Expression Of Fibronectin Research Articles

The suppression of cell motility through the reduction of FAK activity and expression of cell adhesion proteins by hAMSCs secretome in MDA-MB-231 breast cancer cells.

Breast cancer is a leading cause of death in women worldwide. Cancer therapy based on stem cells is considered as a novel and promising platform. In the present study, we explore the therapeutic effects of human amniotic mesenchymal stromal cells (hAMSCs) through the reduction of focal adhesion kinase (FAK) activity, SHP-2, and cell adhesion proteins such as Paxillin, Vinculin, Fibronectin, Talin, and integrin αvβ3 expression in MDA-MB-231 breast cancer cells. For this purpose, we employed a co-culture system using 6-well plate transwell. After 72h, hAMSCs-treated MDA-MB-231 breast cancer cells, the activity of focal adhesion kinase (FAK) and the expression of SHP-2 and cell adhesion proteins such as Paxillin, Vinculin, Fibronectin, Talin, and integrin αvβ3 expression were analyzed using western blot. The shape and migration of cells were also analyzed. Based on our results, a significant reduction in tumor cell motility through downregulation of the tyrosine phosphorylation level of FAK (at Y397 and Y576/577 sites) and cell adhesion expression in MDA-MB-231 breast cancer cells was demonstrated. Our findings indicate that hAMSCS secretome has therapeutic effects on cancer cell migration through downregulation of FAK activity and expression of cell adhesion proteins.

RfxCas13d-mediated inhibition of Circ1647 alleviates renal fibrosis via PI3K/AKT signaling pathway

Background Circular RNAs (CircRNAs) have been shown to be involved in the development of chronic kidney disease (CKD). This study aimed to investigate the role of Circ1647 in renal fibrosis, which is a hallmark of CKD. Methods In this study, we established a unilateral ureteral obstruction (UUO) model and delivered Circ1647 RfxCas13d knockdown plasmid into renal parenchymal cells via retrograde injection through the ureter followed by electroporation. After that, the pathological changes were determined by Hematoxylin and Eosin. Meanwhile, Immunohistochemistry, qRT-PCR and Western blot were conducted to assess the degree of fibrosis. In addition, overexpressing of Circ1647 in renal tubular epithelial cells (TCMK1) was performed to investigate the underlying mechanisms of Circ1647. Results Our results displayed that electroporation-mediated knockdown of Circ1647 by RfxCas13d knockdown plasmid significantly inhibited renal fibrosis in UUO mice as evidenced by reduced expression of fibronectin and α-SMA (alpha-smooth muscle actin). Conversely, overexpression of Circ1647 in TCMK1 cells promoted the fibrosis. In terms of mechanism, Circ1647 may mediate the PI3K/AKT Signaling Pathway as demonstrated by the balance of the phosphorylation of PI3K and AKT in vivo and the aggravated phosphorylation of PI3K and AKT in vitro. These observations were corroborated by the effects of the PI3K inhibitor LY294002, which mitigated fibrosis post Circ1647 overexpression. Conclusion Our study suggests that Circ1647 plays a significant role in renal fibrosis by mediating the PI3K/AKT signaling pathway. RfxCas13d-mediated inhibition of Circ1647 may serve as a therapeutic target for renal fibrosis in CKD.

Abstract 207: Tumor suppressive role of NKX2-1 in advanced thyroid cancer progression

Abstract NKX2-1 is a homeodomain transcription factor, known as a master regulator of thyroid development. NKX2-1 is also crucial for lung epithelium differentiation, and the involvement of NKX2-1 in lung carcinogenesis and cancer progression has been intensely investigated. However, the role of NKX2-1 in thyroid cancer progression remains elusive. The Cancer Genome Atlas (TCGA) dataset analysis demonstrated that papillary thyroid cancer (PTC) patients with low NKX2-1 mRNA expression had significantly poorer prognosis than those with a combination of intermediate and high mRNA expression of NKX2-1. Lentivirus-transduced tetracycline (Tet)-inducible system was used to achieve NKX2-1 overexpression in advanced follicular thyroid cancer (FTC) cell lines, FTC-238 and WRO, and an anaplastic thyroid cancer (ATC) cell line 8305C, in which NKX2-1 expression is barely/not detected. All these thyroid cancer cell lines showed significant suppression of cell proliferation with NKX2-1 overexpression. In addition, NKX2-1 overexpression inhibited migration of FTC-238 cells. The levels of mRNA and protein expression of mesenchymal markers, matrix metalloproteinase-2 (MMP-2) and fibronectin, were decreased by NKX2-1 overexpression in FTC-238 cells, suggesting a possibility that NKX2-1 partially inhibits epithelial-mesenchymal transition (EMT). SNAIL and SLUG are essential EMT transcription factors involved in metastasis in cancer progression. NKX2-1 mRNA expression was significantly lower in high SNAI1 (encoding SNAIL) expressing PTC in TCGA database. Indeed, suppression of SNAIL protein expression was found upon overexpression of NKX2-1 in FTC-238 cells which naturally express high level of SNAIL. These findings suggest that NKX2-1 may inhibit the migration of advanced thyroid cancer cells through SNAIL-induced EMT. Similarly, suppression of SLUG protein expression by overexpression of NKX2-1 was observed in WRO and 8305C cells which express high level of SLUG protein. Whether NKX2-1 overexpression inhibits migration and invasion of WRO and 8305C cells through SLUG-induced EMT is currently under examination. The Tet-inducible NKX2-1 expressing thyroid cancer cells will be used for in vivo evaluation of their metastatic ability using an immunocompromised mouse model. The findings so far suggest that NKX2-1 exerts a tumor suppressive activity in advanced thyroid cancers through suppression of cell proliferation, and cell migration presumably via suppression of EMT transcription factors. Citation Format: Yo-Taro Shirai, Shioko Kimura. Tumor suppressive role of NKX2-1 in advanced thyroid cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 207.

Phillyrin alleviates high glucose-induced oxidative stress and inflammation in HBZY-1 cells through inhibition of the PI3K/Akt signaling pathway.

Phillyrin, the major lignin compound of Forsythia suspense (Thunb.) Vahl, has been shown the effects of anti-inflammatory and antioxidant. Our study was aimed to explore the protective effect of phillyrin on glomerular mesangial cells (HBZY-1) and the potential mechanism. Cell viability, cytokine production, levels of reactive oxygen radicals (ROS), glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD), as well as autophagy and apoptosis levels were determined to verify the mechanism of phillyrin on HBZY-1 cells. Our result indicated that phillyrin significantly inhibited HG-induced HBZY-1 proliferation by inhibiting Bcl-2 expression and upregulating Bad, cleaved caspase-3, and -9 expression. Also, phillyrin suppressed HG-induced mesangial extracellular matrix accumulation by inhibiting the expression of fibronectin and transforming growth factor-β1. Further, phillyrin inhibited oxidative stress and inflammation by decreasing ROS, MDA, TNF-α, IL-1β, and IL-6 contents and increasing SOD and GSH expression. Phillyrin also promoted autophagy by increasing LC3-II/LC3-I ratio and down-regulating p62 expression. Furthermore, WB assay showed that phillyrin inhibited oxidative stress caused by HG via activating Nrf2 signaling pathway, while attenuated proliferation and inflammation in HBZY-1 cells through inactivating PI3K/Akt/mTOR and NF-κB pathways. All results showed that phillyrin might be a promising therapeutic agent for the treatment of DN.

Insights into Disease Progression of Translational Preclinical Rat Model of Interstitial Pulmonary Fibrosis through Endpoint Analysis.

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease characterized by the relentless deposition of extracellular matrix (ECM), causing lung distortions and dysfunction. Animal models of human IPF can provide great insight into the mechanistic pathways underlying disease progression and a means for evaluating novel therapeutic approaches. In this study, we describe the effect of bleomycin concentration on disease progression in the classical rat bleomycin model. In a dose-response study (1.5, 2, 2.5 U/kg i.t), we characterized lung fibrosis at day 14 after bleomycin challenge using endpoints including clinical signs, inflammatory cell infiltration, collagen content, and bronchoalveolar lavage fluid-soluble profibrotic mediators. Furthermore, we investigated fibrotic disease progression after 2 U/kg i.t. bleomycin administration at days 3, 7, and 14 by quantifying the expression of clinically relevant signaling molecules and pathways, epithelial mesenchymal transition (EMT) biomarkers, ECM components, and histopathology of the lung. A single bleomycin challenge resulted in a progressive fibrotic response in rat lung tissue over 14 days based on lung collagen content, histopathological changes, and modified Ashcroft score. The early fibrogenesis phase (days 3 to 7) is associated with an increase in profibrotic mediators including TGFβ1, IL6, TNFα, IL1β, CINC1, WISP1, VEGF, and TIMP1. In the mid and late fibrotic stages, the TGFβ/Smad and PDGF/AKT signaling pathways are involved, and clinically relevant proteins targeting galectin-3, LPA1, transglutaminase-2, and lysyl oxidase 2 are upregulated on days 7 and 14. Between days 7 and 14, the expressions of vimentin and α-SMA proteins increase, which is a sign of EMT activation. We confirmed ECM formation by increased expressions of procollagen-1Aα, procollagen-3Aα, fibronectin, and CTGF in the lung on days 7 and 14. Our data provide insights on a complex network of several soluble mediators, clinically relevant signaling pathways, and target proteins that contribute to drive the progressive fibrotic phenotype from the early to late phase (active) in the rat bleomycin model. The framework of endpoints of our study highlights the translational value for pharmacological interventions and mechanistic studies using this model.

Effect of Air Pollutants on Allergic Inflammation in Structural Cells of the Nasal Mucosa.

Air pollution is an increasing global concern, and its effect on allergic inflammation has attracted the attention of many researchers. Particulate matter (PM) is a major component of ambient air pollution, and heavy metals are the primary toxic constituents of PM. As previous studies on the impact of air pollutants on allergic inflammation did not adequately mimic real-world atmospheric exposure, we developed an experimental model to investigate the effects of aerosolized air pollutants on nasal epithelial cells and fibroblasts. We collected particulate matter 2.5 (PM2.5) samples from ambient 24-hour air samples obtained in Seoul from August 2020 to August 2022, and then conducted component analysis for metallic constituents. Primary nasal epithelial cells and nasal fibroblasts, obtained and cultured from the turbinate tissues of human participants, were treated with PM2.5. The associations of heavy metals identified from the component analysis with cytokine expression were investigated. A three-dimensional (3D)-hybrid culture model, consisting of co-culture of an air-liquid interface and nasal fibroblast spheroids, was constructed to observe the impact of aerosolized air pollutants. Among the heavy metals, Si was the predominant component of PM2.5, and Zn showed the highest correlation with the concentration of PM2.5 in Seoul. PM2.5, Zn, and Si increased the production of epithelial cell-derived cytokines, and PM2.5 and Zn exhibited similar trends with one another. Exposure of the 3D-hybrid model to aerosolized PM2.5 and Zn resulted in elevated periostin, alpha-smooth muscle actin, and fibronectin expression in fibroblast spheroids, and those without an epithelial barrier exhibited a similar increase in periostin expression. Ambient air pollutants in the form of aerosols increase the expression of allergic inflammatory cytokines in both nasal epithelial cells and fibroblasts. Regulations on air pollution will help reduce the global burden of allergic diseases in the future.

COMP promotes pancreatic fibrosis by activating pancreatic stellate cells through CD36-ERK/AKT signaling pathways

BackgroundPancreatic fibrosis is one of the most important pathological features of chronic pancreatitis (CP) and pancreatic stellate cells (PSCs) are the key cells of fibrosis. As an extracellular matrix (ECM) glycoprotein, cartilage oligomeric matrix protein (COMP) is critical for collagen assembly and ECM stability and recent studies showed that COMP exert promoting fibrosis effect in the skin, lungs and liver. However, the role of COMP in activation of PSCs and pancreatic fibrosis remain unclear. We aimed to investigate the role and specific mechanisms of COMP in regulating the profibrotic phenotype of PSCs and pancreatic fibrosis. MethodsELISA method was used to determine serum COMP in patients with CP. Mice model of CP was established by repeated intraperitoneal injection of cerulein and pancreatic fibrosis was evaluated by Hematoxylin-Eosin staining (H&E) and Sirius red staining. Immunohistochemical staining was used to detect the expression changes of COMP and fibrosis marker such as α-SMA and Fibronectin in pancreatic tissue of mice. Cell Counting Kit-8, Wound Healing and Transwell assessed the proliferation and migration of human pancreatic stellate cells (HPSCs). Western blotting, qRT-PCR and immunofluorescence staining were performed to detect the expression of fibrosis marker, AKT and MAPK family proteins in HPSCs. RNA-seq omics analysis as well as small interfering RNA of COMP, recombinant human COMP (rCOMP), MEK inhibitors and PI3K inhibitors were used to study the effect and mechanism of COMP on activation of HPSCs. ResultsELISA showed that the expression of COMP significantly increased in the serum of CP patients. H&E and Sirius red staining analysis showed that there was a large amount of collagen deposition in the mice in the CP model group and high expression of COMP, α-SMA, Fibronectin and Vimentin were observed in fibrotic tissues. TGF-β1 stimulates the activation of HPSCs and increases the expression of COMP. Knockdown of COMP inhibited proliferation and migration of HPSCs. Further, RNA-seq omics analysis and validation experiments in vitro showed that rCOMP could significantly promote the proliferation and activation of HPSCs, which may be due to promoting the phosphorylation of ERK and AKT through membrane protein receptor CD36. rCOMP simultaneously increased the expression of α-SMA, Fibronectin and Collagen I in HPSCs. ConclusionIn conclusion, this study showed that COMP was up-regulated in CP fibrotic tissues and COMP induced the activation, proliferation and migration of PSCs through the CD36-ERK/AKT signaling pathway. COMP may be a potential therapeutic candidate for the treatment of CP. Interfering with the expression of COMP or the communication between COMP and CD36 on PSCs may be the next direction for therapeutic research.

Stop codon variant in EFEMP1 is associated with primary open-angle glaucoma due to impaired regulation of aqueous humor outflow

It is known that the actin cytoskeleton and its associated cellular interactions in the trabecular meshwork (TM) and juxtacanalicular tissues mainly contribute to the formation of resistance to aqueous outflow of the eye. Fibulin-3, encoded by EFEMP1 gene, has a role in extracellular matrix (ECM) modulation, and interacts with enzymatic ECM regulators, but the effects of fibulin-3 on TM cells has not been explored. Here, we report a stop codon variant (c.T1480C, p.X494Q) of EFEMP1 that co-segregates with primary open angle glaucoma (POAG) in a Chinese pedigree. In the human TM cells, overexpression of wild-type fibulin-3 reduced intracellular actin stress fibers formation and the extracellular fibronectin levels by inhibiting Rho/ROCK signaling. TGFβ1 up-regulated fibulin-3 protein levels in human TM cells by activating Rho/ROCK signaling. In rat eyes, overexpression of wild-type fibulin-3 decreased the intraocular pressure and the fibronectin expression of TM, however, overexpression of mutant fibulin-3 (c.T1480C, p.X494Q) showed opposite effects in cells and rat eyes. Taken together, the EFEMP1 variant may impair the regulatory capacity of fibulin-3 which has a role for modulating the cell contractile activity and ECM synthesis in TM cells, and in turn may maintain normal resistance of aqueous humor outflow. This study contributes to the understanding of the important role of fibulin-3 in TM pathophysiology and provides a new possible POAG therapeutic approach.

PARP9 affects myocardial function through TGF-β/Smad axis and pirfenidone.

Cardiac arrhythmias are often linked to the overactivity of cardiac fibroblasts (CFs). Investigating the impact of poly (ADP-ribose) polymerase 9 (PARP9) on Angiotensin II (Ang II)-induced fibroblast activation and the therapeutic effects of pirfenidone (PFD) offers valuable insights into cardiac arrhythmias. This study utilized weighted gene co-expression network analysis (WGCNA), differential gene expression (DEG) analysis, protein-protein interaction (PPI), and receiver operating characteristic (ROC) analysis on the GSE42955 dataset to identify the hub gene with a significant diagnostic value. The ImmuCellAI tool revealed an association between PARP9 and immune cell infiltration. Our in vitro assessments focused on the influence of PFD on myofibroblast differentiation, transforming growth factor-beta (TGF-β) expression, and Ang II-induced proliferation and migration in CFs. Additionally, we explored the impact on fibrosis markers and the TGF-β/Smad signaling pathway in the context of PARP9 overexpression. Analysis of the GSE42955 dataset revealed PARP9 as a central gene with high clinical diagnostic value, linked to seven types of immune cells. The in vitro studies demonstrated that PFD significantly mitigates Ang II-induced CF proliferation, migration, and fibrosis. It also reduces Ang II-induced PARP9 expression and decreases fibrosis markers, including TGF-β, collagen I, collagen III, and α-SMA. Notably, PARP9 overexpression can partially counteract PFD's inhibitory effects on CFs and modify the expression of fibronectin, CTGF, α-SMA, collagen I, collagen III, MMP2, MMP9, TGF-β, and p-Smad2/3 in the TGF-β/Smad signaling pathway. In summary, our findings suggest that PFD effectively counteracts the adverse effects of Ang II-induced CF proliferation and fibrosis, and modulates the TGF-β/Smad signaling pathway and PARP9 expression. This identifies a potential therapeutic approach for managing myocardial fibrosis.

Human umbilical cord/placenta mesenchymal stem cell conditioned medium attenuates intestinal fibrosis in vivo and in vitro

BackgroundA significant unmet need in inflammatory bowel disease is the lack of anti-fibrotic agents targeting intestinal fibrosis. This study aimed to investigate the anti-fibrogenic properties and mechanisms of the conditioned medium (CM) from human umbilical cord/placenta-derived mesenchymal stem cells (UC/PL-MSC-CM) in a murine intestinal fibrosis model and human primary intestinal myofibroblasts (HIMFs).MethodsUC/PL-MSC-CM was concentrated 15-fold using a 3 kDa cut-off filter. C57BL/6 mice aged 7 weeks old were randomly assigned to one of four groups: (1) control, (2) dextran sulfate sodium (DSS), (3) DSS + CM (late-phase treatment), and (4) DSS + CM (early-phase treatment). Chronic DSS colitis and intestinal fibrosis was induced by three cycles of DSS administration. One DSS cycle consisted of 7 days of oral DSS administration (1.75%, 2%, and 2.5% DSS), followed by 14 days of drinking water. UC/PL-MSC-CM was intraperitoneally administered in the late phase (from day 50, 10 times) or early phase (from day 29, 10 times) of DSS cycles. HIMFs were treated with TGF-β1 and co-treated with UC/PL-MSC-CM (10% of culture media) in the cellular model.ResultsIn the animal study, UC/PL-MSC-CM reduced submucosa/muscularis propria thickness and collagen deposition, which improved intestinal fibrosis in chronic DSS colitis. The UC/PL-MSC-CM significantly reduced the expressions of procollagen1A1 and α-smooth muscle actin, which DSS significantly elevated. The anti-fibrogenic effect was more apparent in the UC-MSC-CM or early-phase treatment model. The UC/PL-MSC-CM reduced procollagen1A1, fibronectin, and α-smooth muscle actin expression in HIMFs in the cellular model. The UC/PL-MSC-CM downregulated fibrogenesis by suppressing RhoA, MRTF-A, and SRF expression.ConclusionsHuman UC/PL-MSC-CM inhibits TGF-β1-induced fibrogenic activation in HIMFs by blocking the Rho/MRTF/SRF pathway and chronic DSS colitis-induced intestinal fibrosis. Thus, it may be regarded as a novel candidate for stem cell-based therapy of intestinal fibrosis.

Antifibrotic effects of vitamin D3 on human lung fibroblasts derived from patients with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. Up to now, no treatment can stop the progression of IPF. Vitamin D3 (VD) reduces experimental lung fibrosis in murine models and depletion of vitamin D3 might be associated with the reduced survival of patients with IPF. In this context, we determined if VD can prevent the pro-fibrotic functions of human lung fibroblasts (HLFs) isolated from patients with IPF. IPF and control HLFs were derived from surgical lung biopsies collected from patients with IPF or with primary lung cancer, respectively. VD (3-100 nM) markedly reduced the basal and PDGF-induced proliferation of HLFs. VD also altered cell cycle by increasing the percentage of IPF HLFs arrested in the G0/G1 phase, and by downregulating the expression of various cell cycle regulatory proteins. In addition, VD barely prevented the TGF-β1-induced differentiation in HLFs. At 100 nM, VD slightly reduced the expression of the pro-fibrotic marker α-smooth muscle actin, and had no effect on fibronectin and collagen-1 expression. In contrast, 100 nM VD strongly inhibited the aerobic glycolytic metabolism induced by TGF- β1. Finally, VD reduced both the secretion of lactate, the levels of lactate deshydrogenase mRNA and the activity of intracellular LDH in IPF HLFs. In conclusion, our study shows that VD reduced pro-fibrotic functions of HLFs. These findings suggest that it might be interesting to assess the potential clinical benefits of vitamin D supplementation in patients with IPF, especially on lung function decline.

Attenuating Renal Interstitial Fibrosis by Shenqi Pill viaReducing Inflammation Response Regulated by NF-κBPathway In Vitro and In Vivo.

One of the most significant clinical features of chronic kidney disease is renal interstitial fibrosis (RIF). This study aimed to investigate the role and mechanism of Shenqi Pill (SQP) on RIF. RIF model was established by conducting unilateral ureteral obstruction (UUO) surgery on rat or stimulating human kidney-2 (HK-2) cell with transforming growth factor β1 (TGFβ1). After modeling, the rats in the SQP low dose group (SQP-L), SQP middle dose group (SQP-M) and SQP high dose group (SQP-H) were treated with SQP at 1.5, 3 or 6 g/kg/d, and the cells in the TGFβ1+SQP-L/M/H were treated with 2.5%, 5%, 10% SQP-containing serum. In in vivo assays, serum creatinine (SCr) and blood urea nitrogen (BUN) content were measured, kidney histopathology was evaluated., and α-smooth muscle actin (α-SMA) expression was detected by immunohistochemistry. Interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) content, inhibitor of kappa B alpha (IKBα) and P65 phosphorylation were assessed. Meanwhile, cell viability, inflammatory cytokines content, α-SMA expression, IKBα and P65 phosphorylation were detected in vitro experiment. Results. SQP exhibited reno-protective effect by decreasing SCr and BUN content, improving renal interstitial damage, blunting fibronectin (FN) and α-SMA expression in RIF rats. Similarly, after the treatment with SQP-containing serum, viability and α-SMA expression were remarkably decreased in TGFβ1-stimulated HK-2 cell. Furthermore, SQP markedly down-regulated IL-1β, IL-6, and TNF-α content, IKBα and RelA (P65) phosphorylation both in vivo and in vitro. Conclusion. SQP has a reno-protective effect against RIF in vivo and in vitro, and the effect is partly linked to nuclear factor-kappa B (NF-κB) pathway related inflammatory response, which indicates that SQP may be a candidate drug for RIF. DOI: 10.52547/ijkd.7546.

Advanced Glycation End Products Upregulate CD40 in Human Retinal Endothelial and Müller Cells: Relevance to Diabetic Retinopathy.

CD40 induces pro-inflammatory responses in endothelial and Müller cells and is required for the development of diabetic retinopathy (DR). CD40 is upregulated in these cells in patients with DR. CD40 upregulation is a central feature of CD40-driven inflammatory disorders. What drives CD40 upregulation in the diabetic retina remains unknown. We examined the role of advanced glycation end products (AGEs) in CD40 upregulation in endothelial cells and Müller cells. Human endothelial cells and Müller cells were incubated with unmodified or methylglyoxal (MGO)-modified fibronectin. CD40 expression was assessed by flow cytometry. The expression of ICAM-1 and CCL2 was examined by flow cytometry or ELISA after stimulation with CD154 (CD40 ligand). The expression of carboxymethyl lysine (CML), fibronectin, and laminin as well as CD40 in endothelial and Müller cells from patients with DR was examined by confocal microscopy. Fibronectin modified by MGO upregulated CD40 in endothelial and Müller cells. CD40 upregulation was functionally relevant. MGO-modified fibronectin enhanced CD154-driven upregulation of ICAM-1 and CCL2 in endothelial and Müller cells. Increased CD40 expression in endothelial and Müller cells from patients with DR was associated with increased CML expression in fibronectin and laminin. These findings identify AGEs as inducers of CD40 upregulation in endothelial and Müller cells and enhancers of CD40-dependent pro-inflammatory responses. CD40 upregulation in these cells is associated with higher CML expression in fibronectin and laminin in patients with DR. This study revealed that CD40 and AGEs, two important drivers of DR, are interconnected.

Enhancing Vasculogenesis in Dental Pulp Development: DPSCs-ECs Communication via FN1-ITGA5 Signaling.

Dental pulp regeneration therapy is a challenge to achieve early vascularization during treatment. Studying the regulatory mechanisms of vascular formation during human dental pulp development may provide insights for related therapies. In this study, we utilized single-cell sequencing analysis to compare the gene expression of dental pulp stem cells (DPSCs) and vascular endothelial cells (ECs) from developing and mature dental pulps. Immunohistochemistry, Western blot, and real-time polymerase chain reaction (RT-PCR) were used to detect fibronectin 1 (FN1) expression and molecules, such as PI3K/AKT. Cell proliferation assay, scratch assay, tube formation assay and were used to investigate the effects of DPSCs on the vasculogenetic capability of ECs. Additionally, animal experiments involving mice were conducted. The results revealed that DPSCs exist around dental pulp vasculature. FN1 expression was significantly higher in DPSCs from young permanent pulps than mature pulps, promoting HUVEC proliferation, migration, and tube formation via ITGA5 and the downstream PI3K/AKT signaling pathway. Our data indicate that intercellular communication between DPSCs and ECs mediated by FN1-ITGA5 signaling is crucial for vascularizationduring dental pulp development, laying an experimental foundation for future clinical studies.

The H9N2 avian influenza virus increases APEC adhesion to oviduct epithelia by viral NS1 protein-mediated activation of the TGF-β pathway.

H9N2 avian influenza is a low-pathogenic avian influenza circulating in poultry and wild birds worldwide and frequently contributes to chicken salpingitis that is caused by avian pathogenic Escherichia coli (APEC), leading to huge economic losses and risks for food safety. Currently, how the H9N2 virus contributes to APEC infection and facilitates salpingitis remains elusive. In this study, in vitro chicken oviduct epithelial cell (COEC) model and in vivo studies were performed to investigate the role of H9N2 viruses on secondary APEC infection, and we identified that H9N2 virus enhances APEC infection both in vitro and in vivo. To understand the mechanisms behind this phenomenon, adhesive molecules on the cell surface facilitating APEC adhesion were checked, and we found that H9N2 virus could upregulate the expression of fibronectin, which promotes APEC adhesion onto COECs. We further investigated how fibronectin expression is regulated by H9N2 virus infection and revealed that transforming growth factor beta (TGF-β) signaling pathway is activated by the NS1 protein of the virus, thus regulating the expression of adhesive molecules. These new findings revealed the role of H9N2 virus in salpingitis co-infected with APEC and discovered the molecular mechanisms by which the H9N2 virus facilitates APEC infection, offering new insights to the etiology of salpingitis with viral-bacterial co-infections.IMPORTANCEH9N2 avian influenza virus (AIV) widely infects poultry and is sporadically reported in human infections. The infection in birds frequently causes secondary bacterial infections, resulting in severe symptoms like pneumonia and salpingitis. Currently, the mechanism that influenza A virus contributes to secondary bacterial infection remains elusive. Here we discovered that H9N2 virus infection promotes APEC infection and further explored the underlying molecular mechanisms. We found that fibronectin protein on the cell surface is vital for APEC adhesion and also showed that H9N2 viral protein NS1 increased the expression of fibronectin by activating the TGF-β signaling pathway. Our findings offer new information on how AIV infection promotes APEC secondary infection, providing potential targets for mitigating severe APEC infections induced by H9N2 avian influenza, and also give new insights on the mechanisms on how viruses promote secondary bacterial infections in animal and human diseases.

Crosstalk with renal proximal tubule cells drives acidosis-induced inflammatory response and dedifferentiation of fibroblasts via p38-singaling

BackgroundTubulointerstitial kidney disease associated microenvironmental dysregulation, like acidification, inflammation and fibrosis, affects tubule cells and fibroblasts. Micromilieu homeostasis influences intracellular signaling and intercellular crosstalk. Cell–cell communication in turn modulates the interstitial microenvironment. We assessed the impact of acidosis on inflammatory and fibrotic responses in proximal tubule cells and fibroblasts as a function of cellular crosstalk. Furthermore, cellular signaling pathways involved were identified.MethodsHK-2 (human proximal tubule) and CCD-1092Sk (human fibroblasts), in mono and coculture, were exposed to acidic or control media for 3 or 48 h. Protein expression of inflammation markers (TNF, TGF-ß and COX-2), dedifferentiation markers (N-cadherin, vinculin, ß-catenin and vimentin), fibrosis markers (collagen III and fibronectin) and phospho- as well as total MAPK levels were determined by western blot. Secreted collagen III and fibronectin were measured by ELISA. The impact of MAPK activation was assessed by pharmacological intervention. In addition, necrosis, apoptosis and epithelial permeability were determined.ResultsIndependent of culture conditions, acidosis caused a decrease of COX-2, vimentin and fibronectin expression in proximal tubule cells. Only in monoculture, ß-Catenin expression decreased and collagen III expression increased in tubule cells during acidosis. By contrast, in coculture collagen III protein expression of tubule cells was reduced. In fibroblasts acidosis led to an increase of TNF, COX-2, vimentin, vinculin, N-cadherin protein expression and a decrease of TGF-ß expression exclusively in coculture. In monoculture, expression of COX-2 and fibronectin was reduced. Collagen III expression of fibroblasts was reduced by acidosis independent of culture conditions.In coculture, acidosis enhanced phosphorylation of ERK1/2, JNK1/2 and p38 transiently in proximal tubule cells. In fibroblasts, acidosis enhanced phosphorylation of p38 in a sustained and very strong manner. ERK1/2 and JNK1/2 were not affected in fibroblasts. Inhibition of JNK1/2 and p38 under coculture conditions reduced acidosis-induced changes in fibroblasts significantly.ConclusionsOur data show that the crosstalk between proximal tubule cells and fibroblasts is crucial for acidosis-induced dedifferentiation of fibroblasts into an inflammatory phenotype. This dedifferentiation is at least in part mediated by p38 and JNK1/2. Thus, cell–cell communication is essential for the pathophysiological impact of tubulointerstitial acidosis.

The Effect of Chrysin on the Distribution of Extracellular Matrix Proteins around Leydig Cells under Heat Stress Injury

Objective. In the testicular tissue stroma, matrix proteins such as laminin and fibronectin play a critical role in transmitting signals under normal and stressful conditions to Leydig cells. Heat stress is considered one of the stressful conditions in the testicular tissue. This study has investigated the protective effects of chrysin on the distribution of laminin and fibronectin around Leydig cells under heat stress. Methods. Rats were divided into four groups, including sham (A), heat stress (B), heat stress with chrysin treatment (50 mg/kg) (C), and heat stress with chrysin treatment (75 mg/kg) (D). At the end of the treatment period, on the 21st day, testosterone serum levels were evaluated, and the testes were also assessed to determine the changes in gene expression and distribution of laminin, fibronectin, and caspase-3 proteins. Results. Exposure to heat stress in Group B resulted in increased levels of caspase-3, decreased activity, height, and diameter of seminiferous tubules, and altered distribution of laminin and fibronectin proteins around the Leydig cells when compared to the sham group (p<0.05). Treatment with chrysin 75 resulted in a significant decrease in caspase-3 levels, improved testosterone levels, and increased distribution of laminin and fibronectin around Leydig cells compared to group B (p<0.05). In addition, the group treated with chrysin 75 showed improved seminiferous histological activity markers in comparison to group B (p<0.05). Conclusion. Following heat stress, concurrent with disrupted steroidogenesis, an evident change happens in the distribution pattern of the extracellular matrix (ECM) proteins of the testis, which may indicate the transmission of stress signals from the ECM to the Leydig cells. Treatment of chrysin in 75 mg/kg dose can reduce the damage caused by heat stress on the ECM and Leydig cells.

TGF-β2-induced alterations of m6A methylation in hTERT RPE-1 cells

N6-methyladenosine (m6A) is a major type of RNA modification implicated in various pathophysiological processes. Transforming growth factor β2 (TGF-β2) induces epithelial-mesenchymal transition (EMT) in retinal pigmental epithelial (RPE) cells and promotes the progression of proliferative vitreoretinopathy (PVR). However, the role of m6A methylation in the EMT of human telomerase reverse transcriptase (hTERT) retinal pigmental epithelium (RPE)-1 cells has not been clarified. Here, we extracted RNA from RPE cells subjected to 0 or 20 ng/mL TGF-β2 for 72 h and identified differentially methylated genes (DMGs) by m6A-Seq and differentially expressed genes (DEGs) by RNA-Seq. We selected the genes related to EMT by conjoint m6A-Seq/RNA-Seq analysis and verified them by qRT-PCR. We then confirmed the function of m6A methylation in the EMT of RPE cells by knocking down the methyltransferase METTL3 and the m6A reading protein YTHDF1. Sequencing yielded 5814 DMGs and 1607 DEGs. Conjoint analysis selected 467 genes altered at the m6A and RNA levels that are closely associated with the EMT-related TGF-β, AGE-RAGE, PI3K-Akt, P53, and Wnt signaling pathways. We also identified ten core EMT genes ACTG2, BMP6, CDH2, LOXL2, SNAIL1, SPARC, BMP4, EMP3, FOXM1, and MYC. Their RNA levels were evaluated by qRT-PCR and were consistent with the sequencing results. We observed that METTL3 knockdown enhanced RPE cell migration and significantly upregulated the EMT markers N-cadherin (encoded by CDH2), fibronectin (FN), Snail family transcription repressor (SLUG), and vimentin. However, YTHDF1 knockdown had the opposite effects and decreased both cell migration and the N-cadherin, FN, and SLUG expression levels. The present study clarified TGF-β2-induced m6A- and RNA-level differences in RPE cells, indicated that m6A methylation might regulate EMT marker expression, and showed that m6A could regulate TGF-β2-induced EMT.

Effect of Spray-Type Alginate Hydrogel Dressing on Burn Wounds.

Immediate burn wound care is a critical factor influencing the outcomes of burn treatment. In this study, we developed a spray-type alginate hydrogel dressing that promotes wound healing, reduces pain, and increases the convenience of use in a burn treatment emergency. We investigated the efficiency of newly developed spray-type alginate hydrogel dressing on the wound healing process. We investigated the efficacy of the alginate hydrogel dressing for wound healing in 30 Sprague Dawley rats. Four deep, round second-degree burn wounds (diameter, 1.5 cm) were created bilaterally on the dorsum of the rat's trunk; the rats were divided into four groups, in which different dressing materials were applied as follows: group A, gauze (control); group B, Mepilex™ (control); group C, 2.25% alginate hydrogel; and group D, 2.5% alginate hydrogel. The gross findings of each group were compared by tracing the remaining wound and performing visual and histological observations and biochemical analysis for proteins associated with wound healing at each time period. In burn wounds, groups C and D showed significantly higher contraction, epithelialization, and healing rates. Histologically, groups C and D showed an improved arrangement of collagen fibers and a thick epithelial layer 14 days after initial wound formation. Group C showed higher CD31, TGF-β, and fibronectin expression in Western blot analyses after day 14. This study suggests that the spray-type alginate hydrogel dressing is an effective material for initial burn wound care.

Programming temporal stiffness cues within extracellular matrix hydrogels for modelling cancer niches

Extracellular matrix (ECM) stiffening is a common occurrence during the progression of many diseases, such as breast cancer. To accurately mimic the pathophysiological context of disease within 3D in vitro models, there is high demand for smart biomaterials which replicate the dynamic and temporal mechanical cues of diseased states. This study describes a preclinical disease model, using breast cancer as an example, which replicates the dynamic plasticity of the tumour microenvironment by incorporating temporal (3-week progression) biomechanical cues within a tissue-specific hydrogel microenvironment. The composite hydrogel formulation, integrating adipose-derived decellularised ECM (AdECM) and silk fibroin, was initially crosslinked using a visible light-mediated system, and then progressively stiffened through spontaneous secondary structure interactions inherent between the polymer chains (∼10–15 kPa increase, with a final stiffness of 25 kPa). When encapsulated and cultured in vitro, MCF-7 breast cancer cells initially formed numerous, large spheroids (>1000 μm2 in area), however, with progressive temporal stiffening, cells demonstrated growth arrest and underwent phenotypic changes resulting in intratumoral heterogeneity. Unlike widely-investigated static mechanical models, this stiffening hydrogel allowed for progressive phenotypic changes to be observed, and fostered the development of mature organoid-like spheroids, which mimicked both the organisation and acinar-structures of mature breast epithelium. The spheroids contained a central population of cells which expressed aggressive cellular programs, evidenced by increased fibronectin expression and reduction of E-cadherin. The phenotypic heterogeneity observed using this model is more reflective of physiological tumours, demonstrating the importance of establishing temporal cues within preclinical models in future work. Overall, the developed model demonstrated a novel strategy to uncouple ECM biomechanical properties from the cellular complexities of the disease microenvironment and offers the potential for wide applicability in other 3D in vitro disease models through addition of tissue-specific dECM materials.