Primary Fibroblast Cultures Research Articles

Mitogen-Activated Protein Kinases Mediate Adventitial Fibroblast Activation and Neointima Formation via GATA4/Cyclin D1 Axis.

Activation of mitogen-activated protein kinases (MAPKs) by pathological stimuli participates in cardiovascular diseases. Dysfunction of adventitial fibroblast has emerged as a critical regulator in vascular remodeling, while the potential mechanism remains unclear. In this study, we sought to determine the effect of different activation of MAPKs in adventitial fibroblast contributing to neointima formation. Balloon injury procedure was performed in male 12-week-old Sprague-Dawley rats. After injury, MAPK inhibitors were applied to the adventitia of injured arteries to suppress MAPK activation. Adventitial fibroblasts were stimulated by platelet-derived growth factor-BB (PDGF-BB) with or without MAPK inhibitors. RNA sequencing was performed to investigate the change of pathway and cell function. Wound healing, transwell assay, and flow cytometry were used to analyze adventitial fibroblast function. Phosphorylation of p38, c-Jun N-terminal kinase (JNK), andextracellular regulated kinases 1/2 (ERK1/2) was increased in injured arteries after balloon injury. In primary culture of adventitial fibroblasts, PDGF-BB increased phosphorylation of p38, JNK, ERK1/2, andextracellular regulated kinase 5 (ERK5) in a short time, which was normalized by their inhibitors respectively. Compared with the injury group, perivascular administration of four MAPK inhibitors significantly attenuated neointima formation by quantitative analysis of neointimal area, intima to media (I/M) ratio, and lumen area. RNA sequencing of adventitial fibroblasts treated with PDGF-BB with or without four inhibitors demonstrated differentially expressed genes involved in multiple biological processes, including cell adhesion, proliferation, migration, and inflammatory response. Wound healing and transwell assays showed that four inhibitors suppressed PDGF-BB-induced adventitial fibroblast migration. Cell cycle analysis by flow cytometry demonstrated that JNK, ERK1/2, and ERK5 but not p38 inhibitor blocked PDGF-BB-induced G1 phase release associated with decrease expression of cell cycle protein Cyclin D1 and transcription factor GATA4. Moreover, four inhibitors decreased macrophage infiltration into adventitia and monocyte chemoattractant protein-1 (MCP-1) expression. These results suggest that MAPKs differentially regulate activation of adventitial fibroblast through GATA4/Cyclin D1 axis that participates in neointima formation.

The telomerase activator TA-65 protects from cigarette smoke-induced small airway remodeling in mice through extra-telomeric effects

Small airway remodeling (SAR) is a key phenomenon of airflow obstruction in smokers, leading to chronic obstructive pulmonary disease (COPD). SAR results in an increased thickness of small airway walls, with a combination of peribronchiolar fibrosis with increased fibrous tissue and accumulation of mesenchymal and epithelial cells. SAR pathogenesis is still unclear but recent data suggest that alterations in telomerase activity could represent a possible underlying mechanism of SAR. Our study was dedicated to identify a potential protective role of TA-65, a pharmacological telomerase activator, in a cigarette smoke (CS) model of SAR in mice, and to further precise if extra-telomeric effects of telomerase, involving oxidative stress modulation, could explain it. C57BL/6J mice were daily exposed to air or CS during 4 weeks with or without a concomitant administration of TA-65 starting 7 days before CS exposure. Morphological analyses were performed, and mucus production, myofibroblast differentiation, collagen deposition, as well as transforming growth factor-β1 (TGF-β1) expression in the small airway walls were examined. In addition, the effects of TA-65 treatment on TGF-β expression, fibroblast-to-myofibroblast differentiation, reactive oxygen species (ROS) production and catalase expression and activity were evaluated in primary cultures of pulmonary fibroblasts and/or mouse embryonic fibroblasts in vitro. Exposure to CS during 4 weeks induced SAR in mice, characterized by small airway walls thickening and peribronchiolar fibrosis (increased deposition of collagen, expression of α-SMA in small airway walls), without mucus overproduction. Treatment of mice with TA-65 protected them from CS-induced SAR. This effect was associated with the prevention of CS-induced TGF-β expression in vivo, the blockade of TGF-β-induced myofibroblast differentiation, and the reduction of TGF-β-induced ROS production that correlates with an increase of catalase expression and activity. Our findings demonstrate that telomerase is a critical player of SAR, probably through extra-telomeric anti-oxidant effects, and therefore provide new insights in the understanding and treatment of COPD pathogenesis.

A Biomimetic Polynucleotides–Hyaluronic Acid Hydrogel Promotes the Growth of 3D Spheroid Cultures of Gingival Fibroblasts

(1) Background: Three-dimensional cultures are useful tools to evaluate regenerative approaches in vitro, as they may mimic the spatial arrangement of cells more closely to natural tissues than routine 2D culture methods. (2) Methods: We investigated the effects of a polynucleotide, hyaluronic acid (PN, HA) compound on 3D spheroid cultures of primary gingival fibroblasts, by measuring their morphology over time, cell viability with Calcein-AM, a fluorescent marker, and cell growth potential by re-plating spheroids in attachment-permissive regular culture plates under routine conditions and following them up for 15 days. (3) Results: PN + HA induced an increase in spheroid size and perimeter and a decrease in spheroid circularity, as cells tended to grow and form small peripheral stacks around the spheroid. Levels of cell viability were also higher in this group. After re-plating, only the spheroids previously stimulated with PN + HA dissolved completely during the second week of culture and colonized the plate, thus indicating the retention of a higher level of viability by the cells forming the whole spheroid with this stimulus. (4) Conclusions: Taken together, our data support the idea that the combination of PN and HA has synergic effects on primary fibroblasts and promotes their viability, the growth of 3D cellular constructs, and the retention of a remarkable proliferative potential over the course of the experimental period, making it a promising compound for further investigations.

Changes in Length and Positive Charge of Palindromic Sequence RWQWRWQWR Enhance Cytotoxic Activity against Breast Cancer Cell Lines.

Breast cancer is one of the main causes of premature death in women; current treatments have low selectivity, generating strong physical and psychological sequelae. The palindromic peptide R-1-R (RWQWRWQWR) has cytotoxic activity against different cell lines derived from cancer and selectivity against noncancerous cells. To determine if changes in the charge/length of this peptide increase its activity, six peptides were obtained by SPPS, three of them with addition of Arg at the N, C-terminal or both and three with deletion of Arg at the N, C-terminal or both. The cytotoxic and selective activities were evaluated against MCF-7, MDA-MB-231, and MCF-12 cell lines and fibroblast primary cell culture, evidencing that the RR-1-R peptide with the inclusion of Arg in the N-terminal end maintained selectivity and increased cytotoxicity against lines derived from breast cancer. The effect of this addition regarding the type of induced cell death was evaluated by flow cytometry, showing very low rates of necrosis and a significant majority of apoptotic events with activation of both Caspase 8 and Caspase 9. This work allowed us to find a modification that generates a peptide with greater cytotoxic effects and can be considered a promising molecule for other approaches to improve anticancer peptides.

Isolation and Culture of Primary Fibroblasts from Neonatal Murine Hearts to Study Cardiac Fibrosis.

Cardiac fibroblasts are one of the major constituents of a healthy heart. Cultured cardiac fibroblasts are a crucial resource for conducting studies on cardiac fibrosis. The existing methods for culturing cardiac fibroblasts involve complicated steps and require special reagents and instruments. The major problems faced with primary cardiac fibroblast culture are the low yield and viability of the cultured cells and contamination with other heart cell types, including cardiomyocytes, endothelial cells, and immune cells. Numerous parameters, including the quality of the reagents used for the culture, conditions maintained during digestion of the cardiac tissue, composition of the digestion mixture used, and age of the pups used for culture determine the yield and purity of the cultured cardiac fibroblasts. The present study describes a detailed and simplified protocol to isolate and culture primary cardiac fibroblasts from neonatal murine pups. We demonstrate the transdifferentiation of fibroblasts into myofibroblasts through transforming growth factor (TGF)-β1 treatment, representing the changes in fibroblasts during cardiac fibrosis. These cells can be used to study the various aspects of cardiac fibrosis, inflammation, fibroblast proliferation, and growth.

EFFECTIVENESS OF CRYOPRESERVED CHICKEN EMBRYO FIBROBLAST CELLS FOR PROPAGATION OF DUCK ENTERITIS VIRUS AND FOWLPOX VIRUS

Primary chicken embryo fibroblast (CEF) cultures are excellent model systems for in-vitro virus propagation. However, the cumbersome preparation process of CEF makes cryopreservation highly essential. Cryopreservation in Liquid Nitrogen liquid phase (-196 °C) or vapour phase (-156 °C) are frequently used methods. In the present study, efficiency of cryopreserved primary CEF cells for the propagation of Duck plague virus and Fowl pox virus was evaluated. The cells were preserved for different time periods and different cooling methodologies were employed in order to assess the effects exerted by these variations. The efficiency of the cells was compared based on cell viability/ revival rate, ability to form monolayer and TCID50 of the propagated viruses. Cell count revealed viability of 75.60% and 68% post 2 weeks and 6 months of cryopreservation respectively following step freezing method. On the other hand, cells preserved using insulated cryo-containers exhibited 89% and 80% viability post 2 weeks and 6 months of cryopreservation respectively. Development of complete monolayer could be seen within 24 hours in all the batches irrespective of the cooling method used and the cryopreservation storage time. Infection with DPV and FPV yielded respective cytopathic effect from 48 hours onwards. CPE observed in the freshly prepared CEF and in those revived 2 weeks and 6 months post freezing, were similar in terms of typical characteristics and duration of appearance. From the findings of the present study it is evident that cryopreservation of CEF cells is a convenient and practical approach for virus propagation. Cryopreservation of primary cells offers a definite advantage over repeated culture preparation which is labour intensive, time consuming and dependent on the availability of embryonated eggs.

The role of Notch-dependent differentiation of resident fibroblasts in the development of pulmonary fibrosis

Background. Molecular mechanisms of the development of pulmonary fibrosis are poorly understood. It is known that differentiation of resident lung cells into myofibroblasts plays a key role in this disease. The search for factors capable of regulating such differentiation is an urgent task. Objective. To evaluate the effect of Notch signaling pathway activation on resident lung fibroblasts by introducing the intracellular domains of each of the 4 Notch receptors (N1-4ICD). Design and methods. Primary cultures of pulmonary fibroblasts from donors (n = 4) were used. Notch-dependent activation of fibroblasts was carried out by introducing lentiviral vectors with/ without sequences of N1-4ICD domains. After 8 days, immunocytochemical staining was performed and the relative expression levels of the PDPN, HOPX, SLUG, SNAIL and ACTA2 genes were evaluated by qPCR. Results. Activation of the Notch signaling pathway by N1-4CD resulted in increased expression of SLUG, SNAIL and ACTA2. The most pronounced effect was observed with the introduction of Notch4-activating sequence. Induction of signaling by the introduction of N1-3ICD activating components contributed to an increase in PDPN expression, with the introduction of N4ICD, an increase in the level of HOPX expression was noted. Conclusion. Activation of each of the 4 intracellular Notch receptor domains is able to trigger the differentiation of resident alveolar fibroblasts into myofibroblasts, which are key players in the development of pulmonary fibrosis.

Anti-inflammatory effects of α-humulene and β-caryophyllene on pterygium fibroblasts.

To investigate the anti-inflammatory effects of the sesquiterpenes α-humulene and β-caryophyllene on pterygium fibroblasts. Primary cultures of pterygium fibroblasts were established. Third passage pterygium fibroblasts were exposed to α-humulene and β-caryophyllene separately and together. The cell viability was assessed by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay at 12, 24, 48, and 72h after exposure. The levels of the inflammatory cytokines interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α and IL-10 in the conditioned culture medium were assessed by enzyme-linked immunosorbent assay (ELISA) at 12, 24 and 48h after exposure. Data were statistically analyzed using Friedman repeated measures analysis of variances on ranks. The 25 µmol/L β-caryophyllene induced significant decrease in the IL-6 production by pterygium fibroblasts 48h after the exposure (P=0.041). The levels of IL-1β, IL-8, IL-10, and TNF-α were very low and had no statistically significant variations after exposure to α-humulene, β-caryophyllene, or both compounds together. The exposure to 25 µmol/L of β-caryophyllene significantly reduce the production of IL-6 by pterygium fibroblasts after 48h. This sesquiterpene may be a potential alternative adjuvant agent for the treatment of pterygium.

Selenium Nanoparticles Can Influence the Immune Response Due to Interactions with Antibodies and Modulation of the Physiological State of Granulocytes.

Currently, selenium nanoparticles (SeNPs) are considered potential immunomodulatory agents and as targets for activity modulation are granulocytes, which have the most abundant population of immune blood cells. The present study aims to evaluate the cytotoxic effect and its effect on the functional responses of granulocytes. In addition to the intrinsic activity of SeNPs, we studied the activity of the combination of SeNPs and IgG antibodies. Using laser ablation and fragmentation, we obtained nanoparticles with an average size of 100 nm and a rather narrow size evolution. The resulting nanoparticles do not show acute toxicity to primary cultures of fibroblasts and hepatocytes, epithelial-like cell line L-929 and granulocyte-like culture of HL-60 at a concentration of 109 NPs/mL. SeNPs at a concentration of 1010 NPs/mL reduced the viability of HL-60 cells by no more than 10% and did not affect the viability of the primary culture of mouse granulocytes, and did not have a genotoxic effect on progenitor cells. The addition of SeNPs can affect the production of reactive oxygen species (ROS) by mouse bone marrow granulocytes, modulate the proportion of granulocytes with calcium spikes and enhance fMLF-induced granulocytes degranulation. SeNPs can modulate the effect of IgG on the physiological responses of granulocytes. We studied the expression level of genes associated with inflammation and cell stress. SeNPs increase the expression of catalase, NF-κB, Xrcc5 and some others; antibodies enhance the effect of SeNPs, but IgG without SeNPs decreases the expression level of these genes. This fact can be explained by the interaction between SeNPs and IgG. It has been established that antibodies interact with SeNPs. We showed that antibodies bind to the surface of selenium nanoparticles and are present in aqueous solutions in a bound form from DLS methods, ultraviolet-visible spectroscopy, vibrational-rotational spectrometry, fluorescence spectrometry, and refractometry. At the same time, in a significant part of the antibodies, a partial change in the tertiary and secondary structure is observed. The data obtained will allow a better understanding of the principles of the interaction of immune cells with antibodies and SeNPs and, in the future, may serve to create a new generation of immunomodulators.

Production of growth factors by fibroblasts in the conditions of the wound process and under the exposure of a bacterial biofilm matrix

Aim to analyze the production of growth factors by fibroblasts in the wound process and under the influence of the bacterial biofilm matrix.
 Material and methods. We assessed the levels of such growth factors as: VEGF, TGF-1, GM-CSF, FGF produced by granulation tissue fibroblasts of acute wound (AW) (group 1, n=9) and chronic wound (CW) (group 2, n=17). Skin fibroblasts (n=5) were used for reference. The primary cultures were obtained by the method of explants. The experimental biofilm matrix included S. aureus, E. faecalis, A. baumannii, P. aeruginosa, K. pneumoniae, P. mirabilis (5 of each species).
 Results. The elevated levels of TGF-1 and VEGF, secreted by the primary cultures of fibroblasts, against of the absence of changes in the values of FGF and GM-CSF, are the criteria for the imbalance of growth factors characteristic of CW. The impact of the biofilm matrix P. aeruginosa, K. pneumoniae, A. baumannii, P. mirabilis on skin fibroblasts was accompanied by an increase in the production of VEGF and GM-CSF and a decrease in the synthesis of TGF-1 and FGF. In the experiments with S. aureus and E. faecalis, a similar direction of changes was recorded, but the degree of its severity was lower than for experiments with the gram-negative bacteria.
 Conclusion. The obtained results have demonstrated various effects of the biofilm matrix on the production of growth factors by fibroblasts and updated the information on the pathogenesis of CW formation. The revealed imbalance in the synthesis of growth factors by fibroblasts in the granulation tissue of wounds with borderline lifetimes (2228 days) determines the need for tactical approaches in the treatment of CW.

Оценка методов инактивирования вируса лейкоза птиц при производстве гриппозных вакцин

The process of production of inactivated influenza vaccines involves a stage of inactivation of both the influenza virus and the possible viral contaminants that can come from the raw materials (chicken embryos). One of such contaminants is the avian leukemia virus. The minimum viral contaminant load reduction that the inactivating agents should guarantee is by 4 lg/ml; this or higher level of the deactivating ability ensures the finished vaccine is free from viral contaminants. The purpose of this work was to cultivate the leukemia virus to the titer of 5 lg/ml (minimum) and to measure the reduction of the avian leukemia virus titer in influenza vaccine intermediates upon exposure to the inactivating agents. The RAV-1 and RAV-2 leukemia virus strains and influenza vaccine intermediates such as virus-containing allantoic fluid and virus concentrates were used in the study. Avian leukemia virus titers were determined by enzyme immunoassay. We created conditions for cultivation of the RAV-1 and RAV-2 avian leukemia virus strains in the primary culture of chicken embryo fibroblasts (CEF); the inactivating agents considered were the most commonly used β-propiolactone and UV radiation. It was found that after 12 hours of exposure to β-propiolactone, the RAV-1 avian leukemia virus load decreased by 4.61 ± 0.46 lg, and that of RAV-2 strain - by 4.33 ± 0.33 lg, which indicates that β-propiolactone is an effective inactivating agent. Five minutes of exposure to UV radiation reduces the RAV-1 strain viral load by 4.22 ± 0.31 lg and RAV-2 strain viral load by 4.44 ± 0.48 lg.

Composite Coating for the Food Industry Based on Fluoroplast and ZnO-NPs: Physical and Chemical Properties, Antibacterial and Antibiofilm Activity, Cytotoxicity

Bacterial contamination of meat products during its preparation at the enterprise is an important problem for the global food industry. Cutting boards are one of the main sources of infection. In order to solve this problem, the creation of mechanically stable coatings with antibacterial activity is one of the most promising strategies. For such a coating, we developed a composite material based on "liquid" Teflon and zinc oxide nanoparticles (ZnO-NPs). The nanoparticles obtained with laser ablation had a rod-like morphology, an average size of ~60 nm, and a ζ-potential of +30 mV. The polymer composite material was obtained by adding the ZnO-NPs to the polymer matrix at a concentration of 0.001-0.1% using the low-temperature technology developed by the research team. When applying a composite material to a surface with damage, the elimination of defects on a micrometer scale was observed. The effect of the composite material on the generation of reactive oxygen species (H2O2, •OH), 8-oxoguanine in DNA in vitro, and long-lived reactive protein species (LRPS) was evaluated. The composite coating increased the generation of all of the studied compounds by 50-200%. The effect depended on the concentration of added ZnO-NPs. The antibacterial and antibiofilm effects of the Teflon/ZnO NP coating against L. monocytogenes, S. aureus, P. aeruginosa, and S. typhimurium, as well as cytotoxicity against the primary culture of mouse fibroblasts, were studied. The conducted microbiological study showed that the fluoroplast/ZnO-NPs coating has a strong bacteriostatic effect against both Gram-positive and Gram-negative bacteria. In addition, the fluoroplast/ZnO-NPs composite material only showed potential cytotoxicity against primary mammalian cell culture at a concentration of 0.1%. Thus, a composite material has been obtained, the use of which may be promising for the creation of antibacterial coatings in the meat processing industry.

Polyene phosphatidylcholine ameliorates synovial inflammation: involvement of PTEN elevation and glycolysis suppression.

Synovial inflammation, characterized by the activation of synovial fibroblasts (SFs), is a crucial factor to drive the progression of rheumatoid arthritis (RA). Polyene phosphatidylcholine (PPC), the classic hepatoprotective drug, has been reported to ameliorate arthritis in animals. However, the molecular mechanism remains poorly understood. METHODS ANDRESULTS: Using in vitro primary synovial fibroblast (SFs) culture system, we revealed that phosphatase and tension homolog deleted on chromosome 10 (PTEN), a tumor suppressor, mediates the anti-inflammatory effect of PPC in lipopolysaccharide (LPS)-stimulated primary SFs. PPC decreased the production of TNF-α and IL-6 production while elevating the level of IL-10 and TGF-β. Furthermore, PPC up-regulated the expression of PTEN, but inhibited the expression of p-AKT (ser473) and PI3K-p85α. Moreover, pre-treatment of SF1670 (the inhibitor of PTEN) or 740Y-P (the agonist of AKT/PI3K pathways) partiallyabrogated the anti-inflammatory effect of PPC. In addition, PPC could inhibit the expression of GLUT4, a key transporter of glucose that fuels the glycolysis, which is accompanied by the expression downregualtion of glycolytic enzymes PFKFB3 and PKM2. Furthermore, PPC could reduce ROS production and mitochondrial membrane potential in LPS-stimulated SFs and MH7A cell line. The present study supported that PPC can alleviate synovial inflammation, which involves in the elevation of PTEN and blockage of glycolysis.

Antisense oligonucleotides targeting exon 11 are able to partially rescue the NF2-related schwannomatosis phenotype in vitro

NF2-related schwannomatosis (NF2-related SWN) is an autosomal dominant condition caused by loss of function variants in the NF2 gene, which codes for the protein Merlin and is characterized by the development of multiple tumors of the nervous system. The clinical presentation of the disease is variable and related to the type of the inherited germline variant. Here, we tested if phosphorodiamidate morpholino oligomers (PMOs) could be used to correct the splice signaling caused by variants at±13 within the intron-exon boundary region and showed that the PMOs designed for these variants do not constitute a therapeutic approach. Furthermore, we evaluated the use of PMOs to decrease the severity of the effects of NF2 truncating variants with the aim of generating milder hypomorphic isoforms invitro through the induction of the in-frame deletion of the exon-carrying variant. We were able to specifically induce the skipping of exons 4, 8, and 11 maintaining the NF2 gene reading frame at cDNA level. Only the skipping of exon 11 produced a hypomorphic Merlin (Merlin-e11), which is able to partially rescue the observed phenotype in primary fibroblast cultures from NF2-related SWN patients, being encouraging for the treatment of patients harboring truncating variants located in exon 11.

Loss of primary cilia promotes inflammation and carcinogenesis

Primary cilia (PC) are important signaling hubs, and we here explored their role in colonic pathology. In the colon, PC are mostly present on fibroblasts, and exposure of mice to either chemically induced colitis‐associated colon carcinogenesis (CAC) or dextran sodium sulfate (DSS)‐induced acute colitis decreases PC numbers. We generated conditional knockout mice with reduced numbers of PC on colonic fibroblasts. These mice show increased susceptibility to CAC, as well as DSS‐induced colitis. Secretome and immunohistochemical analyses of DSS‐treated mice display an elevated production of the proinflammatory cytokine IL‐6 in PC‐deficient colons. An inflammatory environment diminishes PC presence in primary fibroblast cultures, which is triggered by IL‐6 as identified by RNA‐seq analysis together with blocking experiments. These findings suggest an activation loop between IL‐6 production and PC loss. An analysis of PC presence on biopsies of patients with ulcerative colitis or colorectal cancer (CRC) reveals decreased numbers of PC on colonic fibroblasts in pathological compared with surrounding normal tissue. Taken together, we provide evidence that a decrease in colonic PC numbers promotes colitis and CRC.

Effects of urinary extracellular vesicles from prostate cancer patients on the transcriptomes of cancer-associated and normal fibroblasts

BackgroundIncreasing evidence suggests that cancer-derived extracellular vesicles (EVs) alter the phenotype and functions of fibroblasts and trigger the reprogramming of normal fibroblasts into cancer-associated fibroblasts (CAFs). Here, we for the first time studied the effects of urinary EVs from PC patients and healthy males on the transcriptional landscape of prostate CAFs and normal foreskin fibroblasts.MethodsPatient-derived prostate fibroblast primary cultures PCF-54 and PCF-55 were established from two specimens of PC tissues. EVs were isolated from urine samples of 3 patients with PC and 2 healthy males and used for the treatment of prostate fibroblast primary cultures and normal foreskin fibroblasts. The EV-treated fibroblasts were subjected to RNA sequencing analysis.ResultsRNA sequencing analysis showed that the fibroblast cultures differed significantly in their response to urinary EVs. The transcriptional response of foreskin fibroblasts to the urinary EVs isolated from PC patients and healthy controls was very similar and mostly related to the normal functions of fibroblasts. On the contrary, PCF-54 cells responded very differently - EVs from PC patients elicited transcriptional changes related to the regulation of the cell division and chromosome segregation, whereas EVs from healthy males affected mitochondrial respiration. In PCF-55 cells, EVs from both, PC-patients and controls induced the expression of a number of chemokines such as CCL2, CCL13, CXCL1, CXCL8, whereas pathways related to regulation of apoptotic signaling and production of cell adhesion molecules were triggered specifically by EVs from PC patients.ConclusionThis study demonstrates that urinary EVs from PC patients and healthy controls elicit distinct transcriptional responses in prostate CAFs and supports the idea that EVs contribute to the generation of functional heterogeneity of CAFs. Moreover, this study suggests that the changes in the gene expression pattern in EV recipient cells might serve as a novel type of functional cancer biomarkers.

Cancer-Associated Fibroblasts Hinder Lung Squamous Cell Carcinoma Oxidative Stress-Induced Apoptosis via METTL3 Mediated m6A Methylation of COL10A1.

Background Cancer-associated fibroblasts (CAFs) within the tumor microenvironment are key players in tumorigenesis and tumor development. Nevertheless, the regulatory mechanisms of CAFs on lung squamous cell carcinoma- (LUSC-) associated remain poorly elucidated. Methods The microarray dataset GSE22874, containing 30 specimens of primary culture of normal fibroblasts (NFs) and 8 specimens of cancer-associated fibroblasts (CAFs) samples derived from LUSC, was retrieved from the Gene Expression Omnibus (GEO) database and then calculated by using the R language (limma package) to identify differentially expressed genes (DEGs). CAF-conditioned medium (CAF-CM) was collected and used to culture LUSC cells, followed by assessment of cell proliferation, apoptosis, and oxidative stress levels by using CCK-8, annexin V-FITC/PI double staining and ELISA assays. Subsequently, COL10A1 was knocked down in CAFs to assess the role of COL10A1 in CAF regulation of LUSC behavior. Bioinformatics online analysis and MeRIP were applied to predict and test the m6A modification of COL10A1 mRNA and the regulatory relationship with METTL3. Rescue experiments were next performed to explore the effects of METTL3 and COL10A1 in CAFs on LUSC cell proliferation, apoptosis, and oxidative stress. LUSC tumor cells with or without (COL10A1-silenced) CAFs were subcutaneously inoculated in nude mice to evaluate the effect of COL10A1 in CAFs on LUSC tumor growth. Results Elevated expression of COL10A1 was found in LUSC-derived CAFs by GSE22874 dataset analysis. We discovered that COL10A1 and METTL3 was expressed in both LUSC cells and matched CAFs, while COL10A1 expression was prominently higher in CAFs than in LUSC cells. CAF-CM memorably encouraged LUSC cell proliferation and suppressed apoptosis-induced oxidative stress, which was reversed by interfering with COL10A1 expression in CAFs, suggesting that COL10A1 might be secreted by CAFs into the culture medium to exert its effects inside LUSC cells. Global m6A modification was decreased in METTL3 knocked down CAFs. M6A modification, expression levels, and stability of COL10A1 mRNA were impaired upon METTL3 knockdown in CAFs. Overexpression of COL10A1 in CAFs partially reversed the effect of METTL3 knockdown on the malignant behavior of LUSC cells. In vivo studies confirmed that CAFs accelerated LUSC tumor growth, and this effect was counteracted by COL10A1 silencing. Conclusions COL10A1 secreted by CAFs could facilitate LUSC cell proliferation and repress apoptosis-induced oxidative stress, and the mechanism was due to elevated expression mediated by METTL3 promoting its mRNA m6A modification, thereby accelerating tumor growth.

Transforming Growth Factor-β Receptor–Mediated, p38 Mitogen-Activated Protein Kinase–Dependent Signaling Drives Enhanced Myofibroblast Differentiation during Skin Wound Healing in Mice Lacking Hyaluronan Synthases 1 and 3

Normal myofibroblast differentiation is critical for proper skin wound healing. Neoexpression of α-smooth muscle actin (α-SMA), a marker for myofibroblast differentiation, is driven by transforming growth factor (TGF)-β receptor-mediated signaling. Hyaluronan and its three synthesizing enzymes, hyaluronan synthases (Has 1, 2, and 3), also participate in this process. Closure of skin wounds is significantly accelerated in Has1/3 double-knockout (Has1/3-null) mice. Herein, TGF-β activity and dermal collagen maturation were increased in Has1/3-null healing skin. Cultures of primary skin fibroblasts isolated from Has1/3-null mice had higher levels of TGF-β activity, α-SMA expression, and phosphorylation of p38 mitogen-activated protein kinase at Thr180/Tyr182, compared with wild-type fibroblasts. p38α mitogen-activated protein kinase was a necessary element in a noncanonical TGF-β receptor signaling pathway driving α-SMA expression in Has1/3-null fibroblasts. Myocardin-related transcription factor (MRTF), a cofactor that binds to the transcription factor serum response factor (SRF), was also critical. Nuclear localization of MRTF was increased, and MRTF binding to SRF was enhanced in Has1/3-null fibroblasts. Inhibition of MRTF or SRF expression by RNA interference suppresses α-SMA expression at baseline and diminished its overexpression in Has1/3-null fibroblasts. Interestingly, total matrix metalloproteinase activity was increased in healing skin and fibroblasts from Has1/3-null mice, possibly explaining the increased TGF-β activation.

Assessing the Cytotoxicity of TiO2-x Nanoparticles with a Different Ti3+(Ti2+)/Ti4+ Ratio.

Titanium dioxide (TiO2) nanoparticles are promising biomedical agents characterized by good biocompatibility. In this study, we explored the cytotoxicity of TiO2-x nanoparticles with a different Ti3+(Ti2+)/Ti4+ ratio and analyzed the efficiency of eryptosis indices as a tool in nanotoxicology. Two types of TiO2-x nanoparticles (NPs) were synthesized by the hydrolysis of titanium alkoxide varying the nitric acid content in the hydrolysis mixture. Transmission electron microscopy (TEM) images show that 1-TiO2-x and 2-TiO2-x NPs are 5nm in size, whereas X-ray photoelectron spectroscopy (XPS) reveals different Ti3+ (Ti2+)/Ti4+ ratios in the crystal lattices of synthesized NPs. 1-TiO2-x nanoparticles contained 54% Ti4+, 38% Ti3+, and 8% Ti2+, while the relative amount of Ti4+ and Ti3+ in the crystal lattice of 2-TiO2-x nanoparticles was 63% and 37%, respectively. Cell viability and cell motility induced by TiO2-x nanoparticles were investigated on primary fibroblast cultures. Eryptosis modulation by the nanoparticles along with cell death mechanisms was studied on rat erythrocytes. We report that both TiO2-x nanoparticles do not decrease the viability of fibroblasts simultaneously stimulating cell migration. Data from in vitro studies on erythrocytes indicate that TiO2-x nanoparticles trigger eryptosis via ROS- (1-TiO2-x) and Ca2+-mediated mechanisms (both TiO2-x nanoparticles) suggesting that evaluation of eryptosis parameters is a more sensitive nanotoxicological approach for TiO2-x nanoparticles than cultured fibroblast assays. TiO2-x nanoparticles are characterized by low toxicity against fibroblasts, but they induce eryptosis, which is shown to be a promising tool for nanotoxicity screening. The Ti3+ (Ti2+)/Ti4+ ratio at least partly determines the cytotoxicity mechanisms for TiO2-x nanoparticles.

SerpinE1 drives a cell-autonomous pathogenic signaling in Hutchinson–Gilford progeria syndrome

Hutchinson–Gilford progeria syndrome (HGPS) is a rare, fatal disease caused by Lamin A mutation, leading to altered nuclear architecture, loss of peripheral heterochromatin and deregulated gene expression. HGPS patients eventually die by coronary artery disease and cardiovascular alterations. Yet, how deregulated transcriptional networks at the cellular level impact on the systemic disease phenotype is currently unclear. A genome-wide analysis of gene expression in cultures of primary HGPS fibroblasts identified SerpinE1, also known as Plasminogen Activator Inhibitor (PAI-1), as central gene that propels a cell-autonomous pathogenic signaling from the altered nuclear lamina. Indeed, siRNA-mediated downregulation and pharmacological inhibition of SerpinE1 by TM5441 could revert key pathological features of HGPS in patient-derived fibroblasts, including re-activation of cell cycle progression, reduced DNA damage signaling, decreased expression of pro-fibrotic genes and recovery of mitochondrial defects. These effects were accompanied by the correction of nuclear abnormalities. These data point to SerpinE1 as a novel potential effector and target for therapeutic interventions in HGPS pathogenesis.