Activity In Human Fibroblasts Research Articles

Chronic activation of human cardiac fibroblasts in vitro attenuates the reversibility of the myofibroblast phenotype

Activation of cardiac fibroblasts and differentiation to myofibroblasts underlies development of pathological cardiac fibrosis, leading to arrhythmias and heart failure. Myofibroblasts are characterised by increased α-smooth muscle actin (α-SMA) fibre expression, secretion of collagens and changes in proliferation. Transforming growth factor-beta (TGF-β) and increased mechanical stress can initiate myofibroblast activation. Reversibility of the myofibroblast phenotype has been observed in murine cells but has not been explored in human cardiac fibroblasts. In this study, chronically activated adult primary human ventricular cardiac fibroblasts and human induced pluripotent stem cell derived cFbs (hiPSC-cFbs) were used to investigate the potential for reversal of the myofibroblast phenotype using either subculture on soft substrates or TGF-β receptor inhibition. Culture on softer plates (25 or 2 kPa Young’s modulus) did not alter proliferation or reduce expression of α-SMA and collagen 1. Similarly, culture of myofibroblasts in the presence of TGF-β inhibitor did not reverse myofibroblasts back to a quiescent phenotype. Chronically activated hiPSC-cFbs also showed attenuated response to TGF-β receptor inhibition and inability to reverse to quiescent fibroblast phenotype. Our data demonstrate substantial loss of TGF-β signalling plasticity as well as a loss of feedback from the surrounding mechanical environment in chronically activated human myofibroblasts.

Fibronectin-coated microgroove titanium surface promotes the behaviors of human gingival fibroblasts.

The present study was designed to clarify the activity of human gingival fibroblasts (HGFs) on the fibronectin (FN)-coated silanized microgroove titanium surface. The surface elemental composition of titanium discs was detected using XPS. HGFs' adhesion to the titanium discs was detected by immunofluorescence staining of vinculin. HGFs' number on the titanium discs was detected using the CCK8 assay. HGFs' secretion of type 1 collagen after five days of culturing was detected using ELISA and qPCR. HGFs could proliferate and spread well on the surface. The viability of HGFs in the experimental group was significantly more than in the control group. The HGFs in the experimental group significantly secreted more type 1 collagen than in the control group. Therefore, FN-coated can improve the morphology, viability, and type 1 collagen secretion of HGFs silanized microgroove titanium surface, which might ameliorate the efficacy of implants.

SPRR1A is a key downstream effector of MiR-150 during both maladaptive cardiac remodeling in mice and human cardiac fibroblast activation

MicroRNA-150 (miR-150) is conserved between rodents and humans, is significantly downregulated during heart failure (HF), and correlates with patient outcomes. We previously reported that miR-150 is protective during myocardial infarction (MI) in part by decreasing cardiomyocyte (CM) apoptosis and that proapoptotic small proline-rich protein 1a (Sprr1a) is a direct CM target of miR-150. We also showed that Sprr1a knockdown in mice improves cardiac dysfunction and fibrosis post-MI and that Sprr1a is upregulated in pathological mouse cardiac fibroblasts (CFs) from ischemic myocardium. However, the direct functional relationship between miR-150 and SPRR1A during both post-MI remodeling in mice and human CF (HCF) activation was not established. Here, using a novel miR-150 knockout;Sprr1a-hypomorphic (Sprr1ahypo/hypo) mouse model, we demonstrate that Sprr1a knockdown blunts adverse post-MI effects caused by miR-150 loss. Moreover, HCF studies reveal that SPRR1A is upregulated in hypoxia/reoxygenation-treated HCFs and is downregulated in HCFs exposed to the cardioprotective β-blocker carvedilol, which is inversely associated with miR-150 expression. Significantly, we show that the protective roles of miR-150 in HCFs are directly mediated by functional repression of profibrotic SPRR1A. These findings delineate a pivotal functional interaction between miR-150 and SPRR1A as a novel regulatory mechanism pertinent to CF activation and ischemic HF.

Lung cancer-derived exosomal miR-132-3p contributed to interstitial lung disease development

PurposeInterstitial lung diseases (ILDs) have high morbidity and mortality and poor prognosis. The significance of microRNAs (miRNAs) was highlighted in ILDs development. Currently, we attempted to confirm the functions of lung cancer-derived exosomal miR-132-3p and reveal the underlying mechanism.MethodCharacteristics of exosomes were verified by transmission electron microscope (TEM), nanoparticle tracking analysis, and Western blot assay. Exosome uptake for the normal human lung fibroblasts (NHLF) was assessed using a PKH67 staining assay. MTT and colony formation assays were applied to examine the proliferation abilities of NHLF. The interaction between miR-132-3p and sprouty1 (SPRY1) was confirmed by a luciferase reporter assay.ResultsLung cancer-derived exosomes promoted normal human lung fibroblast activation. Exosome inhibitor GW4869 reversed the effects of Exo on NHLF. Subsequently, miR-132-3p in lung cancer-derived exosomes activated the normal human lung fibroblast and promoted interstitial lung disease development ex vivo. Next, SPRY1 was verified to be the binding protein of miR-132-3p, and sh-SPRY1 abrogated the effects of the miR-132-3p inhibitor on NHLF.ConclusionExosomal miR-132-3p from A549 cells accelerated the development of interstitial lung disease through binding to SPRY1, which might serve as an important target for ILDs.

IL-33/ST2 axis of human amnion fibroblasts participates in inflammatory reactions at parturition

BackgroundInflammation of the fetal membranes is an indispensable event of labor onset at both term and preterm birth. Interleukin-33 (IL-33) is known to participate in inflammation via ST2 (suppression of tumorigenicity 2) receptor as an inflammatory cytokine. However, it remains unknown whether IL-33/ST2 axis exists in human fetal membranes to promote inflammatory reactions in parturition.MethodsThe presence of IL-33 and ST2 and their changes at parturition were examined with transcriptomic sequencing, quantitative real-time polymerase chain reaction, Western blotting or immunohistochemistry in human amnion obtained from term and preterm birth with or without labor. Cultured primary human amnion fibroblasts were utilized to investigate the regulation and the role of IL-33/ST2 axis in the inflammation reactions. A mouse model was used to further study the role of IL-33 in parturition.ResultsAlthough IL-33 and ST2 expression were detected in both epithelial and fibroblast cells of human amnion, they are more abundant in amnion fibroblasts. Their abundance increased significantly in the amnion at both term and preterm birth with labor. Lipopolysaccharide, serum amyloid A1 and IL-1β, the inflammatory mediators pertinent to labor onset, could all induce IL-33 expression through NF-κB activation in human amnion fibroblasts. In turn, via ST2 receptor, IL-33 induced the production of IL-1β, IL-6 and PGE2 in human amnion fibroblasts via the MAPKs-NF-κB pathway. Moreover, IL-33 administration induced preterm birth in mice.ConclusionIL-33/ST2 axis is present in human amnion fibroblasts, which is activated in both term and preterm labor. Activation of this axis leads to increased production of inflammatory factors pertinent to parturition, and results in preterm birth. Targeting the IL-33/ST2 axis may have potential value in the treatment of preterm birth.

Methacrylated fibrinogen hydrogels for 3D cell culture and delivery.

Methacrylation was performed on fibrinogen to design a new biomedical hydrogel for 3D cell culture or as a biodegradable delivery matrix for in vivo implantation. The methacrylation of denatured fibrinogen in solution was performed using methacrylic anhydride (MAA). The extent of fibrinogen methacrylation was quantified by proton NMR and controlled using stochiometric quantities of MAA during the reaction. The methacrylated fibrinogen (FibMA) hydrogels were formed by light-activated free-radical polymerization in the presence of macromolecular cross-linking polymers made from acrylated poly(ethylene glycol) (PEG). The biocompatibility and biodegradability of the FibMA hydrogels were characterized by in vitro assays and in vivo implantation experiments using quantitative magnetic resonance imaging (MRI) of the implant volume. The FibMA supported the growth and metabolic activity of human dermal fibroblasts in both 2D and 3D cultures. The methacrylation did not alter important biological attributes of the fibrinogen, including the ability to support cell adhesion and 3D cell culture, as well as to undergo proteolysis. Animal experiments confirmed the biodegradability of the FibMA for potential use as a scaffold in tissue engineering, as a bioink for 3D bioprinting, or as a biodegradable matrix for in vivo sustained delivery of bioactive factors. STATEMENT OF SIGNIFICANCE: This paper describes methacrylated fibrinogen (FibMA) and the formation of a biomedical hydrogel from FibMA for cell culture and other biomedical applications. Inspired from methacrylated gelatin (GelMA), the FibMA is made from blood-derived fibrinogen which is more suitable for clinical use. Sharing similar properties to other hydrogels made from methacrylated proteins, the FibMA has yet to be reported in the literature. In this manuscript, we provide the methodology to produce the FibMA hydrogels, we document the mechanical versatility of this new biomaterial, and we show the biocompatibility using 3D cell culture studies and in vivo implantations.

Carbon dioxide-induced decrease in extracellular pH enhances the production of extracellular matrix components by upregulating TGF-β1 expression via CREB activation in human dermal fibroblasts.

Mild acidification caused by transcutaneous administration of carbon dioxide (CO2 ) has been reported to improve some epidermal skin impairments, such as desquamation and inflammation; however, its effects on dermal tissue remain unclear. Here, we examined the effect and mechanism of mild acidity on extracellular matrix (ECM) protein production in normal human dermal fibroblasts (NHDFs). To achieve this, the skin permeability of CO2 and its effect on intradermal pH were evaluated by treating reconstructed human skin equivalents (HSEs) with a CO2 -containing formulation. Additionally, NHDFs were cultured in a pH-adjusted medium (pH 6.5). CO2 successfully permeated HSEs and reduced the intradermal pH. Decreased extracellular pH activated CREB, upregulated TGF-β1 expression, promoted the production of elastic and collagen fibres, and increased hyaluronan concentration in NHDFs. Additionally, the low pH-induced increase in TGF-β1 expression was attenuated via the RNAi-mediated suppression of the expression of CREB1 and proton-sensing G protein-coupled receptors (GPCRs), including GPR4 and GPR65. Moreover, low pH-induced CREB activation was suppressed by the inhibition of the cAMP/PKA and PLC/PKC signalling pathways. Taken together, a CO2 -induced decrease in intradermal pH may promote ECM production in NHDFs via the upregulation of TGF-β1 expression, which was mediated by the activation of the GPCR signalling pathway and CREB, indicating that CO2 could be used to treat ultraviolet radiation-induced photoaging, intrinsic ageing and ECM deterioration.

Polydeoxyribonucleotide exerts opposing effects on ERK activity in human skin keratinocytes and fibroblasts.

Polydeoxyribonucleotide (PDRN) is a mixture of deoxyribonucleotides. It serves as an anti‑inflammatory and tissue‑regenerating agent. The mitogen‑activated protein kinase pathway modulates cell growth and collagen accumulation. It also regulates inflammation by suppressing the expression of proinflammatory cytokines. In the present study, it was attempted to elucidate the molecular mechanism of PDRN in skin healing by confirming the effects of PDRN treatment on skin keratinocytes and fibroblasts, and by assessing the levels of collagen and inflammatory cytokines regulated by the extracellular signal‑regulated kinase (ERK) pathway. The potential effects of PDRN on skin regeneration were investigated. Fibroblast and keratinocyte proliferation and migration were analyzed using the water‑soluble tetrazolium‑8 and wound healing assays. The upregulation of collagen synthesis by PDRN‑induced ERK activation was analyzed in fibroblasts with or without an ERK inhibitor. Inflammatory cytokine expression levels in keratinocytes were determined using reverse transcription‑quantitative polymerase chain reaction. PDRN promoted the proliferation and migration of keratinocytes and fibroblasts. However, PDRN‑induced ERK phosphorylation differed between keratinocytes and fibroblasts; PDRN increased ERK phosphorylation and collagen accumulation in fibroblasts, while it inhibited matrix metalloproteinase expression. By contrast, PDRN inhibited ERK phosphorylation in keratinocytes, and it decreased inflammatory cytokine expression levels. PDRN affects skin cell proliferation and migration, and collagen and inflammatory cytokine expression levels via ERK signaling. Overall, PDRN exerts a positive effect on skin regeneration, but the mechanism by which it promotes skin regeneration varies among different skin cell types.

Cardiac Fibroblast Activation Induced by Oxygen-Glucose Deprivation Depends on the HIF-1α/miR-212-5p/KLF4 Pathway.

It is widely accepted that miRNAs play an important role in the pathogenesis of myocardial fibrosis. This study aimed to identify a new pathway of miR-212-5p in the activation of human cardiac fibroblasts (HCFs) induced by oxygen-glucose deprivation (OGD). First, we found that KLF4 protein was markedly decreased in OGD-induced HCFs. Then, bioinformatics analysis and verification experiments were used to identify the existence of an interaction of KLF4 with miR-212-5p. Functional experiments indicated that OGD significantly upregulated the expression of hypoxia inducible factor-1 alpha (HIF-1α) in HCFs, which positively regulated miR-212-5p transcription by binding to its promoter. MiR-212-5p inhibited the expression of Krüppel-like factor 4 (KLF4) protein by binding to the 3' untranslated coding regions (UTRs) of KLF4 mRNA. Inhibition of miR-212-5p effectively inhibited the activation of OGD-induced HCFs by upregulating KLF4 expression and inhibited cardiac fibrosis in vivo and in vitro.

Pore Structure Tuning of Poly-EGDMA Biomedical Material by Varying the O-Quinone Photoinitiator.

Porous polymer monoliths with thicknesses of 2 and 4 mm were obtained via polymerization of ethylene glycol dimethacrylate (EGDMA) under the influence visible-light irradiation in the presence of a 70 wt% 1-butanol porogenic agent and o-quinone photoinitiators. The o-quinones used were: 3,5-di-tret-butyl-benzoquinone-1,2 (35Q), 3,6-di-tret-butyl-benzoquinone-1,2 (36Q), camphorquinone (CQ), and 9,10-phenanthrenequinone (PQ). Porous monoliths were also synthesized from the same mixture but using 2,2'-azo-bis(iso-butyronitrile) (AIBN) at 100 °C instead o-quinones. According to the results of scanning electron microscopy, all the resulting samples were conglomerates of spherical, polymeric particles with pores between them. Use of mercury porometry showed that the interconnected pore systems of all the polymers were open. The average pore size, Dmod, in such polymers strongly depended on both the nature of the initiator and the method of initiation of polymerization. For polymers obtained in the presence of AIBN, the Dmod value was as low as 0.8 μm. For polymers obtained via photoinitiation in the presence of 36Q, 35Q, CQ, and PQ, the Dmod values were significantly greater, i.e., 9.9, 6.4, 3.6, and 3.7 μm, respectively. The compressive strength and Young's modulus of the porous monoliths increased symbatically in the series PQ < CQ < 36Q < 35Q < AIBN with decreasing proportions of large pores (over 12 μm) in their polymer structures. The photopolymerization rate of the EGDMA and 1-butanol, 30:70 wt% mixture was maximal for PQ and minimal for 35Q. All polymers tested were non-cytotoxic. Based on the data from MTT testing, it can be noted that the polymers obtained via photoinitiation were characterized by their positive effect on the proliferative activity of human dermal fibroblasts. This makes them promising osteoplastic materials for clinical trials.

Influence of Three Laser Wavelengths with Different Power Densities on the Mitochondrial Activity of Human Gingival Fibroblasts in Cell Culture.

Phototherapy plays a key role in wound healing and tissue regeneration. The use of lasers has the potential to become an effective and minimally invasive treatment in periodontal and peri-implant disease. The aim of this study was to evaluate the influence of three laser wavelengths with the combination of parameters such as power density and energy density on human gingival fibroblasts (hGFs) in vitro culture. Isolated cells were seeded in 96-well plates with culture medium (DMEM, Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum (FBS). After 24 h cells were irradiated (1064, 980 and 635 nm, various energy density value). After 24, 48 and 72 h, cells were evaluated for viability. Data were analyzed by ANOVA followed by Tukey's HSD test. We found the best outcomes for hGFs irradiated with laser 1064 nm for all combinations of power output (50/400/1000 mW) and energy dose (3/25/64 J/cm2) after 48 h and 72 h compared with control group. Cell viability increase ranged from 0.6× (3 J/cm2, 50 mW) to 1.3× (64 J/cm2, 1000 mW). Our findings indicate that the appropriate use of low-level laser irradiation (LLLI) can increase the proliferation rate of cultured cells. The use of LLLI can be extremely useful in tissue engineering and regenerative medicine.

Lung endothelial cells regulate pulmonary fibrosis through FOXF1/R-Ras signaling

Pulmonary fibrosis results from dysregulated lung repair and involves multiple cell types. The role of endothelial cells (EC) in lung fibrosis is poorly understood. Using single cell RNA-sequencing we identified endothelial transcription factors involved in lung fibrogenesis, including FOXF1, SMAD6, ETV6 and LEF1. Focusing on FOXF1, we found that FOXF1 is decreased in EC within human idiopathic pulmonary fibrosis (IPF) and mouse bleomycin-injured lungs. Endothelial-specific Foxf1 inhibition in mice increased collagen depositions, promoted lung inflammation, and impaired R-Ras signaling. In vitro, FOXF1-deficient EC increased proliferation, invasion and activation of human lung fibroblasts, and stimulated macrophage migration by secreting IL-6, TNFα, CCL2 and CXCL1. FOXF1 inhibited TNFα and CCL2 through direct transcriptional activation of Rras gene promoter. Transgenic overexpression or endothelial-specific nanoparticle delivery of Foxf1 cDNA decreased pulmonary fibrosis in bleomycin-injured mice. Nanoparticle delivery of FOXF1 cDNA can be considered for future therapies in IPF.

In vitro antioxidant and wound healing activity of Sargassum polycystum hydroethanolic extract in fibroblasts and keratinocytes

Objective: To investigate the in vitro antioxidant and wound healing properties of the hydroethanolic extract of Sargassum polycystum, and elucidate the mechanism of its wound healing activity. Methods: Human dermal fibroblast and HaCaT cells were used to evaluate the proliferation by sulforhodamine B and dsDNA assay after treatment with Sargassum polycystum extracts. Scratch wound healing and phalloidin-rhodamine staining were employed to observe migratory activity and filopodia formation, respectively. Western blot and real-time RT-PCR assays were performed to determine the protein and gene expressions related to wound healing activities. Results: The phytochemical analysis found a higher level of flavonoid than phenolic compound in Sargassum polycystumextracts. In human dermal fibroblast cells, Sargassum polycystum extracts at 50 and 100 µg/mL significantly increased fibroblast proliferation and the gene expressions of hyaluronic acid synthase 1 (HAS1), HAS2, HAS3, collagen type 1 alpha 1 chain (COL1A1), collagen type 3 alpha 1 chain (COL3A1), and elastin. The phosphorylation of Akt, ERK1/2, and p38 MAPK was also significantly upregulated after treatment with Sargassum polycystumextracts. Additionally, 50 and 100 µg/mL of the extracts prominently enhanced the proliferation, migration, and filopodia formation of HaCaT cells, as well as the protein levels of pFAK/FAK, pSrc/Src, pAkt/Akt, pERK1/2/ERK1/2, Rac1 and Cdc42. Conclusions: Sargassum polycystum extracts show promising wound healing activities in human dermal fibroblasts and keratinocytes.

PPARδ Agonist GW501516 Suppresses the TGF-β-Induced Profibrotic Response of Human Bronchial Fibroblasts from Asthmatic Patients.

The airway wall remodeling observed in asthma is associated with subepithelial fibrosis and enhanced activation of human bronchial fibroblasts (HBFs) in the fibroblast to myofibroblast transition (FMT), induced mainly by transforming growth factor-β (TGF-β). The relationships between asthma severity, obesity, and hyperlipidemia suggest the involvement of peroxisome proliferator-activated receptors (PPARs) in the remodeling of asthmatic bronchi. In this study, we investigated the effect of PPARδ ligands (GW501516 as an agonist, and GSK0660 as an antagonist) on the FMT potential of HBFs derived from asthmatic patients cultured in vitro. This report shows, for the first time, the inhibitory effect of a PPARδ agonist on the number of myofibroblasts and the expression of myofibroblast-related markers-α-smooth muscle actin, collagen 1, tenascin C, and connexin 43-in asthma-related TGF-β-treated HBF populations. We suggest that actin cytoskeleton reorganization and Smad2 transcriptional activity altered by GW501516 lead to the attenuation of the FMT in HBF populations derived from asthmatics. In conclusion, our data demonstrate that a PPARδ agonist stimulates antifibrotic effects in an in vitro model of bronchial subepithelial fibrosis. This suggests its potential role in the development of a possible novel therapeutic approach for the treatment of subepithelial fibrosis during asthma.

Effects of Cold Atmospheric Plasma Pre-Treatment of Titanium on the Biological Activity of Primary Human Gingival Fibroblasts

Cold atmospheric plasma treatment (CAP) enables the contactless modification of titanium. This study aimed to investigate the attachment of primary human gingival fibroblasts on titanium. Machined and microstructured titanium discs were exposed to cold atmospheric plasma, followed by the application of primary human gingival fibroblasts onto the disc. The fibroblast cultures were analyzed by fluorescence, scanning electron microscopy and cell-biological tests. The treated titanium displayed a more homogeneous and denser fibroblast coverage, while its biological behavior was not altered. This study demonstrated for the first time the beneficial effect of CAP treatment on the initial attachment of primary human gingival fibroblasts on titanium. The results support the application of CAP in the context of pre-implantation conditioning, as well as of peri-implant disease treatment.

An Antibody Targeting Fibroblast Activation Protein Simultaneously Fused to Interleukin-2 and Tumor Necrosis Factor Selectively Localizes to Neoplastic Lesions

The delivery of specific cytokine payloads to a neoplastic environment employing antibodies able to selectively accumulate at the tumor site represents an attractive strategy to stimulate an immune response to cancer. Whilst conventional antibody-cytokine fusions based on a single payload have shown potent anticancer activity, the concomitant delivery of two cytokine payloads may further improve the therapeutic outcome as the immune system typically adopts multiple signals to reinforce an antitumor strategy. We here describe a potency-matched dual-cytokine antibody fusion protein containing a tumor-targeting antibody fragment specific to human fibroblast activation protein (FAP), simultaneously linked to both interleukin-2 (IL2) and a tumor necrosis factor (TNF) mutant. The resulting fusion protein, termed IL2-7NP2-TNFmut, formed stable non-covalent trimers driven by the interaction of the tumor necrosis factor subunits. Both cytokine payloads retained their biological activity within the fusion protein, as shown by in vitro cellular assays. The tumor-targeting properties and the anticancer activity of IL2-7NP2-TNFmut were investigated in vivo in immunocompromised mice bearing SKRC52 cells transduced with human FAP. The fusion protein preferentially localized to the cancer site and induced partial tumor retardation.

Nanoencapsulated cordyceps extract enhances collagen synthesis and skin cell regeneration through antioxidation and autophagy

Oxidative stress from reactive oxygen species is the main cause of skin ageing. Cordycepin, a bioactive compound of Cordyceps militaris, contains antioxidant activity. This study examined extracellular matrix, antioxidant effect, autophagy activity, and skin regeneration in human dermal fibroblasts (HDFs) under normal and oxidative stress conditions. Slow disintegration was used to create nano-encapsulated cordyceps extract. HDFs were cultured and treated with 1 M cordycepin, 1 M medium, 0.1 M cordyceps medium loaded nanoparticles (CMP), or 1 mM H2O2. HDFs’ senescent phenotypes were assessed, including cell proliferation, ROS scavenging, collagen and elastin synthesis, antioxidant activity, and wound healing. CMP size averaged 184.5 ± 95.2 nm increased cell proliferation and reduced H2O2-induced ROS. Thus, HDFs treated for 48 h increased skin regeneration activity 2.76-fold by expressing extracellular matrix and rescuing H2O2-induced damaged cells. It was significant that this CMP inhibited H2O2-induced oxidative stress and induced autophagy to regenerate HDFs. The developed CMP could be used in cosmetics.

Analysis of povidone iodine, chlorhexidine acetate and polyhexamethylene biguanide as wound disinfectants: in vitro cytotoxicity and antibacterial activity

ObjectivesEven though disinfectants are commonly used in clinical practice and daily life, there are few studies on their antibacterial ability and cytotoxicity, which are closely related to the safety and...

Use of a Cellulase from Trichoderma reesei as an Adjuvant for Enterococcus faecalis Biofilm Disruption in Combination with Antibiotics as an Alternative Treatment in Secondary Endodontic Infection.

Apical periodontitis is an inflammation leading to the injury and destruction of periradicular tissues. It is a sequence of events that starts from root canal infection, endodontic treatment, caries, or other dental interventions. Enterococcus faecalis is a ubiquitous oral pathogen that is challenging to eradicate because of biofilm formation during tooth infection. This study evaluated a hydrolase (CEL) from the fungus Trichoderma reesei combined with amoxicillin/clavulanic acid as a treatment against a clinical E. faecalis strain. Electron microscopy was used to visualize the structure modification of the extracellular polymeric substances. Biofilms were developed on human dental apices using standardized bioreactors to evaluate the antibiofilm activity of the treatment. Calcein and ethidium homodimer assays were used to evaluate the cytotoxic activity in human fibroblasts. In contrast, the human-derived monocytic cell line (THP-1) was used to evaluate the immunological response of CEL. In addition, the secretion of the pro-inflammatory cytokines IL-6 and TNF-α and the anti-inflammatory cytokine IL-10 were measured by ELISA. The results demonstrated that CEL did not induce the secretion of IL-6 and TNF-α when compared with lipopolysaccharide used as a positive control. Furthermore, the treatment combining CEL with amoxicillin/clavulanic acid showed excellent antibiofilm activity, with a 91.4% reduction in CFU on apical biofilms and a 97.6% reduction in the microcolonies. The results of this study could be used to develop a treatment to help eradicate persistent E. faecalis in apical periodontitis.

Standard of care drugs do not modulate activity of senescent primary human lung fibroblasts

Cellular senescence is crucial in the progression of idiopathic pulmonary fibrosis (IPF), but it is not evident whether the standard-of-care (SOC) drugs, nintedanib and pirfenidone, have senolytic properties. To address this question, we performed colorimetric and fluorimetric assays, qRT-PCR, and western blotting to evaluate the effect of SOC drugs and D + Q on senescent normal and IPF lung fibroblasts. In this study, we found that SOC drugs did not provoke apoptosis in the absence of death ligand in normal or IPF senescent lung fibroblasts. Nintedanib increased caspase-3 activity in the presence of Fas Ligand in normal but not in IPF senescent fibroblasts. Conversely, nintedanib enhanced B cell lymphoma 2 expression in senescent IPF lung fibroblasts. Moreover, in senescent IPF cells, pirfenidone induced mixed lineage kinase domain-like pseudokinase phosphorylation, provoking necroptosis. Furthermore, pirfenidone increased transcript levels of FN1 and COL1A1 in senescent IPF fibroblasts. Lastly, D + Q augmented growth differentiation factor 15 (GDF15) transcript and protein levels in both normal and IPF senescent fibroblasts. Taken together, these results establish that SOC drugs failed to trigger apoptosis in senescent primary human lung fibroblasts, possibly due to enhanced Bcl-2 levels by nintedanib and the activation of the necroptosis pathway by pirfenidone. Together, these data revealed the inefficacy of SOC drugs to target senescent cells in IPF.