Medium Of Fibroblasts Research Articles

Feasibility of improving platelet-rich plasma therapy by using chitosan with high platelet activation ability.

Platelet-rich plasma (PRP) is blood plasma containing a high number of platelets that release growth factors for wound healing and tissue regeneration. In the present study, the feasibility of improving PRP therapy by using chitosan that exhibits high platelet activation ability was investigated. A total of 13 chitosan samples with different molecular weight (Mw) and degree of deacetylation (DDA) were individually added to blood samples of rats and the amount of growth factors, albumin and fibrinogen in plasma was measured. To examine the influence of plasma activated by chitosan on the proliferation of fibroblasts and adipose tissue-derived stromal cells (ASCs), the plasma was added to the culture medium of human fibroblasts and adipose tissue-derived stromal cells. Chitosan with a DDA of >75% increased the release of platelet factor 4 into the plasma. The amount of growth factors released into the plasma and platelet activation varied depending on the Mw and DDA, while albumin and fibrinogen were hardly affected. The proliferation rate was highest when using plasma activated by chitosan with a DDA of 75-85% and an Mw of 50,000-190,000 Da. These results suggested that the effectiveness of PRP therapy may be improved by using chitosan with a DDA of 75-85% and an Mw of 50,000-190,000 Da.

Haploinsufficiency of the folliculin gene leads to impaired functions of lung fibroblasts in patients with Birt-Hogg-Dubé syndrome.

Birt–Hogg–Dubé syndrome (BHDS) is an autosomal dominant inherited disorder caused by germline mutations in the FLCN gene, and characterized by skin fibrofolliculomas, multiple lung cysts, spontaneous pneumothorax, and renal neoplasms. Pulmonary manifestations frequently develop earlier than other organ involvements, prompting a diagnosis of BHDS. However, the mechanism of lung cyst formation and pathogenesis of pneumothorax have not yet been clarified. Fibroblasts were isolated from lung tissues obtained from patients with BHDS (n = 12) and lung cancer (n = 10) as controls. The functional abilities of these lung fibroblasts were evaluated by the tests for chemotaxis to fibronectin and three‐dimensional (3‐D) gel contraction. Fibroblasts from BHDS patients showed diminished chemotaxis as compared with fibroblasts from controls. Expression of fibronectin and TGF‐β1 was significantly reduced in BHDS fibroblasts when assessed by qPCR. Addition of TGF‐β1 in culture medium of BHDS lung fibroblasts significantly restored these cells' abilities of chemotaxis and gel contraction. Human fetal lung fibroblasts (HFL‐1) exhibited reduced chemotaxis and 3‐D gel contraction when FLCN expression was knocked down. To the contrary, a significant increase in chemotactic activity toward to fibronectin was demonstrated when wild‐type FLCN was overexpressed, whereas transduction of mutant FLCN showed no effect on chemotaxis. Our results suggest that FLCN is associated with chemotaxis in lung fibroblasts. Together with reduced TGF‐β1 expression by BHDS lung fibroblasts, a state of FLCN haploinsufficiency may cause lung fibroblast dysfunction, thereby impairing tissue repair. These may reveal one mechanism of lung cyst formation and pneumothorax in BHDS patients.

Exosome expression in the skin and sera of systemic sclerosis patients, and its possible therapeutic application against skin ulcer

Exosomes are small vesicles, which are shed from various cells and contain proteins, lipids and nucleic acids. They are recently paid attention as a tool of cell-cell communication. The aim of this study was to elucidate putative role of exosomes in the pathogenesis of systemic sclerosis (SSc). We examined the expression of exosomes, CD63 and type I collagen by real-time PCR, immunohistochemical analysis, ELISA, immunoblotting and flow cytometry. The effect of exosomes on wound healing was tested on full-thickness wounds in the mid-dorsal skin of BALB/c mice. Exosome levels in SSs fibroblasts were up-regulated compared to normal fibroblasts. Increased exosomes in a cultured medium of SSc fibroblasts stimulated type I collagen expression in normal fibroblasts. Expression of microRNAs contained in SSc exosomes was dysregulated, indicating that both the amount and the content of exosomes were altered in SSc, which stimulate collagen expression. Exosomes in SSc sera were significantly decreased compared to those in normal subjects. The frequencies of vascular involvements, including skin ulcers or pitting scars, were significantly increased in patients with decreased serum exosome levels. The healing of mice wounds was accelerated by treatment with serum-derived exosomes. Our study suggests that SSc patients with vascular involvements have decreased serum exosome levels probably due to the disturbed transfer of exosomes from the skin tissue to the blood stream. The decrease in turn causes the delay of wound healing due to down-regulation of collagen, resulting in susceptibility to pitting scars and/or ulcers. Exosome supplementation to skin ulcers may have a great potential as new therapeutic option.

Efficacy of bezafibrate on fibroblasts of glutaric acidemia type II patients evaluated using an in vitro probe acylcarnitine assay

IntroductionWe evaluated the effects of bezafibrate (BEZ) on β-oxidation in fibroblasts obtained from patients with glutaric acidemia type II (GA2) of various clinical severities using an in vitro probe (IVP) assay. MethodsCultured fibroblasts from 12 patients with GA2, including cases of the neonatal-onset type both with and without congenital anomalies (the prenatal- and neonatal-onset forms, respectively), the infantile-onset, and the myopathic forms, were studied. The IVP assay was performed by measuring acylcarnitines (ACs) in the cell culture medium of fibroblasts incubated with palmitic acid for 96h in the presence of 0–800μM BEZ using tandem mass spectrometry. ResultsThe IVP assay showed that 100μM BEZ markedly reduced the level of palmitoylcarnitine (C16) in the neonatal-onset, infantile-onset, and myopathic forms of GA2, either increasing or maintaining a high level of acetylcarnitine (C2), which serves as an index of energy production via β-oxidation. In the prenatal-onset form, although a small reduction of C16 was also observed in the presence of 100μM BEZ, the level of C2 remained low. At concentrations higher than 100μM, BEZ further decreased the level of ACs including C16, but a concentration over 400μM decreased the level of C2 in most cases. DiscussionBEZ at 100μM was effective for all GA2 phenotypes except for the prenatal-onset form, as a reduction of C16 without deterioration of C2 is considered to indicate improvement of β-oxidation. The effects of higher doses BEZ could not be estimated by the IVP assay but might be small or nonexistent.

Abstract B17: Xenospheres: a comprehensive patient-derived in vitro model to study response and resistance to targeted therapies in metastatic colorectal cancer

Abstract Although EGFR targeted therapy has improved the survival outcome of patients with metastatic colorectal cancer, lack of response or emergence of secondary resistance frequently occurs as result of genetic alterations that hyperactivate the RAS pathway or by activation of compensatory pathways by signals from the tumor microenvironmet that can also induce a stem-like phenotype. From patient-derived xenografts (“xenopatients”) of colorectal cancer liver metastases, we generated primary sphere cultures (“xenospheres”) that display the properties of cancer stem-like cells. Indeed, these cells can long-term self-propagate in vitro, and form phenocopies of original patient tumors in vivo (“spheropatients”), maintaining the same response to anti-EGFR therapy. We obtained a panel of xenospheres derived from more than 40 different xenopatients, all genetically characterized for the presence of mutations in genes that are predictive for response to EGFR targeted therapy. The panel included xenospheres harboring mutations in RAS genes (Rasmut), or recently identified rare predictors of resistance: ERBB2 amplification/mutation, MET amplification, EGFR mutation in the extracellular domain, and IGF2 overexpression. Moreover, we conducted an extensive exome-sequencing analysis of 10 xenospheres, for which the exome data of the matched xenopatients were available, thus allowing a complete and robust comparison. Xenospheres displayed a good genetic stability and a remarkable correspondence with xenopatients, in both the mutational and gene expression profiles. While RASmut xenospheres displayed self-sustained proliferative ability, those lacking mutations affecting the RAS pathway (RASwt) were strongly dependent of exogenous growth factors. We thus assessed the response of all RASwt xenospheres to ligands of the EGF family (TGFA, EREG, AREG, HB-EGF, NRG1), or bFGF, or HGF, testing both proliferation and response to EGFR inhibition (cetuximab). We found that TGFA and HB-EGF induced proliferation and resistance to cetuximab with almost the same potency of EGF, while EREG and AREG had a weak mitogenic activity and did not protect against cetuximab treatment, accordingly with observations in patients and xenopatients. The different proliferative activity (and protection against cetuximab) can be at least in part explained by the effect on receptor stabilization and exposure to the cell membrane exerted by different ligands. Interestingly, we found that, among all EGF family ligands, NRG1 (binding ERBB3/4) had the most potent mitogenic activity and protection against cetuximab treatment. NRG1 was able to replace EGF in sustaining xenosphere propagation, without altering the global gene expression profile, implying that it could sustain cancer-stem like cell properties similarly to EGF. To study the role of NRG1 in vivo we generated both an autocrine and a paracrine mode, by inducing its expression infecting either RASwt xenospheres or a cell line of murine fibroblasts. NRG1-expressing RASwt xenospheres became growth factor independent, resistant to cetuximab but sensitive to lapatinib (a dual EGFR/ERBB2 small molecule). Similar results were obtained using the conditioned medium of NRG1-expressing murine fibroblasts. By injecting parental and modified RASwt xenospheres into immunocompromised mice, we found that NRG1-expressing xenospheres had a strongly increased tumor onset, and that these tumors were resistant to cetuximab but sensitive to lapatinib treatment, suggesting its possible use in the clinical practice of RASwt patients. We conclude that xenospheres are a robust patient-derived in vitro model of colorectal cancer-stem like cells, amenable to study molecular mechanisms of response or resistance to targeted therapies. Citation Format: Paolo Luraghi, Viola Bigatto, Gigliola Reato, Elia Cipriano, Francesco Sassi, Claudio Isella, Andrea Bertotti, Livio Trusolino, Paolo Maria Comoglio, Carla Boccaccio. Xenospheres: a comprehensive patient-derived in vitro model to study response and resistance to targeted therapies in metastatic colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B17.

345 Cell culture media that are less proliferative and more homeostatic improve differentiation, responsiveness and co-culture of keratinocytes, fibroblasts and melanocytes in 2D and 3D culture

Combinations of highly proliferative cell culture additives such as serum (FCS), pituitary extract (BPE), insulin and EGF/FGF-family members are routinely used to generate rapid in vitro proliferation of a variety of skin cell types. Highly concentrated proliferative signals such as these are ideal for amplifying cells, however they are poorly suited to many other situations where very different cell behaviours are required. We have investigated a range of less proliferative, more homeostatic media for their suitability when inducing differentiation and co-culturing keratinocytes, fibroblasts and melanocytes. Differentiation was improved in all three cell types, with 2x better barrier (3D keratinocytes), 2.5x more melanin (2D melanocytes), and 5x more ECM (2D fibroblasts). Homeostatic media were also found to increase responsiveness of melanocytes and fibroblasts to external stimuli such as tyrosinase inhibitors and vitamin C treatment respectively. Variants of standard proliferation media were also found to deliver balanced 2D co-culture of keratinocytes and fibroblasts, with each cell type delivering similar proliferation rates when seeded at equal density on standard tissue culture plastic. Finally, a fibroblast medium tailored for ECM production was also found to be ideally suited to the fast and simple establishment of full-thickness skin models with fully natural dermal matrix, thereby avoiding the handling challenges and variability associated with traditional collagen scaffolds. In conclusion, less proliferative, more homeostatic culture media were found to deliver a range of improved cell behaviours that complement the single rapid-growth behaviour created by standard proliferation media.

Endothelium-Derived 5-Methoxytryptophan Protects Endothelial Barrier Function by Blocking p38 MAPK Activation.

The endothelial junction is tightly controlled to restrict the passage of blood cells and solutes. Disruption of endothelial barrier function by bacterial endotoxins, cytokines or growth factors results in inflammation and vascular damage leading to vascular diseases. We have identified 5-methoxytryptophan (5-MTP) as an anti-inflammatory factor by metabolomic analysis of conditioned medium of human fibroblasts. Here we postulated that endothelial cells release 5-MTP to protect the barrier function. Conditioned medium of human umbilical vein endothelial cells (HUVECs) prevented endothelial hyperpermeability and VE-cadherin downregulation induced by VEGF, LPS and cytokines. We analyzed the metabolomic profile of HUVEC conditioned medium and detected 5-MTP but not melatonin, serotonin or their catabolites, which was confirmed by enzyme-linked immunosorbent assay. Addition of synthetic pure 5-MTP preserved VE-cadherin and maintained barrier function despite challenge with pro-inflammatory mediators. Tryptophan hydroxylase-1, an enzyme required for 5-MTP biosynthesis, was downregulated in HUVECs by pro-inflammatory mediators and it was accompanied by reduction of 5-MTP. 5-MTP protected VE-cadherin and prevented endothelial hyperpermeability by blocking p38 MAPK activation. A chemical inhibitor of p38 MAPK, SB202190, exhibited a similar protective effect as 5-MTP. To determine whether 5-MTP prevents vascular hyperpermeability in vivo, we evaluated the effect of 5-MTP administration on LPS-induced murine microvascular permeability with Evans blue. 5-MTP significantly prevented Evans blue dye leakage. Our findings indicate that 5-MTP is a new class of endothelium-derived molecules which protects endothelial barrier function by blocking p38 MAPK.

Transdifferentiation Potency of Fibroblast Cell to Neuron Cell in Vitro

The aim of this study was to examine the potency of fibroblast cells transdifferentiated to neuron cells in vitro. Newborn rat neuron conditioned medium (NBRN CM) was collected from neuron cells cultured with mDMEM without serum for 48 hours. Fibroblast cells werecollected from fetal rat muscle treated with trypsin. Fibroblast cells were culture with 3 kind of culture medium: mDMEM + 0.01 mM β-mercaptoethanol; mDMEM + 50% NBRN-CM and mDMEM + 0.01 mM β-mercaptoethanol + 50% NBRN CM . As control, cells was culturedwith mDMEM +10% newborn calf serum (NBCS). The addition of NBRN CM into culture medium resulted in 12.97% newborn cells in fibroblastculture medium passage I. Newborn rat neuron conditioned medium in fibroblast culture medium resulted 12.97% neuron cells at passage 1. Thepercentage was increased (14.60%) when β- mercaptoethanol added into medium. The same result was found at passage 3 (12.67%; 13.17%). Itshowed that fibroblast cells has potency to transdifferentiated into neuron cells when cultured with NBRN CM. Further research is needed toknow the fibroblast transdifferentiation potency.Key words: fibroblast, transdifferentiation, conditioned medium, neuron cells, in vitro

CAF-like state in primary skin fibroblasts with constitutional BRCA1 epimutation sheds new light on tumor suppressor deficiency-related changes in healthy tissue

ABSTRACTConstitutive epimutations of tumor suppressor genes are increasingly considered as cancer predisposing factors equally to sequence mutations. In light of the emerging role of the microenvironment for cancer predisposition, initiation, and progression, we aimed to characterize the consequences of a BRCA1 epimutation in cells of mesenchymal origin. We performed a comprehensive molecular and cellular comparison of primary dermal fibroblasts taken from a monozygous twin pair discordant for recurrent cancers and BRCA1 epimutation, whose exceptional clinical case we previously reported in this journal. Comparative transcriptome analysis identified differential expression of extracellular matrix-related genes and pro-tumorigenic growth factors, such as collagens and CXC chemokines. Moreover, genes known to be key markers of so called cancer-associated fibroblasts (CAFs), such as ACTA2, FAP, PDPN, and TNC, were upregulated in fibroblasts of the affected twin (BRCA1mosMe) in comparison to those of the healthy twin (BRCA1wt). Further analyses detected CAF-typical cellular features, including an elevated growth rate, enhanced migration, altered actin architecture and increased production of ketone bodies in BRCA1mosMe fibroblasts compared to BRCA1wt fibroblasts. In addition, conditioned medium of BRCA1mosMe fibroblasts was more potent than conditioned medium of BRCA1wt fibroblasts to promote cell proliferation in an epithelial and a cancer cell line. Our data demonstrate, that a CAF-like state is not an exclusive feature of tumor-associated tissue but also exists in healthy tissue with tumor suppressor deficiency. The naturally occurring phenomenon of twin fibroblasts differing in their BRCA1 methylation status revealed to be a unique powerful tool for exploring tumor suppressor deficiency-related changes in healthy tissue, reinforcing their significance for cancer predisposition.

Site-specific fibroblasts regulate site-specific inflammatory niche formation in gastric cancer.

Fibroblasts are the commonest type of cancer stromal cells. Inflammation occurs in cancer tissue, and the inflammatory process has been suggested to be caused by interactions between immune cells and cancer cells. In this study, we clarified that site-specific fibroblasts regulate the formation of a site-specific inflammatory niche according to the depth of gastric cancer cell invasion. Immunohistochemistry was performed with paraffin-embedded tissues. The numbers of immune cells and the fibroblast area were calculated according to the cancer depth. The gene expression patterns of submucosal fibroblasts and subperitoneal fibroblasts stimulated with HSC44PE-conditioned medium were analyzed with a microarray. To examine the effects on the cancer microenvironment of differences in gene expressions between HSC44PE-stimulated submucosal fibroblasts and subperitoneal fibroblasts, assays of HSC44PE proliferation, T cell migration, and M2-like macrophage differentiation were performed. The distributions of immune cells differed between the submucosal layer and the subserosal layer. The number of M2 macrophages was significantly higher and the fibroblast area was significantly larger in the subserosal layer compared with the submucosal layer. High expression levels of IL1B, TNFSF15, and CCL13 were observed in HSC44PE-stimulated submucosal fibroblasts, and higher expression levels of TGFB2, CSF1, CCL8, and CXCL5 were found in HSC44PE-stimulated subperitoneal fibroblasts. HSC44PE-stimulated subperitoneal fibroblast medium promoted the differentiation of monocytes into M2-like macrophages, whereas HSC44PE-stimulated submucosal fibroblasts significantly induced the migration of Jurkat cells and the growth of HSC44PE cells. The dynamic states of immune cells differ between the submucosal and subserosal layers in cancer tissues. Site-specific fibroblasts regulate site-specific inflammatory niche formation according to the depth of cancer cell invasion.

Abstract 11569: Activation of cAMP/CREB/PGC-1α Pathway Plays a Key Role for a Novel Adenosine-like Angiogenic Agent COA-Cl to Induce Production of Vascular Endothelial Growth Factor (VEGF) in Cultured Human Fibroblasts

COA-Cl is a recently developed pro-angiogenic agent (Figure) that promotes tube forming activity of human umbilical vein endothelial cells that are co-cultured with fibroblasts. Recently, a metabolic stress-related co-transcription factor termed PGC-1α was identified as a novel activator of VEGF gene, a key regulator of angiogenesis. In skeletal muscle, the cAMP/CREB signaling pathway plays a central role in inducing PGC-1α expression. We therefore hypothesized that COA-Cl induces PGC-1α to promote VEGF production by elevating cAMP and activating CREB in fibroblasts. COA-Cl (100 μM for 48 h) augmented VEGF secretion into culture medium of normal human dermal fibroblasts from below the level of detection to 54±4 pg/mL (ELISA, p<0.01 vs. basal). This enhancement of VEGF secretion was associated with up-regulation of mRNA encoding VEGF and PGC-1α (RT-PCR, 2.1±0.3 and 9.9±0.8 fold, p<0.05, respectively). Induction of PGC-1α expression by COA-Cl was attenuated by 49±4% (p<0.01 vs. COA-Cl alone) by H-89 (100 μM), an inhibitor of PKA. Conversely, forskolin (25 μM), a direct stimulator of adenylyl cyclase that increases cAMP, increased VEGF production (423±20 pg/mL, p<0.01 vs. basal) and expression of mRNA encoding both VEGF and PGC-1α. COA-Cl induced an increase in cAMP content (ELISA) and phosphorylation of CREB at Ser133 (immunoblot), a PKA site associated with an increased transcriptional activity of CREB, in a time- and dose-dependent manner (4.4±0.3 and 2.3±0.2 fold at 100 μM COA-Cl for 20 min, p<0.05, respectively). In COS-7 fibroblast-like cells, COA-Cl elevated luciferase activity of transfected reporter construct driven by CRE, which is regulated by cAMP/CREB (2.4±0.1 fold at 200 μM COA-Cl for 24 h). Collectively, our results demonstrate that COA-Cl elevates cAMP and activates CREB in human fibroblasts to induce PGC-1α expression, thereby promoting VEGF transcription and secretion, potentially identifying a novel way to induce therapeutic angiogenesis.

Hydrogen sulfide suppresses transforming growth factor-β1-induced differentiation of human cardiac fibroblasts into myofibroblasts.

In heart disease, transforming growth factor-β1 (TGF-β1) converts fibroblasts into myofibroblasts, which synthesize and secrete fibrillar type I and III collagens. The purpose of the present study was to investigate how hydrogen sulfide (H2S) suppresses TGF-β1-induced differentiation of human cardiac fibroblasts to myofibroblasts. Human cardiac fibroblasts were serum-starved in fibroblast medium for 16 h before exposure to TGF-β1 (10 ng mL(-1)) for 24 h with or without sodium hydrosulfide (NaHS, 100 µmol L(-1), 30 min pretreatment) treatment. NaHS, an exogenous H2S donor, potently inhibited the proliferation and migration of TGF-β1-induced human cardiac fibroblasts and regulated their cell cycle progression. Furthermore, NaHS treatment led to suppression of fibroblast differentiation into myofibroblasts, and reduced the levels of collagen, TGF-β1, and activated Smad3 in TGF-β1-induced human cardiac fibroblasts in vitro. We therefore conclude that H2S suppresses TGF-β1-stimulated conversion of fibroblasts to myofibroblasts by inhibiting the TGF-β1/Smad3 signaling pathway, as well as by inhibiting the proliferation, migration, and cell cycle progression of human cardiac myofibroblasts. These effects of H2S may play significant roles in cardiac remodeling associated with heart failure.

Effects of conditioned medium of prematurely senescent fibroblasts induced by ultraviolet B on cellular proliferation, aging and autophagy of human dermal fibroblasts

Objective To investigate the effects of conditioned medium of prematurely senescent fibroblasts induced by ultraviolet B (UVB) on the proliferation, aging and autophagy of human dermal fibroblasts. Methods Human dermal fibroblasts were isolated from the circumcised foreskin of healthy adolescent males, and subjected to primary culture. Premature senescence was induced in some fibroblasts by UVB radiation at 10 mJ/cm2 once daily for 5 consecutive days. Some fibroblasts were classified into two groups: an experimental group cultured in conditioned medium of UVB-induced prematurely senescent fibroblasts, and a control group cultured in conditioned medium of normal fibroblasts. After treatment for 20 consecutive days, cell counting kit-8 (CCK8) assay and 5-ethynyl-2'.-deoxyuridine (EDU) staining were performed to evaluate cellular proliferation, flow cytometry was conducted to estimate cell cycle, β-galactosidase staining to determine the percentage of senescent cells, accridine orange staining to detect the autophagy level, and Western blot and indirect immunofluorescence assay were carried out to determine the expression level of the autophagy-related protein LC3-B. Statistical analysis was done by using a two-sample t test with the Graphpad Prism 5 software. Results Compared with the control group, the proliferative activity of fibroblasts was significantly decreased (0.831 ± 0.017 vs. 0.973 ± 0.017, t=5.850, P < 0.05), while EDU staining and flow cytometry both showed a significant increase in the percentage of S-phase cells (both P < 0.05), in the experimental group. The percentage of β-galactosidase-positive fibroblasts was significantly higher in the experimental group than in the control group (25.710% ± 0.304% vs. 5.257% ± 1.023%, t=19.170, P< 0.05). Accridine orange staining revealed that the red fluorescence intensity of fibroblasts was significantly lower (14.287 ± 2.269 vs. 29.614 ± 2.650, t=4.390, P < 0.05), while Western blot and indirect immunofluorescence assay both showed a significant elevation in the expression level of LC3-B (both P< 0.05), in the experimental group compared with the control group. Conclusions The conditioned medium of prematurely senescent fibroblasts induced by UVB can downregulate autophagy and proliferation of fibroblasts, but accelerate their aging. Key words: Ultraviolet rays; Fibroblasts; Cell aging; Autophagy; Cell proliferation

5-methoxytryptophan is a circulating anti-inflammatory molecule that controls lipopolysaccharide-induced systemic inflammation and sepsis (IRM11P.622)

Abstract We identified in the conditioned medium of human fibroblasts a tryptophan metabolite, 5-methoxytryptophan (5-MTP) which controls cyclooxgenase-2 expression and inhibits NF-κB binding and p300 activity. We postulated that as with fibroblasts, human endothelial cells produce 5-MTP which plays an important role in controlling systemic inflammatory responses. Human endothelial cells produced and released 5-MTP into the conditioned medium. Lipopolysaccharide (LPS) suppressed 5-MTP production in endothelial cells and induced 5-MTP deficiency in the murine sepsis model. Intraperitoneal infusion of 5-MTP restored serum 5-MTP accompanied by inhibition of systemic pro-inflammatory cytokines, chemokines, and pro-inflammatory mediators. 5-MTP administration rescued organ failure and prevented mortality. The beneficial effect of 5-MTP was correlated with inhibition of cytokine production and COX-2 expression in macrophages. 5-MTP inhibited LPS-induced p38 MAPK, p300 HAT and NF-κB activation in macrophages and endotoxemic lung tissues in vivo. Importantly, a considerable amount of 5-MTP was detected in 30 healthy subjects (1.05 ± 0.39 μM) which was significantly reduced in 50 sepsis patients (0.37 ± 0.15 μM, p&amp;lt;0.0001). 5-MTP is a novel circulating anti-inflammatory molecule which protects against excessive systemic inflammatory responses. It ameliorates LPS-induced sepsis and is a valuable lead compound for developing new drugs to treat sepsis and systemic inflammatory disorders.

Upregulation of fibroblast growth factor 1 in the synovial membranes of patients with late stage osteoarthritis.

Osteoarthritis (OA) is a degenerative disease of the systemic joint that involves multiple cytokines and growth factors. Fibroblast growth factor 1 (FGF-1) is increased in patients with rheumatic arthritis. The aim of this study was to determine whether the expression and secretion of FGF-1 differed in synovial tissue from patients with late stage OA from that in normal tissues. We selected eight patients with late stage OA and eight healthy donors for this study. An enzyme-linked immunosorbent assay was used to determine the amount of FGF-1 in the synovial fluid and in the culture medium of synovial fibroblasts. Real time quantitative polymerase chain reaction (qPCR) analysis was performed to examine the expression levels of FGF-1 and FGF receptor 2 (FGFR2) in synovial and cartilage tissues. We detected FGF-1 in the synovial fluid from all eight donors, as well as in the culture medium of synovial fibroblasts. Synovial fluid from patients with OA and culture medium of OA synovial fibroblasts contained significantly more FGF-1 than those from controls. FGF-1 expression was also lower in the synovial membranes of normal donors than in those of OA patients. FGFR2 expression was also higher in OA cartilage than in normal cartilage. Overall, these results demonstrated that FGF-1 synthesis and secretion by synovial fibroblasts were significantly increased in OA. FGFR2 expression was also shown to be upregulated in patients with OA. These findings suggest that increased FGF-1 signaling correlates with an OA pathological condition.

Corrigendum to 'Mechanisms of paracrine cardioprotection by cord blood mesenchymal stromal cells'

The authors regret that an error appeared in the above article. When reading the manufacturer’s certificate of the Luminex cytokine/growth factor screening assay, a comma (thousand separator) was mistaken for a decimal separator. Hence, the analyte concentrations given in the article as pg/ml are in fact ng/ml. The paragraph of the subsection ‘Growth factors and cytokines in conditioned medium’ under RESULTS should read: In fibroblast-conditioned medium, the total protein content was 26 ± 10% lower than in CBMSC medium (CBMSC 27 ± 2 vs fibroblasts 20 ± 2 μg/ml, P < 0.05). The concentrations of selected growth factors and cytokines are shown in Fig. 2. The concentrations of VEGF (CBMSC 6.0 ± 0.9; fibroblast 4.9 ± 0.7 ng/ml), EGF (CBMSC 0.83 ± 0.09; fibroblast 0.65 ± 0.06 ng/ml), and Angiopoetin-2 (CBMSC 0.44 ± 0.04; fibroblast 0.49 ± 0.06 ng/ml) were similar, CBMSC medium contained more HGF (CBMSC 0.36 ± 0.05 vs fibroblast 0.04 ± 0.01 ng/ml, P < 0.001), and fibroblast medium more bFGF (CBMSC 0.08 ± 0.02 vs fibroblast 0.59 ± 0.14 ng/ml, P < 0.05) and IL-6 (CBMSC 0.15 ± 0.03 vs fibroblast 5.8 ± 1.0 ng/ml, P < 0.001). IL-10 and TNFα were not detectable in either medium. In addition, the Y-axis in Figure 2 should indicate ng/ml rather than pg/ml.

Lysophosphatidylinositol: a novel link between ABC transporters and G-protein-coupled receptors.

Lysophosphatidylinositol (LPI) is a well-known bioactive lipid that is able to activate signalling cascades relevant to cell proliferation, migration, survival and tumorigenesis. Our previous work suggested that LPI is involved in cancer progression since it can be released in the medium of Ras-transformed fibroblasts and can function as an autocrine modulator of cell growth. Different research groups have established that LPI is the specific and functional ligand for G-protein-coupled receptor 55 (GPR55) and that this GPR55-LPI axis is able to activate signalling cascades that are relevant for different cell functions. Work in our laboratory has recently unravelled an autocrine loop, by which LPI synthesized by cytosolic phospholipase A₂ (cPLA₂) is pumped out of the cell by ATP-binding cassette (ABC) transporter C1 (ABCC1)/multidrug resistance protein 1 (MRP1), initiating a signalling cascade downstream of GPR55. Our current work suggests that blockade of this pathway may represent a novel strategy to inhibit cancer cell proliferation.

GRP78 clustering at the cell surface of neurons transduces the action of exogenous alpha-synuclein.

Mutation or multiplication of the alpha-synuclein (Syn)-encoding gene is frequent cause of early onset Parkinson's disease (PD). Recent evidences point to the pathogenic role of excess Syn also in sporadic PD. Syn is a cytosolic protein, which has been shown to be released from neurons. Here we provide evidence that extracellular Syn induces an increase in surface-exposed glucose-related protein of 78 kDa (GRP78), which becomes clustered in microdomains of the neuronal plasma membrane. Upon interacting with Syn, GRP78 activates a signaling cascade leading to cofilin 1 inactivation and stabilization of microfilaments, thus affecting morphology and dynamics of actin cytoskeleton in cultured neurons. Downregulation of GRP78 abolishes the activity of exogenous Syn, indicating that it is the primary target of Syn. Inactivation of cofilin 1 and stabilization of actin cytoskeleton are present also in fibroblasts derived from genetic PD patients, which show a dramatic increase in stress fibers. Similar changes are displayed by control cells incubated with the medium of PD fibroblasts, only when Syn is present. The accumulation of Syn in the extracellular milieu, its interaction with the plasma membrane and Syn-driven clustering of GRP78 appear, therefore, responsible for the dysregulation of actin turnover, leading to early deficits in synaptic function that precede neurodegeneration.

Mechanisms of paracrine cardioprotection by cord blood mesenchymal stromal cells

Among the mechanisms by which somatic stem cells may improve left ventricular function in ischaemic heart disease are pro-survival stimuli mediated by secreted factors. This phenomenon is frequently referred to, but remains poorly understood. We therefore investigated the non-regenerative cardioprotective effects of cord blood mesenchymal stromal cells (CBMSCs) in vitro and sought to identify relevant intracellular signalling pathways. Conditioned medium from CBMSCs and fibroblasts was prepared, and secreted factors were analysed by Luminex(®) immunobead assay. Murine cardiomyocyte-derived HL-1 cells were subjected to simulated ischaemia by glucose and serum deprivation and hypoxia in CBMSC-conditioned or cell-free control medium or in medium conditioned by foreskin fibroblasts. The proportions of vital, apoptotic and necrotic cells (poly-caspase activity, annexin V and ethidium homodimer-III staining) were quantified using a high-content imaging system. Metabolic activity and proliferation rate were determined via 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and 5-bromo-2-deoxyuridine assays. Phosphorylation of Akt, extracellular-signal-regulated kinase (ERK)1/2, signal transducer and activator of transcription 3 (STAT3) and glycogen synthase kinase 3β was determined by western blot, and experiments were repeated in the presence of specific small-molecule inhibitors (Wortmannin, UO126 and Stattic). CBMSC medium reduced the proportion of dead HL-1 cardiomyocytes from 39 ± 3 to 28 ± 1% (P < 0.05) and the rate of late apoptotic cells to 68 ± 2% of that in control medium (P < 0.001). Metabolic activity was increased by 12 ± 1% compared with control (P < 0.05), while in fibroblast medium it was not (5 ± 2%, P = 1). This was associated with increased phosphorylation of Akt (2-fold, P < 0.05), ERK1/2 (3-fold, P < 0.01) and STAT3 (12-fold, P < 0.001). Combined blocking of the phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt and mitogen-activated protein kinase/ERK signalling abolished the protective CBMSC effect, while blocking the pathways individually had no effect. Inhibition of STAT3 phosphorylation drastically lowered HL-1 cell viability in control medium, but not in medium conditioned by CBMSCs. The factors released by CBMSCs protect cardiomyocyte-like HL-1 cells from simulated ischaemia more than those released from fibroblasts. While CBMSC-triggered Akt and ERK1/2 activation provides protection in a compensatory manner, STAT3 is crucial for cardiomyocyte survival in ischaemia, but is not a key mediator of cytoprotective stem cell actions.

Differentiation of human embryonic stem cells to hepatocyte-like cells on a new developed xeno-free extracellular matrix

Human embryonic stem cells (hESCs) provide a new source for hepatocyte production in translational medicine and cell replacement therapy. The reported hESC-derived hepatocyte-like cells (HLCs) were commonly generated on Matrigel, a mouse cell line-derived extracellular matrix (ECM). Here, we performed the hepatic lineage differentiation of hESCs following a stepwise application of growth factors on a newly developed serum- and xeno-free, simple and cost-benefit ECM, designated "RoGel," which generated from a modified conditioned medium of human fibroblasts. In comparison with Matrigel, the differentiated HLCs on both ECMs expressed similar levels of hepatocyte-specific genes, secreted α-fetoprotein, and metabolized ammonia, showed glycogen storage activity as well as low-density lipoprotein and indocyanine green uptake. The transplantation of hESC-HLCs into the carbon tetrachloride-injured liver demonstrated incorporation of the cells into the host mouse liver and the expression of albumin. The results suggest that the xeno-free and cost-benefit matrix may be applicable in bioartificial livers and also may facilitating a clinical application of human pluripotent stem cell-derived hepatocytes in the future.