Expression In Fibroblasts Research Articles

12604 Use Of A Novel Program "WordMiner" To Identify Cellular Proliferation Related Genes Induced In FSTL3 KO Mouse Testis

Abstract Disclosure: N. Mukherjee: None. R. Ballesteros: None. A. Mukherjee: None. TGFβ family ligands play crucial roles in promoting cellular proliferation. Moreover, activin and TGFβ are linked with cancer. Deletion of FSTL3, an inhibitor of a subset of TGFβ ligands, in mice leads to increased proliferation in all tissues investigated including the testis. We also find increased SMAD2/3 activation in FSTL3 knockout (KO) compared to wildtype (WT) in these tissues. Therefore, the FSTL3 KO mouse is a model of increased activity of endogenous TGFβ ligands that are inhibited by FSTL3, such as activin. It is likely that increased signalling by these ligands leads to the observed increased cellular proliferation in FSTL3 KO mice. To discover genes that might be involved in activin-dependent mitosis, we studied gene expression datasets linked to cellular proliferation. We hypothesised that genes important in cellular proliferation will be downregulated in senescent cells and upregulated in models of increased cellular proliferation and activin action such as the FSTL3 KO mice. We queried GEO datasets to identify studies related to senescence and selected Hutchinson-Gilford progeria syndrome (HGPS): fibroblast (HG-U133A), GSE3860. We found 4048 transcripts showing significantly (p<0.05) altered expression in HGPS fibroblasts compared to WT. We developed a Python-based program, “WordMiner”, that inputs a keyword and a list of terms (e.g. genes) and then searches a specified database sequentially for publications including the keyword and each term. The output is a table of search result frequencies and hyperlinks to the search result pages, compiled in an auxiliary file, produced with greater speed and efficiency than manual search methods. The greater the search result frequency, the stronger the known link between the keyword and the term. We used WordMiner to query the association of the search term “mitosis” with the 4048 genes above using PubMed as the database and identified 1235 transcripts. We refined this list further using the keyword “activin” to identify 420 transcripts linked to activin and mitosis. Importantly, by comparing against our RNAseq data, we find 20 of these genes upregulated in the FSTL3 KO postnatal day 3 transcriptome. This finding supports our hypothesis, and also demonstrates WordMiner as an efficient and versatile cross-referencing query tool. Among the 20 genes identified are transcripts known to be important in proliferation, such as Akap9 and Wnt2b. These and the remainder in the list of genes highlighted are therefore likely to be involved in pathways induced by activin, promoting cellular proliferation in FSTL3 KO testis. Currently, siRNA transfection experiments are underway to address whether knockdown of these genes in a Sertoli cell line (TM4) limits proliferation. This will help in the elucidation of the molecular pathways involved in postnatal Sertoli cell proliferation. Presentation: 6/3/2024

ADAMTS7 Promotes Smooth Muscle Foam Cell Expansion in Atherosclerosis.

Human genetic studies have repeatedly associated ADAMTS7 with atherosclerotic cardiovascular disease. Subsequent investigations in mice demonstrated that ADAMTS7 is proatherogenic and induced in response to vascular injury and that the proatherogenicity of ADAMTS7, a secreted protein, is due to its catalytic activity. However, the cell-specific mechanisms governing ADAMTS7 proatherogenicity remain unclear. To determine which vascular cell types express ADAMTS7, we interrogated single-cell RNA sequencing of human carotid atherosclerosis and found ADAMTS7 expression in smooth muscle cells (SMCs), endothelial cells (ECs), and fibroblasts. We subsequently created SMC- and EC-specific Adamts7 conditional knockout and transgenic mice. Conditional knockout of Adamts7 in either cell type is insufficient to reduce atherosclerosis, whereas transgenic induction in either cell type increases atherosclerosis. In SMC transgenic mice, this increase coincides with an expansion of lipid-laden SMC foam cells and decreased fibrous cap formation. RNA-sequencing in SMCs revealed an upregulation of lipid uptake genes typically assigned to macrophages. Subsequent experiments demonstrated that ADAMTS7 increases SMC oxLDL uptake through increased CD36 levels. Furthermore, Cd36 expression is increased due to increased levels of PU.1, a transcription factor typically associated with myeloid fate determination. In summary, Adamts7 expression in either SMCs or ECs promotes SMC foam cell formation and atherosclerosis. In SMCs, ADAMTS7 promotes oxLDL uptake via increased PU.1 and Cd36 expression, thereby increasing SMC foam cell formation and atherosclerosis.

Periodic Paralysis in a Child With Thermosensitive Mitochondrial Trifunctional Protein Deficiency.

Mitochondrial trifunctional protein (MTP) deficiency is a fatty acid oxidation disorder associated with a spectrum of phenotypes. Patients with high residual enzyme activity tend to have milder phenotypes, and recently, fever-induced episodic myopathy was reported in association with a thermosensitive form of MTP deficiency. We report a 10-year-old male with recurrent episodes of acute flaccid paralysis involving upper and lower extremities in association with bulbar muscle weakness in the context of febrile illness, a phenotype reminiscent of recurrent periodic paralysis. The episodes started at the age of 3 years and have always been followed by full recovery within 1-2 weeks with no residual weakness. Whole exome sequencing revealed a homozygous c.2132C > T, p.(Pro711Leu) variant in HADHA. The variant leads to mildly reduced long-chain hydroxyacyl-CoA dehydrogenase (LCHAD) and long-chain ketoacyl-CoA thiolase (LCKAT) enzyme activities and reduced MTP protein expression in patient's fibroblasts when cultured at 37°C. Enzyme activities and MTP protein expression diminished when fibroblasts were cultured at 40°C. This is the first published report of confirmed recurrent periodic paralysis as a manifestation of a thermosensitive form of MTP deficiency, and it calls for this condition to be considered when evaluating patients with recurrent periodic paralysis given therapeutic implications.

Influence of titanium nanoparticles on cytotoxicity and inflammatory cytokines expression in gingival fibroblasts - An in vitro study

Background Despite the success of titanium (Ti) implants in dental rehabilitation, emerging evidence implicates the release of titanium oxide nanoparticles (TiO2NPs) from the implant surface as a potential contributor to the initiation of the pathogenic process leading to peri-implantitis and ultimately failure of the implants. However, a comprehensive investigation to elucidate the dose-dependent effects of TiO2NPs on cytotoxicity and inflammatory responses is lacking. The present study was undertaken to evaluate the effect of different concentrations of TiO2NPs on the cytotoxicity and inflammatory cytokine expression in Human Gingival Fibroblasts (HGFs). Methods HGFs were isolated from gingival tissue samples obtained from periodontally and systemically healthy subjects. Ti standard solution for ICP was diluted to create concentrations of (0.001 ppm, 0.01 ppm, 0.1 ppm, 1 ppm, 10 ppm, and 100 ppm) for cell culture media containing titanium. HGFs were then cultured in these varying concentrations for specific time periods (days 1, 3, 5, and 7) to assess cell viability. A cytotoxicity assay was performed to determine the levels and expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and anti-inflammatory cytokines (IL-10, IL-12, TGF-β) using qRT-PCR and ELISA techniques. Results Our findings demonstrate a concentration and duration-dependent decrease in HGF viability upon exposure to titanium nanoparticles. Notably, a 50% reduction in cell viability was observed at the highest concentration (100 ppm). qRT-PCR analysis revealed a significant upregulation of pro-inflammatory cytokines, particularly IL-1β and IL-6, in HGFs exposed to titanium. Interestingly, the expression of anti-inflammatory cytokines (IL-10, IL-12, and TGF-β) remained comparable or even equivalent compared to controls across different titanium concentrations. Conclusions The study revealed a concentration and duration-dependent influence on HGF viability and cytokine profile, suggesting potential cytotoxicity and modulation of the inflammatory response mediated by TiO2NPs. Further research is necessary to elucidate the underlying mechanisms and their implications for dental implant biocompatibility.

Overexpression of the key metabolic protein Carnitine Palmitoyl Transferase 1A (CPT1A) in equine sarcoid

The equine sarcoid is the most common skin neoplasia of fibroblastic origin in horses, characterized by an excessive accumulation of extracellular matrix produced by sarcoid fibroblasts under hypoxic condition. Neoplastic cells can adapt to hypoxia by using alternative energy sources, particularly those that arise from fatty acid oxidation (FAO). The Carnitine Palmitoyl Transferase 1A (CPT1A) belongs to Carnitine System (CS) and promotes the entrance of fatty acids into the mitochondria for β-oxidation. In this study, CPT1A expression was comparatively addressed in 25 equine sarcoids and 5 normal skin samples using immunohistochemistry (IHC). Specificity of CPT1A antibody was validated by Western Blotting (WB). In normal skin samples IHC staining was weak and mainly confined to basal epidermis and few dermal fibroblasts. Sarcoid fibroblast exhibited a strong cytoplasmic and nuclear signal in 60% of the tumor samples. Cytoplasmic CPT1A expression in sarcoid fibroblasts indicates that the protein is actively involved in metabolic reprogramming processes. Nuclear CPT1A expression suggests that the protein may also be involved in the regulation of neoplastic proliferation.

Identification of FGFR4 as a regulator of myofibroblast differentiation in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with limited therapeutic options. Fibroblast growth factor receptor-4 (FGFR4) is a known receptor for several paracrine fibroblast growth factors (FGFs). FGFR4 is also the main receptor for FGF19, an endocrine FGF that was demonstrated by our group to have antifibrotic properties in the lung. We aimed to determine whether FGFR4 could modulate pulmonary fibrogenesis. We assessed FGFR4 mRNA and protein levels in IPF and control lungs. In vitro, we determined the effect of transforming growth factor-β (TGF-β), endothelin-1, and platelet-derived growth factor (PDGF) on FGFR4 expression in human lung fibroblasts. We determined the effect of FGFR4 inhibition, using a specific pharmacological inhibitor (FGF401), or genetic deletion in murine embryonic fibroblasts (MEFs) on TGF-β-induced myofibroblastic differentiation. In vivo, we evaluated the development of bleomycin-induced lung fibrosis in Fgfr4-deficient (Fgfr4-/-) mice compared with wild-type littermates (WT) and after FGF401 treatment in WT mice compared with a control group receiving the solvent only. FGFR4 was decreased in IPF lungs, as compared with control lungs, at mRNA and protein levels. In vitro, FGFR4 was downregulated after treatment with TGF-β, endothelin-1, and PDGF. In vitro, FGFR4 inhibition by FGF401 prevented TGF-β1-induced collagen and ACTA2 increase in lung fibroblasts. Similar results were observed in Fgfr4-/- MEFs. In vivo, FGFR4 genetic deficiency or FGFR4 pharmacological inhibition did not modulate bleomycin-induced pulmonary fibrosis. Our data suggest that FGFR4 exerts profibrotic properties by enhancing TGF-β signaling in vitro. However, the inhibition of FGFR4 is not sufficient to prevent the development of pulmonary fibrosis in vivo.NEW & NOTEWORTHY FGFR4 has been reported to have antifibrotic effects in the liver. We aimed to determine the involvement of FGFR4 during IPF. Our data suggest that FGFR4 exerts profibrotic properties by enhancing TGF-β signaling in vitro. However, the inhibition of FGFR4 is not sufficient to prevent the development of pulmonary fibrosis in vivo. To our knowledge, this is the first study to assess the profibrotic action of FGFR4 during pulmonary fibrosis.

Caveolin-1 expression is a predictor of survival and recurrence patterns in resected pancreatic ductal adenocarcinoma

Background/objectiveCaveolin-1 (Cav1) expressed in cancer cells (cCav1) or cancer-associated fibroblasts (fCav1) exerts either pro- or anti-tumorigenic effects depending on the cancer type or stage of cancer. We aimed to clarify the impact of cCav1 or fCav1 on survival, recurrence patterns, and efficacy of neoadjuvant chemotherapy (NAC) in resected pancreatic ductal adenocarcinoma (PDAC). MethodsTissue microarrays were constructed including 615 patients who underwent curative resection for PDAC. Cav1 expression was evaluated by immunohistochemistry. Patients were divided into two groups based on Cav1 expression in cancer cells (cCav1high vs. cCav1low) or cancer-associated fibroblasts (fCav1high vs. fCav1low). ResultsAmong all 615 patients, 40.7% were cCav1high and 72.7% were fCav1high. cCav1high was associated with worse overall survival (OS) (p = 0.001) and recurrence-free survival (RFS) (p = 0.001) than cCav1low, and was an independent prognostic factor in multivariate analysis of OS and RFS (OS: p = 0.001, hazard ratio [HR] 1.361; RFS: p = 0.001, HR 1.348). Among 596 patients with resectable/borderline resectable PDAC, cCav1high patients with NAC showed better OS than those without, while there was no significant difference between cCav1low patients with NAC and those without. cCav1high was associated with early recurrence (< 6 months) and liver metastasis after resection. Multivariate analysis revealed cCav1high as an independent predictor of liver metastasis. ConclusionscCav1high correlated with worse survival, early recurrence, and liver metastasis after resection for PDAC, while NAC improved survival in cCav1high patients. The Evaluation of cCav1 status could provide additional information contributing to the personalized management of PDAC.

Enzymatic activity of fibroblast activation protein-α is essential for TGF-β1-induced fibroblastic differentiation of human periodontal ligament cells

Human periodontal ligament cells (hPDLCs) contain multipotent postnatal stem cells that can differentiate into PDL fibroblasts, osteoblasts, and cementoblasts. Interaction between the extracellular environment and stem cells is an important factor for differentiation into other progenitor cells. To identify cell surface molecules that induce PDL fibroblastic differentiation, we developed a series of monoclonal antibodies against membrane/ECM molecules. One of these antibodies, an anti-PDL25 antibody, recognizes approximately a 100 kDa protein, and this antigenic molecule accumulates in the periodontal ligament region of tooth roots. By mass spectrometric analysis, we found that the antigenic molecule recognized by the anti-PDL25 antibody is fibroblast activation protein α (FAPα). The expression level of FAPα/PDL25 increased in TGF-β1-induced PDL fibroblasts, and this protein was localized in the cell boundaries and elongated processes of the fibroblastic cells. Ectopic expression of FAPα induced fibroblastic differentiation. In contrast, expression of representative markers for PDL differentiation was decreased by knock down and antibody blocking of FAPα/PDL25. Inhibition of dipeptidyl peptidase activity by a potent FAPα inhibitor dramatically inhibited PDL fibroblastic marker expression but did not affect in cell proliferation and migration.

Effect of Metformin on Interleukin-6 Expression in Human Fibroblast Cell Aging Model

During skin aging, there is excessive secretion of inflammatory cytokines such as interleukin-6 (IL-6), which can make the ageing process more severe. Metformin has been known to have good anti-inflammatory activity through various pathways, one of which reduces the expression of inflammatory cytokines. This study aimed to analyze the effect of metformin treatment on IL-6 gene expression in an in vitro human fibroblast cell. Human fibroblast cells were obtained through foreskin isolation, and 72 cultured human fibroblast cells were divided into six groups based on cell passages, ranging from the third to the eighth. The expression of IL-6 was assessed in three treatments: negative control (normal cells), metformin 100 µM, and positive control (Vitamin E 50 µM). Treatment and measurement of IL-6 gene expression were carried out using qRT-PCR and calculated using the Livak-Schmittgen method. The results were then compared and analyzed using One-Way Analysis of Variance (ANOVA) and followed by post-hoc analysis. The negative control group had the lowest IL-6 gene expression compared to the metformin group and positive control group. The negative control IL-6 expression showed the highest value at passage 3 (0.166 ± 0.04) and the lowest at passage 6 (0.048 ± 0.04). Meanwhile, the expression of IL-6 positive control passage 4 (5.590 ± 3.34) showed the highest value and passage 7 (0.000 ± 0.00) showed the lowest value. In the metformin group, IL-6 gene expression was highest in passage 5 (0.836 ± 0.15) and lowest in passage 3 (0.078 ± 0.02). Based on treatment, there is a difference in IL-6 gene expression at passage 4 (p-value &lt;0.05) and passage 5 (p-value &lt;0.001). Metformin treatment passage 5 showed a significant difference with negative control (p-value &lt;0.05) and with positive control (p-value &lt;0.05). Our study concluded that the administration of metformin had no effect on IL-6 gene expression in human fibroblasts. However, metformin demonstrated anti-aging potential, as evidenced by a statistically significant difference in IL-6 expression between the negative control and positive control treatment groups.

The effects of fulvic acids and low-level laser therapy on orthodontic retention in rats

BackgroundShortening retention time and minimizing relapse rates are ongoing challenges in orthodontics. This study investigated the effects of natural fulvic acids (FAs) and low-level laser therapy (LLLT) on orthodontic retention in rats.MethodsSeventy-two male Sprague-Dawley rats underwent mesial movement of the left maxillary first molar using a 50 g force via a nickel-titanium tension spring. After three weeks of movement, the rats entered the retention phase with retainer wires and were divided into four groups: Control (no intervention), FAs (80 mg/kg orally daily), LLLT (808 nm laser twice weekly), and FAs + LLLT (both treatments). Retainers were removed on days 7, 14, and 21 for a 3-day relapse assessment. Maxillary impressions were analyzed for relapse rates using 3Shape software, alongside histological and immunohistochemical evaluations of bone morphogenetic protein-2 (BMP-2) expression in periodontal tissues, with differences among groups analyzed using an ordinary two-way analysis of variance (ANOVA).ResultsThe relapse rate decreased over time, particularly at 10, 17, and 24 days (p < 0.001). The FAs group did not significantly affect relapse rates compared to the control group (p = 0.084). In contrast, both the LLLT and FAs + LLLT groups significantly reduced relapse rate (p < 0.001), with no significant difference between these groups (p = 0.555). Histological examination revealed active osteoclasts on day 10, decreasing by days 17 and 24. The LLLT and FAs + LLLT groups showed less local cementum resorption and better periodontal fiber arrangement. All treatment groups significantly increased BMP-2 expression (P < 0.05) compared to controls. with LLLT and FAs + LLLT differing significantly from FAs (P < 0.001), though no difference was observed between LLLT and FAs + LLLT (P = 0.578).ConclusionsFAs did not significantly reduce relapse rate with retainers, while LLLT effectively reduced relapse rates, showing no additional benefit from combining FAs with LLLT. Both FAs and LLLT increased BMP-2 expression in PDL fibroblasts but with no synergistic effect.

A New Strategy to Inhibit Scar Formation by Accelerating Normal Healing Using Silicate Bioactive Materials.

Inspired by the scar-free wound healing in infants, an anti-scar strategy is proposed by accelerating wound healing using silicate bioactive materials. Bioglass/alginate composite hydrogels are applied, which significantly inhibit scar formation in rabbit ear scar models. The underlining mechanisms include stimulation of Integrin Subunit Alpha 2 expression in dermal fibroblasts to accelerate wound healing, and induction of apoptosis of hypertrophic scar fibroblasts by directly stimulating the N-Acylsphingosine Amidohydrolase 2 expression in hypertrophic scar fibroblasts, and indirectly upregulating the secretion of Cathepsin K in dermal fibroblasts. Considering specific functions of the bioactive silicate materials, two scar treatment regimes are tested. For severe scars, a regenerative intervention is applied by surgical removal of the scar followed by the treatment with bioactive hydrogels to reduce the formation of scars by activating dermal fibroblasts. For mild scars, the bioactive dressing is applied on the formed scar and reduces scar by inducing scar fibroblasts apoptosis.

Ionic Liquid-Based Grapeseed Oil Emulsion for Enhanced Anti-Wrinkle Treatment.

To address the poor efficacy and percutaneous penetration of grape seed oil, ionic liquids and nanotechnology were combined to prepare a grape seed oil emulsion. A novel Menthol-CoQ10 ionic liquid and ionic liquid based grapeseed oil emulsion were prepared and confirmed. The average size of the grapeseed oil emulsion was 218 nm, and its zeta potential was -33.5 mV. The ionic liquid-based grape seed oil emulsion exhibited a transdermal penetration effect 4.63-fold higher than that of ordinary grape seed oil emulsion. Ionic liquid also displayed enhanced efficiency both in vitro and in vivo. It significantly inhibited the production of DPPH free radicals and tyrosinase, inhibited melanin and matrix metalloproteinase-1 (MMP-1) produced by cells, and promoted type I collagen expression in fibroblasts. After 28 days of continuous use, the grapeseed oil emulsion improved the water content of the stratum corneum and the rate of transepidermal water loss, enhanced the firmness and elasticity of the skin, and significantly improved the total number and length of under-eye lines, tail lines, nasolabial folds, and marionette lines on the face. Menthol-CoQ10 ionic liquid is a promising functional excipient for both transdermal delivery increase and efficient enhancement. Ionic liquid and nanotechnology for grape seed oil facial mask displayed significantly enhanced efficacy and permeability.

Therapeutic role of histone deacetylase inhibition in an in vitro model of Graves' orbitopathy.

Graves' orbitopathy (GO), a manifestation of Graves' disease, is characterized by orbital fibroblast‑induced inflammation, leading to fibrosis or adipogenesis. Histone deacetylase (HDAC) serves a central role in autoimmune diseases and fibrosis. The present study investigated HDAC inhibition in orbital fibroblasts from patients with GO to evaluate its potential as a therapeutic agent. Primary cultured orbital fibroblasts were treated with an HDAC inhibitor, panobinostat, under the stimulation of IL‑1β, TGF‑β or adipogenic medium. Inflammatory cytokines, and fibrosis‑ and adipogenesis‑related proteins were analyzed using western blotting. The effects of panobinostat on HDAC mRNA expression were measured in GO orbital fibroblasts, and specific HDACs were inhibited using small interfering RNA transfection. Panobinostat significantly reduced the IL‑1β‑induced production of inflammatory cytokines and TGF‑β‑induced production of fibrosis‑related proteins. It also suppressed adipocyte differentiation and adipogenic transcription factor production. Furthermore, it significantly attenuated HDAC7 mRNA expression in GO orbital fibroblasts. In addition, the silencing of HDAC7 led to anti‑inflammatory and anti‑fibrotic effects. In conclusion, by inhibiting HDAC7 gene expression, panobinostat may suppress the production of inflammatory cytokines, profibrotic proteins and adipogenesis in GO orbital fibroblasts. The present in vitro study suggested that HDAC7 could be a potential therapeutic target for inhibiting the inflammatory, adipogenic and fibrotic mechanisms of GO.

Non-thermal atmospheric pressure plasma induces selective cancer cell apoptosis by modulating redox homeostasis

BackgroundAnticancer treatments aim to selectively target cancer cells without harming normal cells. While non-thermal atmospheric pressure plasma (NTAPP) has shown anticancer potential across various studies, the mechanisms behind its selective action on cancer cells remain inadequately understood. This study explores the mechanism of NTAPP-induced selective cell death and assesses its application in cancer therapy.MethodsWe treated HT1080 fibrosarcoma cells with NTAPP and assessed the intracellular levels of mitochondria-derived reactive oxygen species (ROS), mitochondrial function, and cell death mechanisms. We employed N-acetylcysteine to investigate ROS’s role in NTAPP-induced cell death. Additionally, single-cell RNA sequencing was used to compare gene expression in NTAPP-treated HT1080 cells and human normal fibroblasts (NF). Western blotting and immunofluorescence staining examined the expression and nuclear translocation of nuclear factor erythroid 2-related factor 2 (NRF2), a key antioxidant gene transcription factor. We also evaluated autophagy activity through fluorescence staining and transmission electron microscopy.ResultsNTAPP treatment increased ROS levels and induced mitochondrial dysfunction, leading to apoptosis in HT1080 cells. The involvement of ROS in selective cancer cell death was confirmed by N-acetylcysteine treatment. Distinct gene expression patterns were observed between NTAPP-treated NF and HT1080 cells, with NF showing upregulated antioxidant gene expression. Notably, NRF2 expression and nuclear translocation increased in NF but not in HT1080 cells. Furthermore, autophagy activity was significantly higher in normal cells compared to cancer cells.ConclusionsOur study demonstrates that NTAPP induces selective cell death in fibrosarcoma cells through the downregulation of the NRF2-induced ROS scavenger system and inhibition of autophagy. These findings suggest NTAPP’s potential as a cancer therapy that minimizes damage to normal cells while effectively targeting cancer cells.

Evaluation of Biotechnological Active Peptides Secreted by Saccharomyces cerevisiae with Potential Skin Benefits.

Biotechnological active peptides are gaining interest in the cosmetics industry due to their antimicrobial, anti-inflammatory, antioxidant, and anti-collagenase (ACE) effects, as well as wound healing properties, making them suitable for cosmetic formulations. The antimicrobial activity of peptides (2-10 kDa) secreted by Saccharomyces cerevisiae Ethanol-Red was evaluated against dermal pathogens using broth microdilution and challenge tests. ACE was assessed using a collagenase activity colorimetric assay, antioxidant activity via spectrophotometric monitoring of nitrotetrazolium blue chloride (NBT) reduction, and anti-inflammatory effects by quantifying TNF-α mRNA in lipopolysaccharides (LPS)-exposed dermal fibroblasts. Wound healing assays involved human fibroblasts, endothelial cells, and dermal keratinocytes. The peptides (2-10 kDa) exhibited antimicrobial activity against 10 dermal pathogens, with the Minimum Inhibitory Concentrations (MICs) ranging from 125 µg/mL for Staphylococcus aureus to 1000 µg/mL for Candida albicans and Streptococcus pyogenes. In the challenge test, peptides at their MICs reduced microbial counts significantly, fulfilling ISO 11930:2019 standards, except against Aspergillus brasiliensis. The peptides combined with MicrocareⓇ SB showed synergy, particularly against C. albicans and A. brasilensis. In vitro, the peptides inhibited collagenase activity by 41.8% and 94.5% at 250 and 1000 µg/mL, respectively, and demonstrated antioxidant capacity. Pre-incubation with peptides decreased TNF-α expression in fibroblasts, indicating anti-inflammatory effects. The peptides do not show to promote or inhibit the angiogenesis of endothelial cells, but are able to attenuate fibrosis, scar formation, and chronic inflammation during the final phases of the wound healing process. The peptides showed antimicrobial, antioxidant, ACE, and anti-inflammatory properties, highlighting their potential as multifunctional bioactive ingredients in skincare, warranting further optimization and exploration in cosmetic applications.

Effect of fibroblasts small- conductance Ca2+ -activated potassium channel subtype 2 (SK2) on myocardial fibrosis in pressure overload mouse

Studies have shown that Small conductance Ca2 + −activated K+ (SK) channel are expressed in fibroblasts. We aimed to determine the expression of SK2 channels in cardiac fibroblasts during myocardial hypertrophy and investigate its relationship with fibrotic remodeling. Myocardial hypertrophy and fibrotic remodeling induced by transverse aortic constriction (TAC) were assessed by echocardiography, Masson's trichrome staining and Western blot. Knockdown and overexpression of the SK2 protein were used to assess relationship between SK2 expression in fibroblasts and myocardial fibrosis. There is a positive correlation between myocardial fibrosis and SK2 channel protein expression during the development of myocardial hypertrophy. The differentiation and secretion of fibroblasts in mice with cardiac hypertrophy are enhanced, and the expression of SK2 channel protein is increased. Manipulating SK2 levels in fibroblasts can either promote or inhibit their differentiation and secretory function. Knocking down SK2 reduces the up-regulation of TGF β1, p-Smad2/3/GAPDH, p-p38/GAPDH, p-ERK1/2/GAPDH, and p-JNK/GAPDH proteins induced by Ang II in cardiac fibroblasts without significantly affecting total protein levels. AAV9-SK2-RNAi injection in mice improves cardiac function. Collectively, our study suggests that the expression of the SK2 channel is significantly increased in fibroblasts of mice with myocardial hypertrophy, potentially impacting myocardial fibrosis remodeling via the TGF-β signaling pathway.

Decreased plasma gelsolin fosters a fibrotic tumor microenvironment and promotes chemoradiotherapy resistance in esophageal squamous cell carcinoma

BackgroundStromal fibrosis is highly associated with therapeutic resistance and poor survival in esophageal squamous cell carcinoma (ESCC) patients. Low expression of plasma gelsolin (pGSN), a serum abundant protein, has been found to correlate with inflammation and fibrosis. Here, we evaluated pGSN expression in patients with different stages of cancer and therapeutic responses, and delineated the molecular mechanisms involved to gain insight into therapeutic strategies for ESCC.MethodsCirculating pGSN level in ESCC patients was determined by enzyme-linked immunosorbent assay analysis, and the tissue microarray of tumors was analyzed by immunohistochemistry staining. Cell-based studies were performed to investigate cancer behaviors and molecular mechanisms, and mouse models were used to examine the pGSN-induced tumor suppressive effects in vivo.ResultsCirculating pGSN expression is distinctively decreased during ESCC progression, and low pGSN expression correlates with poor therapeutic responses and poor survival. Methylation-specific PCR analysis confirmed that decreased pGSN expression is partly attributed to the hypermethylation of the GSN promoter, the gene encoding pGSN. Importantly, cell-based immunoprecipitation and protein stability assays demonstrated that pGSN competes with oncogenic tenascin-C (TNC) for the binding and degradation of integrin αvβ3, revealing that decreased pGSN expression leads to the promotion of oncogenic signaling transduction in cancer cells and fibroblasts. Furthermore, overexpression of pGSN caused the attenuation of TNC expression and inactivation of cancer-associated fibroblast (CAF), thereby leading to tumor growth inhibition in mice.ConclusionsOur results demonstrated that GSN methylation causes decreased secretion of pGSN, leading to integrin dysregulation, oncogenic TNC activation, and CAF formation. These findings highlight the role of pGSN in therapeutic resistance and the fibrotic tumor microenvironment of ESCC.

DC-derived whole cell cytokine nano-regulator for remodelling extracellular matrix and synergizing tumor immunotherapy.

A smart dendritic cell (DC)-derived whole cell cytokine (DWC) nano-regulator of TCPs was developed for tumor cytokine-immunotherapy. The DWCs were purified from activated DC-cultured media and applied as a nano-dosage form. It was found that TCPs could remodel extracellular matrices via the elimination of fibronectin and type I collagen (Col-I) in tumor tissues, as well as the inhibition of α-SMA expression in cancer associated fibroblasts (CAFs). Furthermore, after local TCP treatment, significant tumor inhibition could be achieved combined with radiotherapy.

Anti-Heat Shock Protein 70 Autoantibodies from Patients with Idiopathic Pulmonary Fibrosis Epigenetically Enhance Lung Fibroblast Apoptosis Resistance and Bcl-2 Expression.

IgG autoantibodies to heat shock protein 70 (HSP70) are found in many immune-mediated clinical syndromes, and their presence among patients with idiopathic pulmonary fibrosis (IPF) portends especially poor outcomes. However, pathological effects of IPF anti-HSP70 have not been studied extensively. IPF lung fibroblasts are apoptosis resistant, and this dysregulation contributes to the accumulation of fibroblasts that characterizes the disease. During stress, HSP70 protein is exported extracellularly, where it binds to cognate cell surface receptors that mediate a variety of functional effects, including apoptosis inhibition. We hypothesized anti-HSP70 could engage HSP70-receptor complexes on fibroblasts that alter their apoptosis susceptibility. We found HSP70 is ubiquitously expressed on primary human lung fibroblasts. Treatment with anti-HSP70 isolated from patients with IPF with acute exacerbations increased Bcl-2 expression in human lung fibroblasts and reduced their susceptibility to staurosporine-induced apoptosis. Chromatin immunoprecipitation assays showed Bcl-2 gene promoter regions are enriched with the active histone mark H4 lysine 16 acetylation, and this was increased in the autoantibody-treated fibroblasts. When H4 lysine 16 acetylation was decreased by knocking down its acetyltransferase, MOF (males absent on the first), the anti-HSP70 treatments failed to upregulate Bcl-2. This study describes a heretofore unknown, to our knowledge, pathogenic consequence of autoimmunity in which autoantibodies affect the epigenetic regulation of fibroblast apoptosis. In addition to IPF, this autoimmune process could also have relevance in other immunological syndromes characterized by anti-HSP70 autoimmunity. These findings lend credence to the importance of autoimmunity in IPF and illustrate pathways that could be targeted in innovative therapies for this morbid, medically refractory lung disease.

Effects of low dose skin tissue derived peptides on the function and collagen expression of keloid fibroblasts

This study aims to investigate the effects of the skin tissue derived peptides on proliferation, apoptosis, migration and collagen expressions in keloid fibroblasts. From January 2015 to January 2017, patients with hypertrophic scar who underwent surgical excision in department of plastic surgery of Nanjing maternal and child health hospital were included in this retrospective study. Four peptides were selected from the differential peptides between human hypertrophic scar and normal skin tissue. They were named as peptide deregulated in hypertrophic scar 2-5 (PDHPS2-5). Bioinformatics and functional analysis were performed. A low dose of 10 μmol/L of four peptides were respectively added to the culture medium of human primary keloid fibroblasts for 24 h. Cell counting kit-8 (CCK-8) were used to detect the changes in cell viability. Cell apoptosis was detected by flow cytometry. Cell migration ability was checked by Transwell chamber. The protein expressions of collagen COL1A2 (Collagen type I alpha 2) and the myofibroblast marker gene ACTA2 (Actin alpha 2) were analyzed by Western blot. The results showed that bioinformatics prediction analysis revealed that peptide PDHPS4 has the longest half-life and the highest thermal stability. Compared with the control group, low dose of four peptides had no significant effect on the survival rate and apoptosis of keloid fibroblasts tested by CCK-8 assay and flowcytometry. Transwell analysis showed that one peptides (PDHPS5) can significantly inhibit the cell migration ability (The optical density value in Control is 0.81±0.11, in PDHPS5 is 0.27±0.03, t=8.61, P=0.001). Western blot analysis showed that four peptides (PDHPS2, PDHPS3, PDHPS4, PDHPS5) can significantly inhibit the protein expressions of COL1A2 (The relative protein band intensity in Control is 1.02±0.02, in PDHPS2 is 0.21±0.04, in PDHPS3 is 0.26±0.03, in PDHPS4 is 0.53±0.04, in PDHPS5 is 0.73±0.04, t=31.38, 38.54, 18.88, 11.07 respectively, all P value are less than 0.01). Three peptides (PDHPS2, PDHPS3, PDHPS5) can significantly inhibit the protein expressions of ACTA2 (The relative protein band intensity in Control is 1.02±0.02, in PDHPS2 is 0.64±0.05, in PDHPS3 is 0.77±0.06, in PDHPS5 is 0.47±0.07, t=12.08, 6.38, 14.06 respectively, all P value are less than 0.01). In conclusion, the differentially expressed peptides in human hypertrophic scar tissue can affect the function of keloid fibroblasts and collagen expressions to varying degrees. Among them, two peptides (PDHPS2,PDHPS3) significantly inhibit the protein expressions of COL1A2 and ACTA2. The peptide PDHPS5 has high stability, significantly suppresses cell migration, and reduces the protein expressions of COL1A2 and ACTA2, which may provide a new strategy for scar prevention and treatment.