatment group rats were administered etanercept (10 mg/kg) intraperitoneally (IP) three times per week with or without etanercept (100 μg) intrathecally (IT) three times in total.Results: We found increased plasma TNF-α, soluble tumor necrosis factor receptor 1 (sTNFR1), soluble tumor necrosis factor receptor 2 (sTNFR2), and ADMA levels, increased cortical TNF-α mRNA and protein and ADMA, and hippocampal TNF-α mRNA and protein, and spatial defects in young BDL rats. The increase in cortex TNF-α mRNA and ADMA were reduced by IP etanercept or combined IP and IT etanercept. Dually IP/IT etanercept administration reduced the increased cortical and hippocampal TNF-α mRNA and protein level as well as spatial deficits.Conclusions: We conclude that combined intraperitoneal and intrathecal etanercept reduce increased brain TNF-α and ADMA levels and rescues spatial deficits in young rats after BDL. Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome present in patients with liver disease that is characterized by personality changes, diminished intellectual capacity, motor incoordination, and consciousness disturbance Collie, . Bile duTumor necrosis factor-alpha (TNF-α) is a central proinflammatory cytokine, with pleiotropic effects on inflammatory and immunological processes  is a naturally occurring amino acid that can competitively inhibit nitric oxide synthase (NOS), thereby decreasing the synthesis of NO  mRNA and protein . Male and female Sprague-Dawley rats were obtained  for breeding. The day of delivery was defined as day 0. Male offspring rats at postnatal day (PND) 17 ± 1 weighing approximately 50 g were used. All animals were housed in a room maintained at 24°C with 12-h light/dark cycles. All animals had free access to standard chow and water.N = 10) and the SHAM4W group (N = 10), respectively. Rats that underwent BDL for 2 or 4 weeks at PND 17 ± 1 were designated as the BDL2W group (N = 10) and BDL4W group (N = 10), respectively . Rats that underwent sham ligation for 4 weeks at PND 17 ± 1 and were injected with etanercept (10 mg/kg) intraperitoneally (IP) every other day from PND 18 ± 1 to PND 38 ± 1  and etanercept (100 μg) intrathecally (IT) from PND 30 ± 1 to PND 38 ± 1  were designated as the SHAMPT group (N = 9). Rats that underwent BDL for 4 weeks at PND 17 ± 1 were designated as the BDL4W group (N = 9). Rats that underwent BDL for 4 weeks and were injected IP with etanercept were designated as the BDLP group (N = 9). Since BDL in rat is characterized by both peripheral and central inflammation , alanine aminotransferase (ALT), ammonia, direct bilirubin and total bilirubin according to previously described methods  score proposed by Knodell et al. . BrieflyEnzyme-linked immunosorbent assay (ELISA) analyses were performed using appropriate kits according to the manufacturer’s instructions, as previously reported  analysis was performed on the cortex , as previously described  method was employed for the relative quantification of gene expression. The averaged TC was subtracted from the corresponding averaged β-actin value for each sample (ΔTC). ΔΔTC was obtained by subtracting the average control ΔTC value from the average experimental ΔTC. The fold-increase was established by calculating 2CT−ΔΔ for experimental vs. control samples.PCR analysis was performed as reported previously  for 1 h, and the third was pre-treatment with rat TNF-α for 1 h with etanercept (0.1 μg/ml) added for 30 min. C6 cells were seeded (1 × 106) onto glass slides overnight and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for 15 min. The cells were then rinsed twice with PBS and permeabilized with 1% Triton X-100 for 7 min. Next, the cells were pretreated with 1% bovine serum albumin (BSA) in PBS at 25°C for 60 min, incubated with rabbit anti-ADMA polyclonal antibodies (Millipore) at a dilution of 1:1000 for 1 h, and treated with fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG polyclonal antibodies at a dilution of 1:10,000 for 1 h. Finally, the cells were washed with PBS, mounted in 90% glycerol containing 4′,6-diamidino-2-phenylindole (DAPI), and photographed under an immunofluorescent microscope (Olympus). In addition, the concentrations of ADMA in the whole cell lysate were examined by ELISA.Rat glioma C6 cell lines provided by American Type Culture Collection (ATCC) were grown in RPMI medium 1640 (Life Technologies) containing 4% fetal bovine serum and 8% horse serum at 37°C in a 5% COpost hoc test. For the Morris water maze spatial acquisition test, the average of the sum of the six trials per day was analyzed by ANOVA, considering group and day as independent variables, as well as by repeated-measure ANOVA. All analyses were performed using SPSS. Values were expressed as mean ± SEM and significance was defined as P < 0.05 for all tests.Biochemical parameters, liver pathology, cell culture results, WBs, PCR, ELISA, and retention in the Morris water maze were analyzed by one-way ANOVA with Tukey HSD P = 0.031), sTNFR1 , and sTNFR2  levels as well as higher cortex TNF-α mRNA expression  than the SHAM controls  or hippocampus TNF-α mRNA expression . Interestingly, the BDL4W group rats had significantly higher cortex TNF-α protein levels , and hippocampus TNF-α mRNA expression , as well as higher plasma TNF-α , sTNFR1 , and sTNFR2  levels and higher cortex TNF-α mRNA expression  than the SHAM controls  between the BDL2W group and SHAM controls  and cortex ADMA levels , while there was no significant difference in the hippocampus levels  as compared with the SHAM controls , cortex (58.9 ± 4.0 μg/mg protein vs. 61.9 ± 26.0 μg/mg protein) or hippocampus ADMA levels  and this increase was reduced in the BDLPT group only (P = 0.042). TNF-α mRNA expression in the cortex was increased in the BDL4W group compared with SHAM controls , and this increase was reduced in both the BDLP and BDLPT groups . TNF-α mRNA expression and protein levels in the hippocampus were both increased in the BDL4W group compared with SHAM controls , and these increases were both reduced only in the BDLPT groups ; however, these were not reduced in either the BDLP or BDLPT group  and this increase was reduced in both the BDLP and BDLPT groups . The plasma level of ADMA was increased in the BDL4W group compared with SHAM controls , yet this increase was not reduced in either the BDLP or BDLPT group . Two-factor ANOVA revealed significant differences among the groups for the number of trial blocks needed to learn to escape by swimming with visual cues  = 3.31, P = 0.023). Escape latencies improved over time in all five groups as indicated by a significant effect of day  = 24.24, P < 0.001), indicating that learning occurred .Acquisition: the water maze tests revealed that all rats were able to learn how to find the platform and that there was no significant difference in swim velocity between the different treatment groups at any time  = 3.73, P = 0.014) and 4  = 3.61, P = 0.016) of acquisition. A post hoc test showed that BDLPT rats performed significantly better than BDL4W and BDLP rats on day 1 (P < 0.05) and as well as SHAM and SHAMPT rats (P > 0.05). On day 4, BDLPT rats performed significantly better than BDL4W and BDLP rats (P < 0.05) and as well as SHAM and SHAMPT rats (P > 0.05). Dually IP/IT etanercept administration ameliorated the spatial deficit, as rats in this group demonstrated a similar performance to SHAM group rats and had faster acquisition as compared with BDL4W group rats (P = 0.004). However, IP etanercept had no significant effect on the spatial deficit.There were group differences on day 1  = 0.601; P = 0.665).Retention: the “free swim” trial performed immediately after the last trial on day 5 revealed no significantly retention differences among the five groups (P = 0.014) and etanercept blocked this increase in ADMA  the BDL4W group of young rats exhibited spatial deficits as compared to SHAM and SHAMPT group rats, and combined IP and IT etanercept administration (BDLPT) rescued these effects.The main findings of this study were as follows: (1) the BDL4W group of young rats had higher plasma TNF-α, sTNFR1, sTNFR2, ADMA levels, higher hippocampus TNF-α mRNA expressions and protein levels, and cortex ADMA, TNF-α mRNA expressions and protein levels than SHAM controls; (2) the increased cortex In the central nervous system, microglia and astrocytes are believed to be the primary sources of TNF-α; TNF-α (through activation of TNFR1) disrupts learning and memory and regulates neuronal death. TNF-α has been implicated in the pathogenesis of a wide number of neurological disorders that develop both acutely, as in traumatic brain injury and stroke, and chronically, as in Alzheimer’s disease and Parkinson’s disease  pathway is impaired in the brain of chronic moderate hyperammonemia and HE (Corbalán et al., In this study, dually IP/IT etanercept injections reduced the TNF-α mRNA and protein levels in both the cortex and the hippocampus. However, IP-administered etanercept reversed the BDL-induced increases only in the cortical TNF-α mRNA and ADMA protein levels. It affected neither the cortical TNF-α protein expression nor the hippocampal TNF-α mRNA and protein levels. The discordant response of TNF-α mRNA and protein expression to the treatment may be related to differences in mRNA export, ribosome recruitment to mRNA affecting translation efficiency, protein stability, or microRNAs (Carpenter et al., It is intriguing that the levels of both ADMA and TNF-α were more readily decreased in the brain than the plasma by peripheral or central etanercept administration. There are several possibilities. First, we measured plasma TNF-α by ELISA and cortex TNF-α by WB through recognizing the epitope in the cohort 2 study. TNF bound to etanercept can still be present and detectable but be unable to mediate biological or inflammatory effects (Bhatia and Kast, HE is a frequent and serious complication of chronic liver failure. This study demonstrated that young BDL rats exhibited increased circulating and brain TNF-α and ADMA levels, as well as spatial deficits, as in adult BDL rats, indicating that increases in and interaction between TNF-α and ADMA play an important role in HE. Moreover, combined IP and IT etanercept reduce spatial deficits in young BDL rats. Appropriate targeting of TNF-α and ADMA may offer a new treatment avenue for HE.J-MS: performed the majority of the experiments and wrote this manuscript. Y-CC: aided in experimental design and behavioral testing. M-HH: assisted with experimental design and aided in serum analysis. Y-LT and H-RY: assisted with planning and interpreting behavioral testing. L-TH: designed the study, edited and provided final approval of this manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Heart failure with preserved ejection fraction (HFpEF) represents a complex and heterogeneous clinical syndrome, which is increasingly prevalent and associated with poor outcome. In contrast to heart failure with reduced ejection fraction (HFrEF), modern heart failure pharmacotherapy did not improve outcome in HFpEF, which was attributed to incomplete understanding of HFpEF pathophysiology, patient heterogeneity and lack of insight into primary pathophysiological processes. HFpEF patients are frequently elderly females and patients demonstrate a high prevalence of non-cardiac comorbidities, which independently adversely affect myocardial structural and functional remodelling. Furthermore, although diastolic left ventricular dysfunction represents the dominant abnormality in HFpEF, numerous ancillary mechanisms are frequently present, which also negatively impact on cardiovascular reserve. Over the past decade, clinical and translational research has improved insight into HFpEF pathophysiology and the importance of comorbidities and patient heterogeneity. Recently, a new paradigm for HFpEF was proposed, which states that comorbidities drive myocardial dysfunction and remodelling in HFpEF through coronary microvascular inflammation. Regarding the conceptual framework of HFpEF treatment, emphasis may need to shift from a ‘one fits all’ strategy to an individualised approach based on phenotypic patient characterisation and diagnostic and pathophysiological stratification of myocardial disease processes. This review will describe these novel insights from a pathophysiological standpoint. In the Under physiological conditions, LV pressure rapidly decreases after systole, allowing fast diastolic LV filling at maintained low filling pressures. Diastolic LV dysfunction in HFpEF is evident from slow LV relaxation and elevated diastolic LV stiffness, which increase diastolic filling pressures and limit cardiac performance at rest, during atrial pacing and exercise , 10. InsHFpEF patients are generally older, more often female and have a high prevalence of cardiovascular and non-cardiovascular comorbidities, such as obesity, metabolic syndrome, diabetes mellitus type 2, salt-sensitive hypertension, atrial fibrillation (AF), chronic obstructive pulmonary disease, anaemia and renal dysfunction –26. SystThe new HFpEF paradigm proposes that comorbidities drive structural and functional remodelling in HFpEF through systemic endothelial inflammation . BecauseMicrovascular endothelial inflammation is also associated with myocardial capillary rarefaction , which wA recent autopsy study demonstrated reduced myocardial capillary density (MCD) in HFpEF patients regardless of the severity of epicardial coronary disease, while the severity of myocardial fibrosis was inversely associated with MCD . Both fiHFpEF is difficult to define as illustrated by various diagnostic classifications and inclusion criteria of clinical trials . These fAlthough ejection fraction is preserved, both chamber and myocardial contractility were subtly but significantly depressed in HFpEF, compared with hypertensive and healthy controls . The extMaintaining low ventricular and arterial elastance allows a dynamic range of volume transfer to be achieved during ejection with minimal change in pressure. Ventricular and vascular stiffening increase with ageing, hypertension and diabetes and are abnormally elevated in HFpEF patients , 39. ComPatients with HFpEF display attenuated exercise-mediated reductions in mean vascular resistance and arterial elastance, coupled with abnormalities in endothelial function and dynamic ventricular-arterial coupling compared with hypertensive subjects and controls . The extChronotropic reserve is depressed in HFpEF , 35, whiIn a community-based study, the prevalence of pulmonary hypertension, defined as pulmonary artery systolic pressure (PASP) > 35 mmHg, amounted to 83 % with a median PASP of 48 mmHg in a group of 244 HFpEF patients. In this study, PASP was significantly higher in HFpEF patients than in hypertensives without heart failure, whereas PASP strongly predicted mortality in HFpEF . ChronicIn an invasive study, right and left heart filling pressures, pulmonary artery pressures and right-sided chamber dimensions were higher in HFpEF compared with controls, while LV size and ejection fraction were similar. RV dysfunction was present in 33 % of HFpEF patients and was caused by both RV contractile impairment and afterload mismatch from pulmonary hypertension . RV dysfThe underlying phenotypic heterogeneity is likely far greater in HFpEF than in HFrEF and may be an important reason for the failure of HFpEF clinical trials . For insRecent HFpEF trials demonstrated structural cardiac remodelling in many HFpEF patients including concentric LV remodelling and hypertrophy 59–77 %) and left atrial (LA) dilatation (59–66 %) 9–77 % an. FurtherCurrently, only NPs are routinely used for diagnosis and risk stratification in HFpEF patients. Because HFpEF is characterised by inflammation, oxidative stress, endothelial dysfunction, alterations in intramyocardial signalling and matrix remodelling and capillary rarefaction, biomarkers reflecting these processes could aid in diagnostic and pathophysiological stratification in HFpEF patients. Recently, several biomarkers were identified, which appeared to be promising diagnostic and prognostic tools in patients with HFpEF .