Metabolism Of Fibroblasts Research Articles

Molecular pathways: trafficking of metabolic resources in the tumor microenvironment.

A model of tumor metabolism is proposed that describes how the complementary metabolic functions of the local stroma and the tumor cells contribute to cancer progression. Cancer cells alter the metabolism of cancer-associated fibroblasts to obtain lactate and amino acids, which are utilized for energy production, rapid growth, and resistance to chemotherapy drugs. Cancer cells use glutamine supplied by cancer-associated fibroblasts to replenish tricarboxylic acid cycle intermediates and as a nitrogen source for nucleotide synthesis. Moreover, adipocytes in the microenvironment attract cancer cells through the secretion of inflammatory cytokines and proteases. The cancer cells then induce metabolic changes in the adipocytes to acquire free fatty acids that are oxidized by cancer cells to generate energy for proliferation. Increasing knowledge about the metabolic symbiosis within the tumor has led to novel therapeutic strategies designed to restrict metabolic adaptation, including inhibiting lactate transporters and repurposing antidiabetic drugs (thiazolidinediones, metformin).

In Vitro Comparison of Biological and Synthetic Materials for Skeletal Chest Wall Reconstruction

Various biological and synthetic materials have been proposed for use in skeletal chest wall reconstruction (SCWR). Because of the lack of studies allowing a direct comparison of SCWR materials, their clinical use often depends on the surgeon's preference and experience. The aim of this study was to analyze 6 synthetic and 3 biological materials frequently used in SCWR with respect to their cytotoxicity, bacterial adhesion, surface characteristics, and mechanical properties to facilitate data-driven decisions. The effect of the SCWR materials and their extracts on the metabolism of human skeletal muscle cells (SkMCs), dermal fibroblasts, adipose cells, and osteoblasts was analyzed invitro. Bacterial adhesion was quantified by incubating samples in bacterial suspensions (Staphylococcus epidermidis, S aureus, and Escherichia coli), followed by counting colony-forming units and performing scanning electron microscopy. Moreover, the mechanical properties of the materials were analyzed under uniaxial tensile loading to failure. The metabolism of all cell types seeded ontheSCWR materials was reduced compared with untreated cells. With the exception of Vypro (Ethicon, Somerville, NJ), whose extracts significantly reduced fibroblast viability, no cytotoxic leachable substances were detected. Biological materials were less cytotoxic compared with synthetic ones, but they demonstrated increased bacterial adhesion. Synthetic materials demonstrated higher elongation to failure than did biological materials. Biological and synthetic SCWR materials showed significant differences in their cytotoxicity, bacterial adhesion, and biomechanical properties, suggesting that they may be used for different indications in SCWR. Further comparable invivo studies are needed to analyze their performance in different indications of clinical application.

Abstract B08: HMGB1 regulates natural killer (NK) cell metabolism and cross-talk with dendritic cells (DCs)

Abstract HMGB1 is an evolutionarily ancient, highly abundant and conserved chromatin-associated protein which regulates transcription factor access to DNA in the nucleus. Within the cytosol, it promotes autophagy, and when extracellular promotes chemotaxis and induces inflammation. Under conditions of cellular stress, it translocates to the cytosol where it enhances autophagy and can be actively or passively secreted and serve as a damage associated molecular pattern (DAMP) molecule. HMGB1 is a critical regulator of metabolism in murine embryonic fibroblasts and tumor cells lacking the receptor for advanced glycation endproducts (RAGE) by modulation of autophagy and promotion of mitochondrial quality control. To determine if HMGB1 also plays a role in NK and DC metabolic regulation, NKp46-Cre and CD11c-Cre mice were crossed with HMGB1 flox/flox mice to generate NK- or DC-specific HMGB1-KO mice (NKH and DCH respectively). NK and DC derived from such mice were evaluated for metabolic flux using real time assessment of oxidative phosphorylation (OCR) and glycolysis (ECR) at basal levels and following sequential addition of oligomycin, FCCP, 2-deoxyglucose, and rotenone. DCH demonstrated depressed basal and CD40L-stimulated OCR. NKH cells similarly were significantly less metabolically active in response to IL-2 stimulation. Since HMGB1 secreted from NK cells play a critical role in DC maturation, wildtype DCs were briefly co-cultured with NK cells, markedly increasing their metabolic activity. This was substantially less in DCs cocultured with NKH. These findings suggest that NK HMGB1 enhances DC metabolic activity, promoting NK:DC crosstalk. References. 1: Hou W, Zhang Q, Yan Z, Chen R, Zeh Iii HJ, Kang R, Lotze MT, Tang D. Strange attractors: DAMPs and autophagy link tumor cell death and immunity. Cell Death Dis. 2013 Dec 12;4:e966. 2: Rubartelli A, Lotze MT, Latz E, Manfredi A. Mechanisms of sterile inflammation. Front Immunol. 2013 Nov 22;4:398. doi: 10.3389/fimmu.2013.00398. 3: Li G, Tang D, Lotze MT. Ménage à Trois in stress: DAMPs, redox and autophagy. Semin Cancer Biol. 2013 Oct;23(5):380-90. 4: Kang R, Tang D, Schapiro NE, Loux T, Livesey KM, Billiar TR, Wang H, Van Houten B, Lotze MT, Zeh HJ. The HMGB1/RAGE inflammatory pathway promotes pancreatic tumor growth by regulating mitochondrial bioenergetics. Oncogene. 2013 Jan 14. doi: 10.1038/onc.2012.631 5: Tang D, Billiar TR, Lotze MT. A Janus tale of two active high mobility group box 1 (HMGB1) redox states. Mol Med. 2012 Dec 20;18:1360-2. 6: Tang D, Kang R, Coyne CB, Zeh HJ, Lotze MT. PAMPs and DAMPs: signal 0s that spur autophagy and immunity. Immunol Rev. 2012 Sep;249(1):158-75. 7: Kang R, Loux T, Tang D, Schapiro NE, Vernon P, Livesey KM, Krasinskas A, Lotze MT, Zeh HJ 3rd. The expression of the receptor for advanced glycation endproducts (RAGE) is permissive for early pancreatic neoplasia. Proc Natl Acad Sci U S A. 2012 May 1;109(18):7031-6. doi: 10.1073/pnas.1113865109 8: Buchser WJ, Laskow TC, Pavlik PJ, Lin HM, Lotze MT. Cell-mediated autophagy promotes cancer cell survival. Cancer Res. 2012 Jun 15;72(12):2970-9. doi: 10.1158/0008-5472.CAN-11-3396. 9: Liang X, De Vera ME, Buchser WJ, Romo de Vivar Chavez A, Loughran P, Beer Stolz D, Basse P, Wang T, Van Houten B, Zeh HJ 3rd, Lotze MT. Inhibiting systemic autophagy during interleukin 2 immunotherapy promotes long-term tumor regression. Cancer Res. 2012 Jun 1;72(11):2791-801. 10: Weiner LM, Lotze MT. Tumor-cell death, autophagy, and immunity. N Engl J Med. 2012 Mar 22;366(12):1156-8. 11: Livesey KM, Kang R, Vernon P, Buchser W, Loughran P, Watkins SC, Zhang L, Manfredi JJ, Zeh HJ 3rd, Li L, Lotze MT, Tang D. p53/HMGB1 complexes regulate autophagy and apoptosis. Cancer Res. 2012 Apr 15;72(8):1996-2005. 12: Kang R, Livesey KM, Zeh HJ 3rd, Lotze MT, Tang D. Metabolic regulation by HMGB1-mediated autophagy and mitophagy. Autophagy. 2011 Oct;7(10):1256-8. 13: Tang D, Kang R, Livesey KM, Kroemer G, Billiar TR, Van Houten B, Zeh HJ 3rd, Lotze MT. High-mobility group box 1 is essential for mitochondrial quality control. Cell Metab. 2011 Jun 8;13(6):701-11. Citation Format: Guanqiao Li, Philip J. Vernon, Bennett van Houten, Xiaoyan Liang, Michael T. Lotze. HMGB1 regulates natural killer (NK) cell metabolism and cross-talk with dendritic cells (DCs). [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B08. doi:10.1158/1538-7445.CHTME14-B08

Effect of Bevacizumab on Human Tenon's Fibroblasts Cultured from Primary and Recurrent Pterygium.

The purpose of this study was to compare the inhibitory effect of bevacizumab on human Tenon's fibroblasts (HTFs) cultured from primary and recurrent pterygium. Cultured HTFs were exposed to 2.0, 5.0, 7.5, and 15.0 mg/mL concentration of bevacizumab for 24 hours. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase leakage assays were then performed to assess fibroblast metabolism and viability. The matrix metalloproteinase (MMP), procollagen type I C terminal propeptide (PIP), and laminin immunoassays were performed to examine extracellular matrix production. Changes in cellular morphology were examined by phase-contrast and transmission electron microscopy. Both metabolic activity and viability of primary and recurrent pterygium HTFs were inhibited by bevacizumab in a dose-dependent manner, especially at concentrations greater than 7.5 mg/mL. Both types of HTFs had significant decreases in MMP-1, PIP, and laminin levels. Distinctly, the inhibitory effect of bevacizumab on MMP-1 level related with collagenase in primary pterygium HTFs was significantly higher than that of recurrent pterygium. Significant changes in cellular density and morphology both occurred at bevacizumab concentrations greater than 7.5 mg/mL. Only primary pterygium HTFs had a reduction in cellular density at a bevacizumab concentration of 5.0 mg/mL. Bevacizumab inhibits primary and recurrent pterygium HTFs in a dose-dependent manner, especially at concentrations greater than 7.5 mg/mL. As the primary HTFs produces larger amounts of MMP-1 compared to recurrent HTFs, significant reduction in MMP-1 level in primary pterygium HTFs after exposure to bevacizumab is likely to be related to the faster cellular density changes in primary pterygium HTFs.

Mitochondrial Ca2+ uptake correlates with the severity of the symptoms in autosomal dominant optic atrophy

The most frequent form of hereditary blindness, autosomal dominant optic atrophy (ADOA), is caused by the mutation of the mitochondrial protein Opa1 and the ensuing degeneration of retinal ganglion cells. Previously we found that knockdown of OPA1 enhanced mitochondrial Ca2+ uptake (Fülöp et al., 2011). Therefore we studied mitochondrial Ca2+ metabolism in fibroblasts obtained from members of an ADOA family. Gene sequencing revealed heterozygosity for a splice site mutation (c. 984+1G>A) in intron 9 of the OPA1 gene. ADOA cells showed a higher rate of apoptosis than control cells and their mitochondria displayed increased fragmentation when forced to oxidative metabolism. The ophthalmological parameters critical fusion frequency and ganglion cell–inner plexiform layer thickness were inversely correlated to the evoked mitochondrial Ca2+ signals. The present data indicate that enhanced mitochondrial Ca2+ uptake is a pathogenetic factor in the progress of ADOA.

GW25-e5133 Phosphatase and tensin homologue on chromosome 10 regulates angiotensin II-induced proliferation and collagen metabolism in neonatal rat cardiac fibroblasts through the Akt/P27 signaling pathway

GW25-e5133 Phosphatase and tensin homologue on chromosome 10 regulates angiotensin II-induced proliferation and collagen metabolism in neonatal rat cardiac fibroblasts through the Akt/P27 signaling pathway

Hyaluronan Metabolism in Human Keratinocytes and Atopic Dermatitis Skin Is Driven by a Balance of Hyaluronan Synthases 1 and 3

Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.

ABCC6- a new player in cellular cholesterol and lipoprotein metabolism?

BackgroundDysregulations in cholesterol and lipid metabolism have been linked to human diseases like hypercholesterolemia, atherosclerosis or the metabolic syndrome. Many ABC transporters are involved in trafficking of metabolites derived from these pathways. Pseudoxanthoma elasticum (PXE), an autosomal-recessive disease caused by ABCC6 mutations, is characterized by atherogenesis and soft tissue calcification.MethodsIn this study we investigated the regulation of cholesterol biosynthesis in human dermal fibroblasts from PXE patients and healthy controls.ResultsGene expression analysis of 84 targets indicated dysregulations in cholesterol metabolism in PXE fibroblasts. Transcript levels of ABCC6 were strongly increased in lipoprotein-deficient serum (LPDS) and under serum starvation in healthy controls. For the first time, increased HMG CoA reductase activities were found in PXE fibroblasts. We further observed strongly elevated transcript and protein levels for the proprotein convertase subtilisin/kexin type 9 (PCSK9), as well as a significant reduction in APOE mRNA expression in PXE.ConclusionIncreased cholesterol biosynthesis, elevated PCSK9 levels and reduced APOE mRNA expression newly found in PXE fibroblasts could enforce atherogenesis and cardiovascular risk in PXE patients. Moreover, the increase in ABCC6 expression accompanied by the induction of cholesterol biosynthesis supposes a functional role for ABCC6 in human lipoprotein and cholesterol homeostasis.

Artocarpin-enriched extract reverses collagen metabolism in UV-exposed fibroblasts

In previous studies, the Artocarpus incisus extract containing 45% w/w artocarpin showed activities of antioxidation, antimelanogenesis and restoration of wrinkled-skin fibroblasts. Here, extract containing 90% w/w artocarpin was tested for its antioxidant activity and in ultraviolet (UV) A-irradiated fibroblasts, its ability to restore type I collagen and inhibit matrix metalloproteinase-1 (MMP-1) elevation. This extract was a less effective antioxidant of EC50 of 116.0 ± 5.1 μg/mL than L-ascorbic acid (9.7 ± 0.01 μg/mL). The extract (0.625–50 μg/mL) showed no cytotoxicity toward primary human skin fibroblasts. MMP-1 was markedly elevated at 72 h after UVA irradiation compared to non-irradiation cells (p < 0.01). This UVA-induced elevation was inhibited by 50 μg/mL extract or 50 ng/mL all-trans retinoic acid. In an aged and sun-exposed skin tissue culture model, the increase of epidermal thickness in the 250 μg/mL artocarpin-enriched extract or 75 μg/mL all-trans retinoic acid-treated group when compared to the non-treated group was markedly observed since day 1 of treatment. Moreover, the extract or all-trans retinoic acid-treated groups exhibited higher density of immunofluorescence staining of type I collagen than non-treated group. This coincides with significantly higher (p < 0.05) collagen content, as indicated by measuring hydroxyproline. Our results firstly revealed that the artocarpin-enriched extract reversed the activities of UVA-irradiated fibroblasts and improved the type I collagen deposition in aged/photoaged skin.

Short and prolonged exposure to hyperglycaemia in human fibroblasts and endothelial cells: Metabolic and osmotic effects

High blood glucose levels are the main feature of diabetes. However, the underlying mechanism linking high glucose concentration to diabetic complications is still not fully elucidated, particularly with regard to human physiology. Excess of glucose is likely to trigger a metabolic response depending on the cell features, activating deleterious pathways involved in the complications of diabetes. In this study, we aim to elucidate how acute and prolonged hyperglycaemia alters the biology and metabolism in human fibroblasts and endothelial cells.We found that hyperglycaemia triggers a metabolic switch from oxidative phosphorylation to glycolysis that is maintained over prolonged time. Moreover, osmotic pressure is a major factor in the early metabolic response, decreasing both mitochondrial transmembrane potential and cellular proliferation. After prolonged exposure to hyperglycaemia we observed decreased mitochondrial steady-state and uncoupled respiration, together with a reduced ATP/ADP ratio. At the same time, we could not detect major changes in mitochondrial transmembrane potential and reactive oxygen species.We suggest that the physiological and metabolic alterations observed in healthy human primary fibroblasts and endothelial cells are an adaptive response to hyperglycaemia. The severity of metabolic and bioenergetics impairment associated with diabetic complications may occur after longer glucose exposure or due to interactions with cell types more sensitive to hyperglycaemia.

Pharmacological Inhibition of Poly(ADP-Ribose) Polymerases Improves Fitness and Mitochondrial Function in Skeletal Muscle

We previously demonstrated that the deletion of the poly(ADP-ribose)polymerase (Parp)-1 gene in mice enhances oxidative metabolism, thereby protecting against diet-induced obesity. However, the therapeutic use of PARP inhibitors to enhance mitochondrial function remains to be explored. Here, we show tight negative correlation between Parp-1 expression and energy expenditure in heterogeneous mouse populations, indicating that variations in PARP-1 activity have an impact on metabolic homeostasis. Notably, these genetic correlations can be translated into pharmacological applications. Long-term treatment with PARP inhibitors enhances fitness in mice by increasing the abundance of mitochondrial respiratory complexes and boosting mitochondrial respiratory capacity. Furthermore, PARP inhibitors reverse mitochondrial defects in primary myotubes of obese humans and attenuate genetic defects of mitochondrial metabolism in human fibroblasts and C. elegans. Overall, our work validates in worm, mouse, and human models that PARP inhibition may be used to treat both genetic and acquired muscle dysfunction linked to defective mitochondrial function.

Impaired Metabolic Reactivity to Oxidative Stress in Early Psychosis Patients

Because increasing evidence point to the convergence of environmental and genetic risk factors to drive redox dysregulation in schizophrenia, we aim to clarify whether the metabolic anomalies associated with early psychosis reflect an adaptation to oxidative stress. Metabolomic profiling was performed to characterize the response to oxidative stress in fibroblasts from control individuals (n = 20) and early psychosis patients (n = 30), and in all, 282 metabolites were identified. In addition to the expected redox/antioxidant response, oxidative stress induced a decrease of lysolipid levels in fibroblasts from healthy controls that were largely muted in fibroblasts from patients. Most notably, fibroblasts from patients showed disrupted extracellular matrix- and arginine-related metabolism after oxidative stress, indicating impairments beyond the redox system. Plasma membrane and extracellular matrix, 2 regulators of neuronal activity and plasticity, appeared as particularly susceptible to oxidative stress and thus provide novel mechanistic insights for pathophysiological understanding of early stages of psychosis. Statistically, antipsychotic medication at the time of biopsy was not accounting for these anomalies in the metabolism of patients' fibroblasts, indicating that they might be intrinsic to the disease. Although these results are preliminary and should be confirmed in a larger group of patients, they nevertheless indicate that the metabolic signature of reactivity to oxidative stress may provide reliable early markers of psychosis. Developing protective measures aimed at normalizing the disrupted pathways should prevent the pathological consequences of environmental stressors.

Cytokines in asthma: effects on human pulmonary fibroblasts (835.8)

In the asthmatic patient, CD4+ T lymphocytes elevate chemokines in airway fluids and blood. This study assayed effects of serum from asthmatic adults on the metabolism of healthy human lung fibroblasts in culture, comparing effects to those seen with sera from lung‐healthy volunteers. WI38 human lung fibroblasts (ATCC) were cultured in MEM + 15% fetal calf serum. At assay, 1500 cells in 100 µL were added to 96‐well plates, and after attachment, media was replaced with serum‐free MEM with 15 µL asthma or control sera. After 24 hr incubation, MTT formazan cell proliferation was assayed at 570 nm. Tests were repeated 3x.MTT of 24 asthmatics averaged 1.37 ± 0.12 vs 1.28 ± 0.11 for 16 controls, p=0.03 with 3 replications. Sera with high IgE (ELISA) were compared to low by MTT values and were not different. Serum and breath condensate have recently been assayed for multiple chemokines but not for effects of these effluents on healthy lung cells. Our tests, while not identifying components demonstrate the expression of substances in asthma that raise the inflammatory response of healthy cells (measured by MTT upregulation). We showed the upregulation was significantly greater by asthmatic sera than by control sera. The use of asthma serum‐sensitized cells in culture offers high possibility of clarifying the disease of asthma and its humoral processes beyond cell‐related triggers.Grant Funding Source: Sarah Morrison Student Grant to SL

Cytotoxicity of peracetic acid in L929 fibroblasts (1139.2)

Recently peracetic acid (PA) has been reported in the dental literature as a possible root canal irrigating solution. It has the advantage of bringing together in one product antibacterial activity and ability to remove smear layer. For selection of root canal irrigating solution it should take into account not only its therapeutic benefits, but also the possible cytotoxic effects, since it can contact the periapical tissues. The aim of this study was to evaluate the cytotoxicity of PA at different concentrations in comparison with 2.5% sodium hypochlorite (NaOCl) through the study of the L929 fibroblasts metabolism and the cytoskeleton. L929 fibroblasts in culture were exposed to different concentrations of PA and NaOCl and to the culture medium for 24 hours (control). After this period, it was evaluated the cell metabolism by methyl tetrazolium (MTT) assay and the cytoskeleton by staining actin and α‐tubulin. Data were analyzed by two‐way ANOVA and Bonferroni post test (α = 0.05). The solutions had an effect on cell viability/metabolism in a dose‐dependent manner. The cell viability decreased progressively as the concentration of irrigating solutions was increased. 0.5% AP showed similar cytotoxicity to 2.5% NaOCl (p&gt;0.5); the AP at concentrations of 1%, 2% and 2.5% showed higher cytotoxicity than 2.5% NaOCl (p&lt;0.5). The cytoskeleton suffered progressive structural changes as the concentrations of the solutions were increased. It was concluded that the AP is more cytotoxic than NaOCl. However, further studies evaluating other cell parameters and employing other PA‐based solutions need to be performed.Grant Funding Source: Supported by São Paulo Research Foundation (FAPESP) #2012/18203‐2

Association between pelvic organ prolapse and stress urinary incontinence with collagen.

The aim of the present study was to investigate the ultrastructure and content of collagen in uterosacral ligaments and paraurethral tissues in patients with pelvic organ prolapse (POP) and stress urinary incontinence (SUI), analyzing the association between POP and collagen dysfunction. The study comprised three groups: Control, POP and POP with SUI (n=30 per group). Histological characteristics of collagen fiber were observed and the diameters were measured using light and electron microscopy to determine the Type I and Type III collagen content of the main ligament in the urethral specimens. In the POP and POP with SUI groups, observations included diffuse atrophy of smooth muscles, active fibroblast metabolism, swollen mitochondria and visible Golgi apparatus. The collagen fibril diameters in the cardinal ligaments, uterosacral ligaments and paraurethral tissues were significantly greater in the POP and POP with SUI groups compared with those in the control group (P<0.01). In addition, the expression levels of Type I and Type III collagen were significantly lower in the POP and POP with SUI groups when compared with the control group (P<0.01). In the POP with SUI group, pelvic tissues were frail, resulting in smooth muscle bundles comminuting and arranging in a disorganized pattern. Fibroblast and myoblast metabolisms were active and new microvascular cells were weak. However, the collagen fibril diameter increased. Thus, collagen and ultrastructural changes in the pelvic floor may be associated with the development of POP and SUI.

STAT3, a key regulator of cell-to-cell communication in the heart

The signal transducer and activator of transcription 3 (STAT3) is fundamental for physiological homeostasis and stress-induced remodelling of the heart as deregulated STAT3 circuits are sufficient to induce dilated and peripartum cardiomyopathy and adverse remodelling after myocardial infarction. STAT3 activity depends on multiple post-translational modifications (phosphorylation, acetylation, and dimerization). It is regulated by multiple receptor systems, which are coupled to positive and negative feedback loops to ensure physiological and beneficial action. Its intracellular functions are diverse as it acts as a signalling protein, a transcription factor but also participates in mitochondria energy production and protection. STAT3 modulates proliferation, differentiation, survival, oxidative stress, and/or metabolism in cardiomyocytes, fibroblasts, endothelial cells, progenitor cells, and various inflammatory cells. By regulating the secretome of these cardiac cells, STAT3 influences a broad range of intercellular communication systems. It thereby impacts on the communication between cardiomyocytes, the plasticity of the cardiac microenvironment, the vasculature, the extracellular matrix, and the inflammation in response to physiological and pathophysiological stress. Here, we sum up current knowledge on STAT3-mediated intra- and intercellular communication within the heterogeneous cellular network of the myocardium to co-ordinate complex biological processes and discuss STAT3-dependent targets as novel therapeutic concepts to treat various forms of heart disease.

Effects of Direct Renin Inhibition on Myocardial Fibrosis and Cardiac Fibroblast Function

Myocardial fibrosis, a major pathophysiologic substrate of heart failure with preserved ejection fraction (HFPEF), is modulated by multiple pathways including the renin-angiotensin system. Direct renin inhibition is a promising anti-fibrotic therapy since it attenuates the pro-fibrotic effects of renin in addition to that of other effectors of the renin-angiotensin cascade. Here we show that the oral renin inhibitor aliskiren has direct effects on collagen metabolism in cardiac fibroblasts and prevented myocardial collagen deposition in a non-hypertrophic mouse model of myocardial fibrosis. Adult mice were fed hyperhomocysteinemia-inducing diet to induce myocardial fibrosis and treated concomitantly with either vehicle or aliskiren for 12 weeks. Blood pressure and plasma angiotensin II levels were normal in control and hyperhomocysteinemic mice and reduced to levels lower than observed in the control group in the groups treated with aliskiren. Homocysteine-induced myocardial matrix gene expression and fibrosis were also prevented by aliskiren. In vitro studies using adult rat cardiac fibroblasts also showed that aliskiren attenuated the pro-fibrotic pattern of matrix gene and protein expression induced by D,L, homocysteine. Both in vivo and in vitro studies demonstrated that the Akt pathway was activated by homocysteine, and that treatment with aliskiren attenuated Akt activation. In conclusion, aliskiren as mono-therapy has potent and direct effects on myocardial matrix turnover and beneficial effects on diastolic function.

Sarcoid-derived fibroblasts: Links between genomic instability, energy metabolism and senescence

Bovine papillomavirus 1 (BPV-1) is a well recognized etiopathogenetic factor in a cancer-like state in horses, namely equine sarcoid disease. Nevertheless, little is known about BPV-1-mediated cell transforming effects. It was shown that BPV-1 triggers genomic instability through DNA hypomethylation and oxidative stress. In the present study, we further characterized BPV-1-positive fibroblasts derived from sarcoid tumors. The focus was on cancer-like features of sarcoid-derived fibroblasts, including cell cycle perturbation, comprehensive DNA damage analysis, end-replication problem, energy metabolism and oncogene-induced premature senescence. The S phase of the cell cycle, polyploidy events, DNA double strand breaks (DSBs) and DNA single strand breaks (SSBs) were increased in BPV-1-positive cells compared to control fibroblasts. BPV-1-mediated oxidative stress may contribute to telomere dysfunction in sarcoid-derived fibroblasts. Loss of mitochondrial membrane potential and concurrent elevation in intracellular ATP production may be a consequence of changes in energy-supplying pathways in BPV-1-positive cells which is also typical for cancer cells. Shifts in energy metabolism may support rapid proliferation in cells infected by BPV-1. Nevertheless, sarcoid-derived fibroblasts representing a heterogeneous cell fraction vary in some aspects of metabolic phenotype due to a dual role of BPV-1 in cell transformation and oncogene-induced premature senescence. This was shown with increased senescence-associated β-galactosidase (SA-β-gal) activity. Taken together, metabolic phenotypes in sarcoid-derived fibroblasts are plastic, which are similar to greater plasticity of cancer tissues than normal tissues.

Role of long-chain acyl-coenzyme A synthetases in the regulation of arachidonic acid metabolism in interleukin 1β-stimulated rat fibroblasts

Acyl coenzyme A synthetase long-chain family members (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their acyl-CoAs and play an important role in fatty acid metabolism. Here we show the role of ACSL isozymes in interleukin (IL)-1β-induced arachidonic acid (AA) metabolism in rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with triacsin C, an ACSL inhibitor, markedly enhanced the IL-1β-induced prostaglandin (PG) biosynthesis. Small interfering RNA-mediated knockdown of endogenous Acsl4 expression increased significantly the release of AA metabolites, including PGE2, PGD2, and PGF2α, compared with replicated control cells, whereas knockdown of Acsl1 expression reduced the IL-1β-induced release of AA metabolites. Experiments with double knockdown of Acsl4 and intracellular phospholipase A2 (PLA2) isozymes revealed that cytosolic PLA2α, but not calcium-independent PLA2s, is involved in the Acsl4 knockdown-enhanced PG biosynthesis. Electrospray ionization mass spectrometry of cellular phospholipids bearing AA showed that the levels of some, if not all, phosphatidylcholine (PC) and phosphatidylinositol species in Acsl4 knockdown cells were decreased after IL-1β stimulation, while those in control cells were not so obviously decreased. In Acsl1 knockdown cells, the levels of some AA-bearing PC species were reduced even in the unstimulated condition. Collectively, these results suggest that Acsl isozymes play distinct roles in the control of AA remodeling in rat fibroblasts: Acsl4 acts as the first step of enzyme for AA remodeling following IL-1β stimulation, and Acsl1 is involved in the maintenance of some AA-containing PC species.

Conjugation of succinic acid to non-ionogenic amphiphilic polymers modulates their interaction with cell plasma membrane and reduces cytotoxic activity

Pluronic block copolymers L61 and L121 were reacted with succinic anhydride to produce, respectively, their mono- and bisderivatives with succinic acid. The critical micelle concentration of Pluronics decreased after modification. The modification of Pluronic L61 promoted its association with the plasma membrane of human cells and increased membrane damage, while the membranotropic activity of modified Pluronic L121 reduced compared to the initial copolymer. Modified Pluronics interfered with the viability, apoptosis induction and metabolism of A549 cells and skin fibroblasts to a much lesser extent presumably due to the introduction of succinic acid residue inhibited intracellular penetration of copolymers. Modified Pluronic L121 promoted the cellular uptake of doxorubicin and rhodamine 123 in A549 cells attributed to the inhibition of membrane P-glycoprotein. Our study provides an approach to assessing the mechanism of interaction of amphiphilic polymers with living cells and demonstrates that Pluronic–succinic acid conjugates can be used as safe and efficient modulators of intracellular drug delivery.