Metabolism Of Fibroblasts Research Articles

Dihydrosphingosine driven enrichment of sphingolipids attenuates TGFβ induced collagen synthesis in cardiac fibroblasts.

The sphingolipid de novo synthesis pathway, encompassing the sphingolipids, the enzymes and the cell membrane receptors, are being investigated for their role in diseases and as potential therapeutic targets. The intermediate sphingolipids such as dihydrosphingosine (dhSph) and sphingosine (Sph) have not been investigated due to them being thought of as precursors to other more active lipids such as ceramide (Cer) and sphingosine 1 phosphate (S1P). Here we investigated their effects in terms of collagen synthesis in primary rat neonatal cardiac fibroblasts (NCFs). Our results in NCFs showed that both dhSph and Sph did not induce collagen synthesis, whilst dhSph reduced collagen synthesis induced by transforming growth factor β (TGFβ). The mechanisms of these inhibitory effects were associated with the increased activation of the de novo synthesis pathway that led to increased dihydrosphingosine 1 phosphate (dhS1P). Subsequently, through a negative feedback mechanism that may involve substrate-enzyme receptor interactions, S1P receptor 1 expression (S1PR1) was reduced.

Abstract 2896: Effects of hypoxia on normal prostate fibroblast and prostate cancer associated fibroblast metabolism and matrix degradation

Abstract Synopsis: Fibroblasts are versatile cells that produce several ECM proteins such as collagen 1 as well as degradative enzymes such as matrix metalloproteinases. In cancer, fibroblasts play a significant role in tumor progression and dissemination, immunosuppression and metabolic support of cancer cells. We have identified increased cancer associated fibroblasts (CAFs) in more metastatic prostate cancers. Here we investigated the influence of hypoxia in modifying fibroblast metabolism and matrix degradation. We characterized metabolic changes triggered by hypoxia in normal prostate fibroblasts and prostate CAFs using magnetic resonance spectroscopy (MRS). We assessed changes in prostate fibroblast invasion and ECM degradation with our MR-compatible cell perfusion system (MR-CPS). Methods: Experiments were performed using human prostate fibroblasts (WPMY-1, ATCC, Manassas, VA) and human prostate cancer associated fibroblasts (PCAFs, Asterand Bioscience, Detroit, MI). WPMY-1 were derived from stromal cells from the peripheral zone of the histologically normal adult prostate. PCAFs were obtained from an adenocarcinoma of the prostate gland. To induce hypoxia, WPMY-1 cells were incubated for 48h under hypoxic conditions (0% O2). For MRS, cell extracts were obtained using a dual-phase extraction method, and HR-MRS performed and analyzed as previously described. MR-CPS experiments were carried out with WPMY-1 cells plated on ECM chamber under well-oxygenated (70% O2) or hypoxic (1% O2) conditions as previously detailed by us. Results: Under normoxia, compared with WPMY-1, PCAFs displayed significantly higher levels of glutamine, glutamate, arginine, lactate, myo-inositol, creatine (Cr), phosphocreatine (PCr), choline (cho) and lower levels of phosphocholine (PC). The response to low oxygenation was completely different between WPMY-1 and PCAFs. WPMY-1 responded to hypoxia with increased levels of glutamine, glutamate, myo-inositol, arginine, PCr and glycerophosphocholine (GPC). PCAFs, on the other hand, responded to hypoxia with decreased levels of arginine, PC, Cr, glutamine, glutamate and lactate. Finally, hypoxia triggered a significantly faster degradation of the ECM by prostate fibroblasts early in the course of the experiment, but not at later time points. Discussion: We found that hypoxia significantly altered the metabolism of both normal and cancer associated fibroblasts. The metabolic profile of WPMY-1 under hypoxia became similar to PCAFs under normoxia. ECM degradation by normal fibroblast increased under hypoxic conditions. Some of the metabolic changes can be related to supporting cancer cell metabolism and creating an immunosuppressive tumor microenvironment. These data suggest that hypoxia plays an important role in the metabolic transformation of fibroblasts to a malignant metabolic phenotype. Supported by NIH R35 CA209960. Citation Format: Jesus Pacheco-Torres, Tariq Shah, W. Nathaniel Brennen, Flonne Wildes, Zaver M. Bhujwalla. Effects of hypoxia on normal prostate fibroblast and prostate cancer associated fibroblast metabolism and matrix degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2896.

Abstract 2591: Characterizing the metabolic landscape of dermal fibroblasts in Li-Fraumeni Syndrome for the prediction of cancer risk and drug response

Abstract Purpose: Li-Fraumeni Syndrome (LFS) is a genetic disorder associated with a significant risk of early-onset cancer. This condition affects 1 in 5000 individuals and is largely driven by germline mutations in the TP53 tumor suppressor gene, which has a broad spectrum of functions including metabolic regulation. While LFS is highly penetrant, there is a wide degree of variability in clinical phenotype, including age of onset and tumor type. This variability suggests a role for patient-specific genetic factors, such as the type of TP53 mutation, which may define each individual's cancer risk and response to therapy. There remains a significant clinical need for better prognostication of LFS patients to predict disease outcomes and improve treatment options. An emerging body of literature is focused on the identification of non-invasive biomarkers to stratify patient populations. To this end, dermal skin fibroblasts (DSFs) have been shown to contain patient-specific disease correlates in a variety of conditions. Importantly, it has been shown that different TP53 mutations may underlie differential metabolic patterns in LFS fibroblasts; hence, we hypothesize that the metabolic signatures of DSFs from LFS patients can be utilized as prognostic biomarkers of cancer risk and response to treatment. Methods and Results: To understand the phenotypic diversity of LFS fibroblasts, our lab created a mouse xenograft model and co-cultured human LFS-derived DSFs with a sarcoma cancer cell line. LFS fibroblasts initiated earlier tumor onset in the mice compared to DSFs from healthy individuals, suggesting that these fibroblasts may secrete tumorigenic factors. Moreover, this effect was abrogated by exposure to rapamycin, an inhibitor of the mTORC1 protein kinase, suggesting that mTORC1 activity may govern the paracrine activity of these divergent fibroblast phenotypes. Next, to explore the role of mTORC1 in LFS, we used inducible expression of mutant p53 in DSFs. p53 mutants promoted mTORC1 hyperactivation, leading to increased anabolic activity, basal respiration and ATP production, suggesting that mTORC1 may alter fibroblast metabolism in a p53-dependent manner. Overall, these data provide evidence for divergent metabolic profiles of LFS skin fibroblasts which may reflect LFS phenotype variability. Ongoing work in our lab aims to further characterize the metabolic profiles of LFS fibroblasts through RNA sequencing and metabolomic profiling. Machine learning techniques will then be employed to identify molecular signatures correlating with clinical features such as age of tumor onset. Significance: This work will advance our understanding of how metabolism may underpin the clinical heterogeneity of LFS. The discovery of metabolic biomarkers will provide prognostic information with the potential to improve the early detection and treatment of cancers in LFS patients. Citation Format: Pamela Psarianos, Camilla Giovino, Sangeetha Paramathas, Nish Patel, Rajesh Gupta, Ran Kafri, David Malkin. Characterizing the metabolic landscape of dermal fibroblasts in Li-Fraumeni Syndrome for the prediction of cancer risk and drug response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2591.

Enhanced Wound Healing Activity of Undenatured Type I Collagen Isolated from Discarded Skin of Black Sea Gilthead Bream (Sparus aurata) Conditioned as 3D Porous Dressing.

Acid-soluble, undenatured, type I collagen (BSC) isolated, for the first time, from gilthead bream skin and the novel fabricated 3D porous wound dressing were analyzed for physicochemical and biological properties, in order to offer a safe alternative to commercial bovine collagen (BC) products. SDS-polyacrylamide analysis confirmed the purity of BSC preparation. The hydroxyproline content and temperature of denaturation of BSC were lower than those of BC, in accordance with the structural data recorded by FT-IR spectroscopy. However, certain concentrations of BSC stimulated the cell metabolism of L929 fibroblasts in a higher proportion than BC. The 3D wound dressing presented high porosity and low surface hydrophobicity that could help cell attachment and growth. The rapid biodegradation of BSC wound dressing could explain the improved in vitro cell migration and wound closure rate. In conclusion, the skin of gilthead bream from the Black Sea coast represented a valuable source for the biomedical industry, providing biocompatible, biodegradable collagen and 3D porous wound dressing, as novel material with enhanced wound healing activity.

Sulforaphane improves mitochondrial metabolism in fibroblasts from patients with fragile X-associated tremor and ataxia syndrome

CGG expansions between 55 and 200 in the 5′-untranslated region of the fragile-X mental retardation gene (FMR1) increase the risk of developing the late-onset debilitating neuromuscular disease Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS). While the science behind this mutation, as a paradigm for RNA-mediated nucleotide triplet repeat expansion diseases, has progressed rapidly, no treatment has proven effective at delaying the onset or decreasing morbidity, especially at later stages of the disease. Here, we demonstrated the beneficial effect of the phytochemical sulforaphane (SFN), exerted through NRF2-dependent and independent manner, on pathways relevant to brain function, bioenergetics, unfolded protein response, proteosome, antioxidant defenses, and iron metabolism in fibroblasts from FXTAS-affected subjects at all disease stages. This study paves the way for future clinical studies with SFN in the treatment of FXTAS, substantiated by the established use of this agent in clinical trials of diseases with NRF2 dysregulation and in which age is the leading risk factor.

Dimethyl fumarate preserves left ventricular infarct integrity following myocardial infarction via modulation of cardiac macrophage and fibroblast oxidative metabolism

Myocardial infarction (MI) is one of the leading causes of mortality and cardiovascular disease worldwide. MI is characterized by a substantial inflammatory response in the infarcted left ventricle (LV), followed by transition of quiescent fibroblasts to active myofibroblasts, which deposit collagen to form the reparative scar. Metabolic shifting between glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) is an important mechanism by which these cell types transition towards reparative phenotypes. Thus, we hypothesized that dimethyl fumarate (DMF), a clinically approved anti-inflammatory agent with metabolic actions, would improve post-MI remodeling via modulation of macrophage and fibroblast metabolism. Adult male C57BL/6J mice were treated with DMF (10 mg/kg) for 3–7 days after MI. DMF attenuated LV infarct and non-infarct wall thinning at 3 and 7 days post-MI, and decreased LV dilation and pulmonary congestion at day 7. DMF improved LV infarct collagen deposition, myofibroblast activation, and angiogenesis at day 7. DMF also decreased pro-inflammatory cytokine expression (Tnf) 3 days after MI, and decreased inflammatory markers in macrophages isolated from the infarcted heart (Hif1a, Il1b). In fibroblasts extracted from the infarcted heart at day 3, RNA-Seq analysis demonstrated that DMF promoted an anti-inflammatory/pro-reparative phenotype. By Seahorse analysis, DMF did not affect glycolysis in either macrophages or fibroblasts at day 3, but enhanced macrophage OXPHOS while impairing fibroblast OXPHOS. Our results indicate that DMF differentially affects macrophage and fibroblast metabolism, and promotes anti-inflammatory/pro-reparative actions. In conclusion, targeting cellular metabolism in the infarcted heart may be a promising therapeutic strategy.

Effects of EGF-coated titanium surfaces on adhesion and metabolism of bisphosphonate-treated human keratinocytes and gingival fibroblasts

To assess the effects of epidermal growth factor (EGF)-coated titanium (Ti) discs on the adhesion and metabolism of keratinocytes and gingival fibroblasts exposed to nitrogen-containing bisphosphonates. Keratinocytes and fibroblasts were seeded (1 × 105 cells/disc) on Ti discs coated with EGF (100 nM). After 24 h, cells were exposed or not to sodium alendronate (SA) or zoledronic acid (ZA) at different concentrations (0 = control, 0.5, 1, or 5 μM) for 48 h. Cell adhesion to the substrates was evaluated by fluorescence microscopy. Cell viability (alamarBlue, n = 6) and synthesis of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2), and keratinocytes growth factor (KGF) (ELISA, n = 6) were assessed. Data were statistically analyzed by one-way ANOVA and Tukey tests (α = 0.05). Higher cell adhesion rate was observed when keratinocytes and fibroblasts were seeded onto EGF-coated discs in comparison to uncoated discs. ZA treatment hindered the adhesion of both cell lines on the Ti discs as well as reduced the viability and synthesis of VEGF, KGF and MMP-2 by cells (p < 0.05). SA treatment did not affect cell viability, but interfered negatively on the adhesion and synthesis of EGF and KGF by the cells (p < 0.05). EGF-coated surface increased cell viability and synthesis of growth factors as well as downregulated the synthesis of MMP-2 in comparison to control (p < 0.05). EGF applied on Ti surface improves the biological responses of oral mucosa cells exposed to SA and ZA. EGF-coating on titanium may be a suitable strategy to improve oral mucosa cellular events related to biological sealing, especially for patients under bisphosphonate therapy.

Increase of aerobic glycolysis mediated by activated T helper cells drives synovial fibroblasts towards an inflammatory phenotype: new targets for therapy?

BackgroundA dysregulated glucose metabolism in synovial fibroblasts (SF) has been associated with their aggressive phenotype in rheumatoid arthritis (RA). Even though T helper (Th) cells are key effector cells in the propagation and exacerbation of synovitis in RA, little is known about their influence on the metabolism of SF. Thus, this study investigates the effect of Th cells on the glucose metabolism and phenotype of SF and how this is influenced by the blockade of cytokines, janus kinases (JAKs) and glycolysis.MethodsSF from patients with RA or osteoarthritis (OA) were cultured in the presence of a stable glucose isotopomer ([U-13C]-glucose) and stimulated with the conditioned media of activated Th cells (ThCM). Glucose consumption and lactate production were measured by proton nuclear magnetic resonance (1H NMR) spectroscopy. Cytokine secretion was quantified by ELISA. The expression of glycolytic enzymes was analysed by PCR, western blot and immunofluorescence. JAKs were blocked using either baricitinib or tofacitinib and glycolysis by using either 3-bromopyruvate or FX11.ResultsQuiescent RASF produced significantly higher levels of lactate, interleukin (IL)-6 and matrix metalloproteinase (MMP) 3 than OASF. Stimulation by ThCM clearly changed the metabolic profile of both RASF and OASF by inducing a shift towards aerobic glycolysis with strongly increased lactate production together with a rise in IL-6 and MMP3 secretion. Interestingly, chronic stimulation of OASF by ThCM triggered an inflammatory phenotype with significantly increased glycolytic activity compared to unstimulated, singly stimulated or re-stimulated OASF. Finally, in contrast to cytokine-neutralizing biologics, inhibition of JAKs or glycolytic enzymes both significantly reduced lactate production and cytokine secretion by Th cell-stimulated SF.ConclusionsSoluble mediators released by Th cells drive SF towards a glycolytic and pro-inflammatory phenotype. Targeting of JAKs or glycolytic enzymes both potently modulate SF’s glucose metabolism and decrease the release of IL-6 and MMP3. Thus, manipulation of glycolytic pathways could represent a new therapeutic strategy to decrease the pro-inflammatory phenotype of SF.

Assessment of cytotoxicity and antioxidant properties of berry leaves as by-products with potential application in cosmetic and pharmaceutical products

Vaccinum myrtillus L., Ribes nigrum L., Rubus fruticosus L., Fragaria vesca L. leaves are considered an agro-waste of the berry industry. Although numerous studies indicate fruit is a rich source of bioactive compounds, the authors prove leaves can also be a valuable source of compounds used in the pharmaceutical and cosmetic industries. The study attempts to assess and compare the antioxidant and cytotoxic properties of berry leaves extracts. The total phenolic compounds, flavonoids, anthocyanins and procyanidins content were determined. Antioxidant potential was evaluated using the DPPH radical scavenging method. Cytotoxicity studies were conducted to evaluate the effect of the extracts on the metabolism and proliferation of keratinocytes and fibroblasts in vitro. The effect on the migration capacity of these cells was also assessed. The obtained results show that the examined extracts are a source of valuable bioactive agents. All tested extracts show significant ability to remove free radicals in higher concentrations. Cytotoxicity assessments have shown that leaf extracts of the analyzed plants differ in cytotoxicity, both for keratinocytes and fibroblasts. The results of the assessment of cell migration capacity correlate with cytotoxicity tests, because the concentration of extracts showing cytotoxic activity towards the tested cells also inhibited their migration.

Mitochondrial P2X7 Receptor Localization Modulates Energy Metabolism Enhancing Physical Performance.

Basal expression of the P2X7 receptor (P2X7R) improves mitochondrial metabolism, Adenosine 5′-triphosphate (ATP) synthesis, and overall fitness of immune and non-immune cells. We investigated P2X7R contribution to energy metabolism and subcellular localization in fibroblasts (mouse embryo fibroblasts and HEK293 human fibroblasts), mouse microglia (primary brain microglia, and the N13 microglia cell line), and heart tissue. The P2X7R localizes to mitochondria, and its lack (1) decreases basal respiratory rate, ATP-coupled respiration, maximal uncoupled respiration, resting mitochondrial potential, mitochondrial matrix Ca2+ level, (2) modifies expression pattern of oxidative phosphorylation enzymes, and (3) severely affects cardiac performance. Hearts from P2rx7-deleted versus wild-type mice are larger, heart mitochondria smaller, and stroke volume, ejection fraction, fractional shortening, and cardiac output, are significantly decreased. Accordingly, the physical fitness of P2X7R-null mice is severely reduced. Thus, the P2X7R is a key modulator of mitochondrial energy metabolism and a determinant of physical fitness.

Glycolysis-derived acidic microenvironment as a driver of endothelial dysfunction in systemic sclerosis.

SSc is an autoimmune disease characterized by peripheral vasculopathy and skin and internal organ fibrosis. Accumulating evidence underlines a close association between a metabolic reprogramming of activated fibroblasts and fibrosis. This prompted us to determine the metabolism of SSc dermal fibroblasts and the effect on the vasculopathy characterizing the disease. A Seahorse XF96 Extracellular Flux Analyzer was used to evaluate SSc fibroblast metabolism. In vitro invasion and capillary morphogenesis assays were used to determine the angiogenic ability of endothelial cells (ECs). Immunofluorescence, flow cytometry and real-time PCR techniques provided evidence of the molecular mechanism behind the impaired vascularization that characterizes SSc patients. SSc fibroblasts, compared with controls, showed a boosted glycolytic metabolism with increased lactic acid release and subsequent extracellular acidification that in turn was found to impair EC invasion and organization in capillary-like networks without altering cell viability. A molecular link between extracellular acidosis and endothelial dysfunction was identified as acidic ECs upregulated MMP-12, which cleaves and inactivates urokinase-type plasminogen activator receptor, impairing angiogenesis in SSc. Moreover, the acidic environment was found to induce the loss of endothelial markers and the acquisition of mesenchymal-like features in ECs, thus promoting the endothelial-to-mesenchymal transition process that contributes to both capillary rarefaction and tissue fibrosis in SSc. This study showed the relationship of the metabolic reprogramming of SSc dermal fibroblasts, extracellular acidosis and endothelial dysfunction that may contribute to the impairment and loss of peripheral capillary networks in SSc disease.

Metabolic requirements of pulmonary fibrosis: role of fibroblast metabolism.

Fibrosis is a pathologic condition characterized by excessive deposition of extracellular matrix and chronic scaring that can affect every organ system. Organ fibrosis is associated with significant morbidity and mortality, contributing to as many as 45% of all deaths in the developed world. In the lung, many chronic lung diseases may lead to fibrosis, the most devastating being idiopathic pulmonary fibrosis (IPF), which affects approximately 3 million people worldwide and has a median survival of 3.8 years. Currently approved therapies for IPF do not significantly extend lifespan, and thus, there is pressing need for novel therapeutic strategies to treat IPF and other fibrotic diseases. At the heart of pulmonary fibrosis are myofibroblasts, contractile cells with characteristics of both fibroblasts and smooth muscle cells, which are the primary cell type responsible for matrix deposition in fibrotic diseases. Much work has centered around targeting the extracellular growth factors and intracellular signaling regulators of myofibroblast differentiation. Recently, metabolic changes associated with myofibroblast differentiation have come to the fore as targetable mechanisms required for myofibroblast function. In this review, we will discuss the metabolic changes associated with myofibroblast differentiation, as well as the mechanisms by which these changes promote myofibroblast function. We will then discuss the potential for this new knowledge to lead to the development of novel therapies for IPF and other fibrotic diseases.

Pro-inflammatory cytokines suppress HYBID (hyaluronan (HA) -binding protein involved in HA depolymerization/KIAA1199/CEMIP) -mediated HA metabolism in human skin fibroblasts

In the skin, the metabolism of hyaluronan (HA) is highly regulated. Aging leads to chronic low-grade inflammation, which is characterized by elevated levels of pro-inflammatory cytokines; however, the relationship between inflammation and HA metabolism is not clear. Herein, we investigated the effects of a mixture of pro-inflammatory cytokines containing TNF-α, IL-1β, and IL-6 on HA metabolism in human skin fibroblasts. Treatment with the cytokine mixture for 24 h suppressed HA depolymerization via downregulation of HYBID (HA-binding protein involved in HA depolymerization/KIAA1199/CEMIP) and promoted HA synthesis via upregulation of HAS2 in human skin fibroblasts. Moreover, HAS2-dependent HA synthesis was driven mainly by IL-1β with partial contribution from TNF-α. Transmembrane protein 2 (TMEM2/CEMIP2), which was previously reported as a candidate hyaluronidase, was upregulated by the cytokine mixture, suggesting that TMEM2 might not function as a hyaluronidase in human skin fibroblasts. Furthermore, the effects of the cytokine mixture on HA metabolism were observed in fibroblasts after 8 days of treatment with cytokines during three passages. Thus, we have shown that HYBID-mediated HA metabolism is negatively regulated by the pro-inflammatory cytokine mixture, providing novel insights into the relationship between inflammation and HA metabolism in the skin.

Photobiomodulation of Human Fibroblasts and Keratinocytes with Blue Light: Implications in Wound Healing.

In recent years, photobiomodulation (PBM) has been recognized as a physical therapy in wound management. Despite several published research papers, the mechanism underlying photobiomodulation is still not completely understood. The investigation about application of blue light to improve wound healing is a relatively new research area. Tests in selected patients evidenced a stimulation of the healing process in superficial and chronic wounds treated with a blue LED light emitting at 420 nm; a study in animal model pointed out a faster healing process in superficial wound, with an important role of fibroblasts and myofibroblasts. Here, we present a study aiming at evidencing the effects of blue light on the proliferation and metabolism in fibroblasts from healthy skin and keratinocytes. Different light doses (3.43, 6.87, 13.7, 20.6, 30.9 and 41.2 J/cm) were used to treat the cells, evidencing inhibitory and stimulatory effects following a biphasic dose behavior. Electrophysiology was used to investigate the effects on membrane currents: healthy fibroblasts and keratinocytes showed no significant differences between treated and not treated cells. Raman spectroscopy revealed the mitochondrial Cytochrome C (Cyt C) oxidase dependence on blue light irradiation: a significant decrease in peak intensity of healthy fibroblast was evidenced, while it is less pronounced in keratinocytes. In conclusion, we observed that the blue LED light can be used to modulate metabolism and proliferation of human fibroblasts, and the effects in wound healing are particularly evident when studying the fibroblasts and keratinocytes co-cultures.

Lipid Metabolism in Tumor-Associated Fibroblasts.

Tumor- or cancer-associated fibroblasts (TAFs), one of the most abundant stromal cell types in various carcinomas, consist of a heterogeneous cell population. Typically, TAFs are assigned with pro-tumor activities to promote tumor growth and progression. One of the key features of solid tumors is the metabolic reprogramming that induces alterations of bioenergetics and biosynthesis in both tumor cells and TAFs. Therefore, this review emphasizes TAFs lipid metabolism related to both TAFs differentiation process and TAFs crosstalk with cancer cells. We hope that this review will help understand lipid metabolism in tumor microenvironment, and support the rational design of metabolism-based approaches to improve the efficacy of cancer therapy.

Metabolic dysfunction and inflammatory disease: the role of stromal fibroblasts.

Mesenchymal stromal fibroblasts have emerged as key mediators of the inflammatory response and drivers of localised inflammation, in part through their interactions with resident and circulating immune cells at inflammatory sites. As such, they have been implicated in a number of chronic inflammatory conditions as well as in tumour progression through modifying the microenvironment. The connection between metabolic changes and altered phenotype of fibroblasts in inflammatory microenvironments has clear implications for our understanding of how chronic inflammation is regulated and for the development of new anti-inflammatory therapeutics. In this review, we consider the evidence that changes to fibroblast metabolic state underpin chronic inflammation. We examine recent research on fibroblast metabolism in inflammatory microenvironments and consider their involvement in inflammation, providing insight into the role of fibroblasts and metabolism in mediating inflammatory disease progression namely cancer, arthritis and fibrotic disorders including chronic kidney disease, pulmonary fibrosis, heart disease and liver disease.

Altered glucose metabolism and cell function in keloid fibroblasts under hypoxia

Keloids exhibit metabolic reprogramming including enhanced glycolysis and attenuated oxidative phosphorylation. Hypoxia induces a series of protective responses in mammalian cells. However, the metabolic phenotype of keloid fibroblasts under hypoxic conditions remains to be elucidated. The present study aimed to investigate glycolytic activity, mitochondrial function and morphology, and the HIF1α and PI3K/AKT signaling pathways in keloid fibroblasts (KFB) under hypoxic conditions. Our results showed that hypoxia promoted proliferation, migration invasion and collagen synthesis and inhibited apoptosis in KFB. The mRNA levels, protein expressions and enzyme activities of glycolytic enzymes in KFB were higher than those in normal skin fibroblasts (NFB) under normoxia. Moreover, hypoxia remarkedly upregulated glycolysis in KFB. Decreased activities of mitochondrial complexes and abnormal mitochondria were detected in KFB under normoxic conditions and the damage was aggravated by hypoxia. An intracellular metabolic profile assay suggested hypoxia increased glycolytic parameters except glycolytic reserve but inhibited the key parameters of mitochondrial function apart from H+ leak. Protein levels of HIF1α and phosphorylation levels of the PI3K/AKT signaling pathway were upregulated in the context of 3% oxygen. Enhanced total reactive oxygen species (ROS), mitochondrial ROS (mitoROS) and antioxidant activities of KFB were observed in response to hypoxia. Additionally, autophagy was induced by hypoxia. Our data collectively demonstrated potentiated glycolysis and attenuated mitochondrial function under hypoxia, indicating that altered glucose metabolism regulated by hypoxia could be a therapeutic target for keloids.

In vitro assessment of triterpenoids NVX-207 and betulinyl-bis-sulfamate as a topical treatment for equine skin cancer.

Equine sarcoid (ES) is the most prevalent skin tumor in equids worldwide. Additionally, aging grey horses frequently suffer from equine malignant melanoma (EMM). Current local therapies targeting these skin tumors remain challenging. Therefore, more feasible topical treatment options should be considered. In order to develop a topical therapy against ES and EMM, betulinyl-bis-sulfamate and NVX-207, derivatives of the naturally occurring betulin and betulinic acid, respectively, were evaluated for their antiproliferative (crystal violet staining assay), cytotoxic (MTS assay) and apoptotic (AnnexinV staining, cell cycle investigations) effects on primary ES cells, EMM cells and equine dermal fibroblasts in vitro. The more potent derivative was assessed for its in vitro penetration and permeation on isolated equine skin within 30 min and 24 h using Franz-type diffusion cells and HPLC analysis. Betulinyl-bis-sulfamate and NVX-207 inhibited the proliferation and metabolism in ES cells, EMM cells and fibroblasts significantly (p < 0.001) in a time- and dose-dependent manner. NVX-207 had superior anticancer effects compared to betulinyl-bis-sulfamate. Both compounds led to the externalization of phosphatidylserines on the cell membrane and DNA fragmentation, demonstrating that the effective mode of action was apoptosis. After 48 h of treatment with NVX-207, the number of necrotic cells was less than 2% in all cell types. Detected amounts of NVX-207 in the different skin layers exceeded the half-maximal inhibitory concentrations calculated by far. Even though data obtained in vitro are auspicious, the results are not unconditionally applicable to the clinical situation. Consequently, in vivo studies are required to address the antitumoral effects of topically applied NVX-207 in ES and EMM patients.

Natural Exogenous Antioxidant Defense against Changes in Human Skin Fibroblast Proteome Disturbed by UVA Radiation.

Daily exposure of the skin to UVA radiation causes oxidative modifications to cellular components and biomolecules. These include proteins involved in the metabolism and cytoprotection of fibroblasts, and their modification can contribute to the disruption of cell function and the development of skin disorders. Therefore, there remains a need for highly active cytoprotective compounds with antioxidant properties. The purpose of this study was to investigate the effect of ascorbic acid on the activity of rutin against UVA-induced changes in the proteome of human fibroblasts. All analyses were carried out on fibroblasts cultured in a three-dimensional system exposed to UVA radiation and incubated with rutin and ascorbic acid. Their proteomic profile was analyzed using nano-HPLC, which revealed 150 proteins whose expression was significantly altered between treatment conditions. UVA radiation led to changes in the expression of 82 proteins. However, some of these changes were mitigated by rutin and ascorbic acid separately (23 and 25 proteins, respectively) and rutin and ascorbic acid together (23 proteins). UVA radiation has led to the upregulation of proteins involved in gene expression, catalytic processes and antioxidant pathways, and downregulation of proteins with binding activity. Nevertheless, rutin and ascorbic acid used separately or together have countered these changes to varying degrees. Moreover, rutin and ascorbic acid stimulated fibroblasts irradiated by UVA to increase the expression of the signalling molecules responsible for the opening of the transmembrane channels. In the context of the results obtained, the observed cytoprotective effect of the cooperation of rutin and ascorbic acid results not only from the overlapping properties of the compounds. The effect of rutin alone is probably inhibited by its limited bioavailability. Therefore, its interaction with ascorbic acid increases membrane penetration and improves the cytoprotective effect on skin fibroblasts.

Gingival fibroblasts dynamically reprogram cellular metabolism during infection of Porphyromonas gingivalis

ObjectiveThe purpose of the present study was to explore the sequential changes in the cellular metabolism in gingival fibroblasts (GFs) in response toPorphyromonas gingvalis (P. gingivalis) ATCC33277 infection. DesignGFs were treated withP. gingivalis at the MOI of 50 for 4, 24 and 48 h to mimic the early, medium, and late phase in the bacterial infection. LDH assay and cell counting kit-8 were utilized to explore cell death and proliferation. Real-time PCR was utilized to explore the gene transcription of pro-inflammatory genes. The relative levels of biomolecules in GFs were measured by gas chromatography-mass spectrometry. Principal component analysis and orthogonal partial least-squares-discriminant analysis were performed to visualize the metabolic difference among experimental groups. In addition, pathway analysis was conducted regarding differential metabolites in GFs. ResultsP. gingivalis infection triggered significant gene transcription of IL-1β, IL 6, MCP 1, and MMP 1 in GFs. In addition, P. gingivalis stimulated cell proliferation of GFs at MOI of 10, 50 and 250. Moreover, P. gingivalis triggered significant cell death at higher MOI. 69, 173 and 148 metabolites were qualitatively detected at 4, 24 and 48 h after P. gingivalis infection respectively in GFs, showing a sequential change of different phase. Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that ATP-binding cassette transporters, glutathione, purine and pyrimidine metabolism was significantly altered in different phase. ConclusionsHuman GFs may sequentially rewire metabolomics to shape the inflammatory responses and support the proliferation of host cells during P. gingivalis infection.