Upregulation Of TGF-β1 Research Articles

Mesenchymal Stem Cell-conditioned Medium Protecting Renal Tubular Epithelial Cells by Inhibiting Hypoxia-inducible Factor-1α and Nuclear Receptor Coactivator-1.

Acute kidney injury (AKI) is characterized by inflammatory infiltration and damage and death of renal tubular epithelial cells (RTECs), in which hypoxia plays an important role. Deferoxamine (DFO) is a well-accepted chemical hypoxia-mimetic agent. Mesenchymal stem cell-conditioned medium (MSC-CM) can reduce local inflammation and repair tissue. In this study, we explored the effect and molecular mechanism of MSC-CM-mediated protection of RTECs under DFO-induced hypoxia. Rat renal proximal tubule NRK-52E cells were treated with different concentrations of DFO for 24 hours, followed by evaluation of RTEC injury, using a Cell Counting Kit-8 (CCK-8) to detect cell viability and western blotting to evaluate the expression of transforming growth factor- beta 1 (TGF-β1), α-smooth muscle actin (α-SMA), and hypoxia-inducible factor-1 alpha (HIF-1α) in NRK-52E cells. Then, three groups of NRK-52E cells were used in experiments, including normal control (NC), 25 μM DFO, and 25 μM DFO + MSC-CM. MSC-CM was obtained from the human umbilical cord. MSC-CM was used to culture cells for 12 hours before DFO treatment, then fresh MSC-CM and 25 μM DFO were added, and cells were cultured for another 24 hours before analysis. Western blotting and cellular immunofluorescence staining showed culture of NRK-52E cells in 25 μM DFO for 24 hours induced HIF-1α and nuclear receptor coactivator-1 (NCoA-1), simulating hypoxia. MSC-CM could inhibit the DFO-induced up-regulation of α-SMA, TGF-β1, HIF-1α and NCoA-1. Our results suggest that MSC-CM has a protective effect on RTECs by down-regulating HIF-1α and NCoA-1, which may be the harmful factors in renal injury.

The mitigating role of lysophospholipids in hepatic lipid metabolism and intestinal immunity in juvenile black seabream (Acanthopagrus schlegelii) fed a high-fat diet

An 8-week feeding experiment investigated the effects of lysophospholipids (LP) on growth performance, hepatic lipid metabolism, intestinal immunity and antioxidation in juvenile black seabream (Acanthopagrus schlegelii) (initial weight 4.60 ± 0.01 g) on a high-fat diet. Five experimental diets were established: normal fat diet (13 %, CON), high-fat diet (18 %, HFD) and HFD with different levels of LP (0.05 %, 0.1 %, and 0.2 %). The results indicated that dietary LP had no impact on the specific growth rate and survival rate of juvenile black seabream (P > 0.05), while 0.2 %LP supplementation reduced the intraperitoneal fat ratio (IPF) (P < 0.05). Dietary 0.1 % and 0.2 %LP supplementation reduced the total lipid content and the size and number of lipid droplets in the liver (P < 0.05). The diets supplemented with LP reduced the contents of TG and LDL-C in the serum (P < 0.05), without affecting T-CHO and HDL-C (P > 0.05). The expression of lipid transport genes including apoa, apob, fatp1, and fatp4, were markedly up-regulated with increased dietary LP supplementation (P < 0.05). Moreover, dietary 0.2 %LP supplementation notably increased mRNA expression levels of scarb1, ffar3, acsl5 and glp-1 in the intestine (P < 0.05). Dietary LP notably down-regulated the mRNA expression of genes related to the lipogenesis pathway including fas, aco and srebp-1c, while up-regulating mRNA expression of lipogenesis-related genes including cpt1α, lpl, and pparα (P < 0.05). Additionally, dietary 0.2 %LP supplementation notably increased total antioxidant capacity in the intestine and up-regulated expression levels of intestinal antioxidant genes, including mn sod, cu-zn sod, gpx, and cat (P < 0.05), fish fed diets with 0.2 %LP down-regulation of pro-inflammatory genes, including tnfα and nf-κb, and up-regulation of anti-inflammatory factors, tgfβ-1, il-10 and tight-junction protein genes including (oclna, cldni, cldn3, and tjp1b) in the intestine (P < 0.05). In conclusion, dietary LP improved intestinal fatty acid transport and liver lipolysis, alleviating intestinal oxidative damage and inflammatory reactions caused by HFD in black seabream.

Cancer Cell-Selective Inhibition of Migration by Styrenic Catiomer Emulsions.

Cancer metastasis remains the most formidable cause of mortality and morbidity in cancer patients. Developing an effective and economical method toward cancer antimetastatic strategy demands immediate attention in anticancer therapy. Herein, we followed a cost-effective greener method for preparing a small family of amphiphilic catiomers with varied styrene content (45, 63, and 83%), which revealed the unique potential of promoting normal cell migration while retarding cancer metastasis. The styrenic polymers formed micellar self-assembly in aqueous phase and exhibited a cationic charge. Polymers were quite nontoxic up to 200 μg/mL concentration toward human embryonic kidney cell HEK293 as well as human, triple negative breast cancer cell MDAMB-231, mouse melanoma cell B16F10, and human oral squamous carcinoma cell FaDu. Confocal imaging and fluorescence activated cell sorting (FACS) showed effective incorporation of polymers within cells. Interestingly, the polymer-treated HEK293 cells underwent prominent wound healing in scratch assay. However, the as-synthesized polymer-treated cancer cells resisted migration as analyzed from the scratch assay. A mechanistic study using immunoblotting assay established upregulation of migratory proteins vimentin and TGF-β and downregulation of E-cadherin in normal HEK293 cells. Remarkably, this trend was completely reversed in cancer cell MDAMB-231. This study describes the extraordinary potential of styrenic catiomers as wound healers for normal cells while inhibiting cancer metastasis.

The Role of TGF-β1 and Mutant SMAD4 on Epithelial-Mesenchymal Transition Features in Head and Neck Squamous Cell Carcinoma Cell Lines.

The aim of the present study was to investigate possible differences in the sensitivity of HNSCC cells to known EMT regulators. Three HNSCC cell lines (UM-SCC-1, -3, -22B) and the HaCaT control keratinocyte cell line were exposed to transforming growth factor beta 1 (TGF-β1), a known EMT master regulator, and the cellular response was evaluated by real-time cell analysis (RTCA), Western blot, quantitative PCR, flow cytometry, immunocytochemistry, and the wound closure (scratch) assay. Targeted sequencing on 50 cancer-related genes was performed using the Cancer Hotspot Panel v2. Mutant, and wild type SMAD4 cDNA was used to generate recombinant SMAD4 constructs for expression in mammalian cell lines. The most extensive response to TGF-β1, such as cell growth and migration, β-actin expression, or E-cadherin (CDH1) downregulation, was seen in cells with a more epithelial phenotype. Lower response correlated with higher basal p-TGFβ RII (Tyr424) levels, pointing to a possible autocrine pre-activation of these cell lines. Targeted sequencing revealed a homozygous SMAD4 mutation in the UM-SCC-22B cell line. Furthermore, PCR cloning of SMAD4 cDNA from the same cell line revealed an additional SMAD4 transcript with a 14 bp insertion mutation, which gives rise to a truncated SMAD4 protein. Overexpression of this mutant SMAD4 protein in the highly epithelial control cell line HaCaT resulted in upregulation of TGF-β1 and vimentin. Consistent with previous reports, the invasive and metastatic potential of HNSCC tumor cells appears associated with the level of autocrine secretion of EMT regulators such as TGF-β1, and it could be influenced by exogenous EMT cytokines such as those derived from immune cells of the tumor microenvironment. Furthermore, mutant SMAD4 appears to be a significant contributor to the mesenchymal transformation of HNSCC cells.

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.

Mechanism of cigarette smoke in promoting small airway remodeling in mice via STAT 3 / PINK 1-Parkin / EMT

BackgroundAirway remodeling is an important pathological of airflow limitation in chronic obstructive pulmonary disease (COPD).However,its mechanism still needs to be further clarify. MethodsAnimals:Healthy male C57BL/6 mice aged 4–6 weeks were randomly divided into control group and cigarette smoke(CS)group. Mice in the CS group were placed in a homemade glass fumigator, 5 cigarettes/time, 40 min/time, 4 times/day, 5 days/week, for 24 weeks. Mice in the control group were placed in a normal air environment.Cells:BEAS-2B cells were stimulated with 0.1%cigarette smoke extract(CSE).HE staining, immunohistochemical staining and Masson staining were used to observe the pathological of lung tissues, transmission electron microscopy was used to observe the structural of mitochondria in bronchial epithelial cells.Western blotting was used to detect the expression of STAT3,transforming growth factor-β1(TGF-β1),microtubule-associated protein 1A/1B-light chain3(LC3),PINK1,Parkin,E-cadherin,zonula occludens1(ZO-1),vimentin and snail family transcriptional inhibitor1 (Snail1),and MitoSOX Red was used to detect mitochondrial reactive oxygen species(mtROS). ResultsCS exposure causes lung parenchymal destruction and airway remodeling in mice.Compared to the control group,the expression of p-STAT3,TGF-β1 and EMT in the whole lung homogenate of the CS group was increased.Mitochondrial architecture disruption in bronchial epithelial cells of CS mice, with impaired PINK1-Parkin-dependent mitophagy.In vitro experiments showed that CSE exposure led to STAT3 activation, increased TGF-β1,EMT and enhanced PINK1-Parkin-mediated mitophagy.STAT3 inhibition reversed TGF-β1 upregulation induced by CSE and improved CSE-induced EMT and mitophagy.Inhibition of mitophagy improves EMT induced by CSE. Inhibition of mitophagy reduces STAT3-induced EMT. ConclusionCS activates the STAT3,and activated STAT3 promotes EMT in bronchial epithelial cells by enhancing PINK1-Parkin-mediated mitophagy and TGF-β1 signaling.Moreover, activated STAT3 can promote EMT directly.This may be one of the mechanisms by which CS causes small airway remodeling in COPD.

Iron-laden macrophage-mediated paracrine profibrotic signaling induces lung fibroblast activation.

Idiopathic pulmonary fibrosis (IPF) is a devastating condition characterized by progressive lung scarring and uncontrolled fibroblast proliferation, inevitably leading to organ dysfunction and mortality. Although elevated iron levels have been observed in patients and animal models of lung fibrosis, the mechanisms linking iron dysregulation to lung fibrosis pathogenesis, particularly the role of macrophages in orchestrating this process, remain poorly elucidated. Here we evaluate iron metabolism in macrophages during pulmonary fibrosis using both in vivo and in vitro approaches. In murine bleomycin- and amiodarone-induced pulmonary fibrosis models, we observed significant iron deposition and lipid peroxidation in pulmonary macrophages. Intriguingly, the ferroptosis regulator glutathione peroxidase 4 (GPX4) was upregulated in pulmonary macrophages following bleomycin instillation, a finding corroborated by single-cell RNA sequencing analysis. Moreover, macrophages isolated from fibrotic mouse lungs exhibited increased transforming growth factor (TGF)-β1 expression that correlated with lipid peroxidation. In vitro, iron overload in bone marrow-derived macrophages triggered lipid peroxidation and TGF-β1 upregulation, which was effectively suppressed by ferroptosis inhibitors. When cocultured with iron-overloaded macrophages, lung fibroblasts exhibited heightened activation, evidenced by increased α-smooth muscle actin and fibronectin expression. Importantly, this profibrotic effect was attenuated by treating macrophages with a ferroptosis inhibitor or blocking TGF-β receptor signaling in fibroblasts. Collectively, our study elucidates a novel mechanistic paradigm in which the accumulation of iron within macrophages initiates lipid peroxidation, thereby amplifying TGF-β1 production, subsequently instigating fibroblast activation through paracrine signaling. Thus, inhibiting iron overload and lipid peroxidation warrants further exploration as a strategy to suppress fibrotic stimulation by disease-associated macrophages. NEW & NOTEWORTHY This study investigates the role of iron in pulmonary fibrosis, specifically focusing on macrophage-mediated mechanisms. Iron accumulation in fibrotic lung macrophages triggers lipid peroxidation and an upregulation of transforming growth factor (TGF)-β1 expression. Coculturing iron-laden macrophages activates lung fibroblasts in a TGF-β1-dependent manner, which can be mitigated by ferroptosis inhibitors. These findings underscore the potential of targeting iron overload and lipid peroxidation as a promising strategy to alleviate fibrotic stimulation provoked by disease-associated macrophages.

TGF-β Signaling-Mediated Upregulation of Podoplanin: A Promising Biomarker for Diagnosis and Prognosis in Acute Promyelocytic Leukemia

TGF-β Signaling-Mediated Upregulation of Podoplanin: A Promising Biomarker for Diagnosis and Prognosis in Acute Promyelocytic Leukemia

Expression of TGFβ-1 and CTGF in the Implanted Cochlea and its Implication on New Tissue Formation.

Transforming growth factor beta-1 (TGFβ-1) and connective tissue growth factor (CTGF) are upregulated in the implanted human cochlea. Cochlear implantation can lead to insertion trauma and intracochlear new tissue formation, which can detrimentally affect implant performance. TGFβ-1 and CTGF are profibrotic proteins implicated in various pathologic conditions, but little is known about their role in the cochlea. The present study aimed to characterize the expression of these proteins in the human implanted cochlea. Archival human temporal bones (HTB) acquired from 12 patients with previous CI and histopathological evidence of new tissue formation as well as surgical samples of human intracochlear scar tissue surrounding the explanted CI were used in this study. Histopathologic analysis of fibrosis and osteoneogenesis was conducted using H&E. Protein expression was characterized using immunofluorescence. RNA expression from surgical specimens of fibrotic tissue surrounding the CI was quantified using qRT-PCR. TGFβ-1 and CTGF protein expressions were upregulated in the areas of fibrosis and osteoneogenesis surrounding the CI HTB. Similarly, surgical samples demonstrated upregulation of protein and mRNA expression of TGFβ-1 and mild upregulation of CTGF compared with control. TGFβ-1 was expressed diffusely within the fibrous capsule, whereas CTGF was expressed in the thickened portion toward the modiolus and the fibrosis-osteoneogensis junction. To our knowledge, this is the first study to demonstrate increased expression of TGFβ-1 and CTGF in the human implanted cochlea and may provide better understanding of the mechanism behind this pathogenic process to better develop future mitigating interventions.

Local vascular Klotho mediates diabetes-induced atherosclerosis via ERK1/2 and PI3-kinase-dependent signaling pathways

Background and aimsDiabetes is one of the major causes of cardiovascular disease (CVD). As high as 29 % of patients with diabetes develop atherosclerosis. Vascular Smooth Muscle Cells (VSMCs) are a key mediator in the pathogenesis of atherosclerosis, generating pro-inflammatory and proliferative characteristics in atherosclerotic lesions. MethodsWe used human atherosclerotic samples, developed diabetes-induced atherosclerotic mice, and generated loss of function and gain of function in Klotho human aortic smooth muscle cells to investigate the function of Klotho in atherosclerosis. ResultsWe found that Klotho expression is decreased in smooth muscle actin-positive cells in patients with diabetes and atherosclerosis. Consistent with human data, we found that Apoe knockout mice with streptozotocin-induced diabetes fed on a high-fat diet showed decreased expression of Klotho in SMCs. Additionally, these mice showed increased expression of TGF-β, MMP9, phosphorylation of ERK and Akt. Further, we utilized primary Human Aortic Smooth Muscle Cells (HASMCs) with d-glucose under dose-response and in time-dependent conditions to study the role of Klotho in these cells. Klotho gain of function and loss of function studies showed that Klotho inversely regulated the expression of atherosclerotic markers TGF-β, MMP2, MMP9, and Fractalkine.Further, High Glucose (HG) induced Akt, and ERK1/2 phosphorylation were enhanced or mitigated by endogenous Klotho deficiency or its overexpression respectively. PI3K/Akt and MAPK/ERK inhibition partially abolished the HG-induced upregulation of TGF-β, MMP2, MMP9, and Fractalkine. Additionally, Klotho knockdown increased the proliferation of HASMCs and enhanced α-SMA and TGF-β expression. ConclusionsTaken together, these results indicate that local vascular Klotho is involved in diabetes-induced atherosclerosis, which is via PI3K/Akt and ERK1/2-dependent signaling pathways.

TGFβ in Pancreas and Colorectal Cancer: Opportunities to Overcome Therapeutic Resistance.

TGFβ is a pleiotropic signaling pathway that plays a pivotal role in regulating a multitude of cellular functions. TGFβ has a dual role in cell regulation where it induces growth inhibition and cell death; however, it can switch to a growth-promoting state under cancerous conditions. TGFβ is upregulated in colorectal cancer and pancreatic cancer, altering the tumor microenvironment and immune system and promoting a mesenchymal state. The upregulation of TGFβ in certain cancers leads to resistance to immunotherapy, and attempts to inhibit TGFβ expression have led to reduced therapeutic resistance when combined with chemotherapy and immunotherapy. Here, we review the current TGFβ inhibitor drugs in clinical trials for pancreatic and colorectal cancer, with the goal of uncovering advances in improving clinical efficacy for TGFβ combinational treatments in patients. Furthermore, we discuss the relevance of alterations in TGFβ signaling and germline variants in the context of personalizing treatment for patients who show lack of response to current therapeutics.

Network pharmacology, molecular docking study, and in vivo validation of the wound healing activity of the Red Sea soft coral Paralemnalia thyrsoides (Ehrenberg 1834) ethanolic extract and bioactive metabolites

BackgroundWounds are a major health issue on a global scale, putting a great deal of financial, commercial, and social strain on healthcare organizations, patients, and individuals. So, this study aims to investigate the in vitro antioxidant activity of Paralemnalia thyrsoides soft coral total ethanolic extract. Also, bio-guided in vivo wound healing validation enhanced by the evaluation of related gene expression of Paralemnalia thyrsoides total extract, derived fractions, and three known metabolites was done. Furthermore, utilizing network pharmacology, we identified ten hub target genes associated with wound healing, including AKT1(RAC-alpha serine/threonine–protein kinase), IL6 (interleukin-6), MAPK3 (mitogen-activated protein kinase 3), MMP9 (matrix metalloproteinase 9), and APP (amyloid P protein precursor). We conducted molecular docking to assess how the three compounds interact with these hub genes and inflammatory cytokines (IL-1β (interleukin-1 beta), TGF-β (transforming growth factor-beta), TNF-α (tumor necrosis factor-alpha), and NF-KB (nuclear factor-kappa B) linked to wound healing.ResultsIn vitro antioxidant activity of the total ethanolic extract of Paralemnalia thyrsoides revealed potent scavenging activity against H2O2 with IC50 of 178.62 μg/mL. Additionally, the bio-guided scheme of the in vivo wound healing assay leads to the most active fraction, petroleum ether, with a healing percentage of 85% ± 4. Several chromatographic procedures upon petroleum ether fraction led to the isolation of three known compounds with significant in vivo wound healing potential which are recognized as triacontan-1-ol (1), 24-methylcholesterol (2) 6α-acetyl-7α-acetate-1(10)-α-13-nornardosine [C16H24O4] (3). Noteworthy downregulation in Cox-2 (Cyclooxygenase-2), Cox-1 (Cyclooxygenase-1), IL-1β, TGF-β, TNF-α, NF-KB, and INF-ϒ (interferon-ϒ) relative genes expression and upregulation in TGF-β, and IL-10 (interleukin-10) relative genes expression proved that compounds (3), (2), and (1) were, respectively, significant. The in silico study suggests that both C16H24O4 and 24-methyl cholesterol have potential in wound healing, possibly involving the regulation of RAC-alpha serine/threonine-protein kinase (AKT1).ConclusionOur study highlights the antioxidant and wound-healing potential of Paralemnalia thyrsoides soft coral that can be attributed to its diverse chemical metabolites. The in vivo and in silico findings highlighted that P. thyrsoides can be an effective remedy for wound restoration with the need for extensive future detailed clinical studies to prove these results.

Final analysis of the BIMES trial, a phase II single arm study assessing efficacy and safety of bintrafusp alfa in previously treated advanced malignant pleural mesothelioma (GECP 20/09).

8084 Background: Transforming growth factor β (TGF-β) is involved in tumor immune evasion and epithelial–mesenchymal transition (EMT). As TGF-β upregulation and EMT are associated with resistance to anti-PD1 therapy, we evaluated the efficacy of bintrafusp alfa, a bifunctional fusion protein targeting TGF-β and PD-L1, in malignant pleural mesothelioma (MPM). Methods: Patients with advanced MPM who were previously treated with platinum-based chemotherapy were enrolled and received bintrafusp alfa 1200mg/m2 every 2 weeks until progression or for a maximum of two years. The primary objective was to determine the progression-free survival (PFS) by modified RECIST v1.1. assessed by the investigators. The expected median PFS was 4.5 months. Secondary objectives were overall response rate, duration of response, overall survival (OS) and safety. Results: Between October 2021 to March 2023, 47 patients were enrolled. Mean age was 70 years (41-84) and 36 (78.3%) were males. Most patients had epithelioid histology (82.9%) and had received only 1 prior line of therapy (84.8%). With a median follow-up of 11.5 months, 43 patients had disease progression and 24 patients died. The median number of doses administered was 4 (1-21) and reasons for treatment discontinuation were disease progression (82.6%), toxicity (6.5%), investigator decision (4.4%) and death (6.5%) by tumor progression. The median PFS was 1.9 months (95% CI 1.7 – 5.4) and the 6 month-PFS rate was 15.9%. Disease control rate was 34.8% (95% CI 22.2-49.9) consisting of 2 patients with partial response and 13 patients with stable disease as best response. The median OS was 11.9 months (95% CI 4.4 – not reached) and 6, 12 and 18-month OS rate was 65.3%, 46.5% and 26.6%, respectively. No significant differences in OS and PFS were observed based on MPM histological subtype. Grade 3-4 treatment-related adverse events occurred in 16 (34%) patients, anemia being the most common (n = 5); skin toxicity (n = 3); colitis (n = 2); adrenal insufficiency, acute kidney injury, allergic reaction, lipase, and amylase increased (n = 1 each). Conclusions: Bintrafusp alfa did not reach the expected efficacy in patients with advanced malignant pleural mesothelioma previously treated with platinum-based chemotherapy. Clinical trial information: NCT05005429 .

GATA3 and TGF-β in normal placenta and pre-eclampsia

GATA3 plays critical roles in the development and function of various tissues and organs throughout the body. Likewise, TGF-β signaling is critical for placental development and can interact with GATA3. We aimed to investigate the involvement of the multifunctional cytokine and transcription factor in trophoblast development. By using immunohistochemistry, we evaluated the localization and expression level of GATA3 and TGF-β in placentas at term of normal pregnancy and with pre-eclampsia. Up-regulation of both GATA3 and TGF-β was observed in pathological placentas, with localization in the villus epithelium (syncytiotrophoblast) stroma and decidua. Our data show altered expression of TGF-β and GATA3, which downstream could lead to a cascade of events that negatively influence trophoblast development and contribute to the pathogenesis of pre-eclampsia.

Comprehensive analysis of m6A modification in immune infiltration, metabolism and drug resistance in hepatocellular carcinoma

N6-methyladenosine (m6A) is important in regulating mRNA stability, splicing, and translation, and it also contributes to tumor development. However, there is still limited understanding of the comprehensive effects of m6A modification patterns on the tumor immune microenvironment, metabolism, and drug resistance in hepatocellular carcinoma (HCC). In this study, we utilized unsupervised clustering based on the expression of 23 m6A regulators to identify m6A clusters. We identified differential m6A modification patterns and characterized m6A-gene-cluster A, which exhibited poorer survival rates, a higher abundance of Treg cells, and increased expression of TGFβ in the tumor microenvironment (TME). Additionally, m6A-gene-cluster A demonstrated higher levels of glycolysis activity, cholesterol metabolism, and fatty acid biosynthesis. We also found that the m6A score was associated with prognosis and drug resistance. Patients with a low m6A score experienced worse prognoses, which were linked to an abundance of Treg cells, upregulation of TGFβ, and increased metabolic activity. HCC patients with a higher m6A score showed improved prognosis following sorafenib treatment and immunotherapy. In conclusion, we reveals the association between m6A modification patterns and the tumor immune microenvironment, metabolism, and drug resistance in HCC. Furthermore, the m6A score holds potential as a predictive factor for the efficacy of targeted therapy and immunotherapy in HCC.

Abstract 5122: Modulating anti-GD2 immunotherapy via dual DFMO and TGF-β inhibition in neuroblastoma

Abstract Neuroblastoma (NB) is an often-lethal childhood solid tumor. Anti-GD2 immunotherapy improves survival but not all responses are durable. Transforming growth factor-beta (TGFβ) has been implicated in solid tumor immunoresistance. High-risk NBs harbor activated MYC or MYCN to coordinately expand intratumoral polyamines (PA), oncometabolites with immunomodulatory roles, to support tumor progression. Difluoromethylornithine (DFMO) is an inhibitor of ornithine decarboxylase, the rate-limiting enzyme in PA synthesis, and has shown activity in preclinical NB models, including TH-MYCN+/+ transgenic mice, and is in clinical trials in children. We sought to augment anti-GD2 therapy using DFMO and the small molecule TGFβ-receptor inhibitor, galunisertib. We randomized TH-MYCN+/+ mice in two experiments (10 therapy arms; n=6-10 mice/arm) to receive combinations of DFMO, galunisertib, and anti-GD2 antibody m14G2a and to assess impact on survival. We measured 770 genes using nCounter Immuno-Oncology 360 (Nanostring), 36 cytokines using xMap bead-arrays (Luminex), and tumor-infiltrating leukocytes using flow cytometry in harvested tumors. Cell line TGFβ signaling was evaluated in tissue culture. In vitro, galunisertib consistently suppressed the upregulated TGFβ-Smad2/3 signaling cascade in MYCN-amplified neuroblastoma cell lines. In vivo, DFMO alone extends mouse survival and increases intra-tumoral neutrophils and natural killer (NK) cells. These NK cells increased surface expression of activation receptors like NKG2D (p&amp;lt;0.05), while tumor cells increased surface expression of NK-ligands. Tumor cells also expressed increased latency-associated peptide (LAP; p&amp;lt;0.05), a TGFβ precursor, suggesting NK cells were poised to kill but restrained by TGFβ. Cytotoxicity assays showed reversal of this inhibition with TGFβR1 inhibitor galunisertib in vitro. In a proof-of-concept trial, DFMO and galunisertib (DFMO/gal) demonstrated a clinical anti-tumor synergistic effect. DFMO/gal treated tumors had upregulated NK cell activity genes and downregulated TGFβ signaling genes. Cytokine analysis confirmed DFMO induced upregulation of TGFβ1 and TGFβ2 (p&amp;lt;0.01) that was modulated by galunisertib. IL6, GCSF, and GMCSF were markedly downregulated in tumors treated with both DFMO and galunisertib (p&amp;lt;0.05). An expanded trial showed DFMO/gal added to m14G2a showed no significant survival benefit over the DFMO and m14G2a combination. Ultimately, DFMO creates a pro-inflammatory cellular and cytokine milieu in TH-MYCN+/+ NBs. DFMO and TGFβR1 inhibition via galunisertib activate NK cells further and decrease the anti-inflammatory cytokine IL6. Ongoing studies are further characterizing the gene expression and cytokine levels of the NB TME under these treatment conditions. Citation Format: Om H. Gandhi, Christina S. Turn, Kangning Liu, Annette T. Vu, Hamid Bassiri, Michael D. Hogarty. Modulating anti-GD2 immunotherapy via dual DFMO and TGF-β inhibition in neuroblastoma [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 5122.

Fast skin healing chitosan/PEO hydrogels: In vitro and in vivo studies

Due to its outstanding qualities, particularly when it takes the shape of hydrogels, chitosan is a well-known biological macromolecule with many applications. When chitosan hydrogels are modified with other polymers, the desirable function as skin regeneration hydrogels is compromised; nevertheless, the mechanical properties can be improved, which is crucial for commercialization. In this study, for the first time, bimetallic zinc silver metal-organic frameworks (ZAg MOF) loaded with ascorbic acid were added to chitosan/polyethylene oxide (PEO) based interpenetrating polymer network (IPN) hydrogels that were crosslinked with biotin to improve their antimicrobial activity, mechanical characteristics, and sustainable treatment of wounds. Significant changes in the microstructure, hydrophilicity level, and mechanical properties were noticed. Ascorbic acid release patterns were upregulated in an acidic environment pH (5.5) that mimics the initial wound pH. Impressive cell viability (98 %), antimicrobial properties, and almost full skin healing in a short time were achieved for the non-replaceable chitosan/PEO developed hydrogels. Enhancing the wound healing of the treated animals using the prepared CS/PEO hydrogel dressing was found to be a result of the inhibition of dermal inflammation via decreasing IL-1β, suppressing ECM degradation (MMP9), stimulating proliferation through upregulation of TGF-β and increasing ECM synthesis as it elevates collagen 1 and α-SMA contents. The findings support the implementation of developed hydrogels as antimicrobial hydrogels dressing for fast skin regeneration.

Empagliflozin and pirfenidone confer renoprotection through suppression of glycogen synthase kinase-3β and promotion of tubular regeneration in rats with induced metabolic syndrome

Metabolic syndrome (MetS) is largely coupled with chronic kidney disease (CKD). Glycogen synthase kinase-3β (GSK-3β) pathway drives tubular injury in animal models of acute kidney injury; but its contribution in CKD is still elusive. This study investigated the effect empagliflozin and/or pirfenidone against MetS-induced kidney dysfunction, and to clarify additional underpinning mechanisms particularly the GSK-3β signaling pathway. Adult male rats received 10%w/v fructose in drinking water for 20 weeks to develop MetS, then treated with either drug vehicle, empagliflozin (30 mg/kg/day) and/or pirfenidone (100 mg/kg/day) via oral gavage for subsequent 4 weeks, concurrently with the high dietary fructose. Age-matched rats receiving normal drinking water were used as controls. After 24 weeks, blood and kidneys were harvested for subsequent analyses. Rats with MetS showed signs of kidney dysfunction, structural changes and interstitial fibrosis. Activation of GSK-3β, decreased cyclinD1 expression and enhanced apoptotic signaling were found in kidneys of MetS rats. There was abundant alpha-smooth muscle actin (α-SMA) expression along with up-regulation of TGF-β1/Smad3 in kidneys of MetS rats. These derangements were almost alleviated by empagliflozin or pirfenidone, with evidence that the combined therapy was more effective than either individual drug. This study emphasizes a novel mechanism underpinning the beneficial effects of empagliflozin and pirfenidone on kidney dysfunction associated with MetS through targeting GSK-3β signaling which can mediate the regenerative capacity, anti-apoptotic effects and anti-fibrotic properties of such drugs. These findings recommend the possibility of using empagliflozin and pirfenidone as promising therapies for management of CKD in patients with MetS.

Mechanism underlying the anti-diabetic potential of bee venom as compared to bone marrow mesenchymal stem cells in the diabetic rat tongue.

Diabetes mellitus (DM) is a critical chronic metabolic disease. Several treatment modalities are currently under investigation. Both bee venom (BV) and bone marrow mesenchymal stem cells (BMSCs) can possibly offer an approach for treating type I diabetes. The aim of the present study was to investigate the mechanism underlying the anti-diabetic effect of BV as compared to BMSCs on the tongue mucosa of diabetic rats. A total of 52 male albino rats were used in the current study. The rats were randomly assigned into 4 groups: group 1 (control); group 2 (streptozocin (STZ)); group 3 (BV-treated); and group 4 (BMSC-treated). Diabetes mellitus was induced via an intraperitoneal (IP) injection of STZ in the rats from groups 2, 3 and 4. Following the diagnosis of DM, the rats in group 3 were injected with a daily dose of 0.5 mg/kg of BV, while the rats in group 4 were treated with a single injection of BMSCs. All rats were euthanized after 4 weeks, and their tongues were dissected and divided into halves. The right halves of the tongues were utilized for the histological examination, followed by morphometric analysis. In contrast, the left halves were used to detect the local gene expression of transforming growth factor beta 1 (TGF-β1) and vascular endothelial growth factor (VEGF). Group 2 revealed marked disruption in the morphology of the fungiform and filiform papillae, and atrophic epithelial changes in both dorsal and ventral surface epithelium as compared to other groups. Group 4 showed a significantly larger number of taste buds, and a higher gene expression of TGF-β1 and VEGF as compared to groups 2 and 3. Additionally, BV and BMSCs effectively increased the thickness of dorsal and ventral surface epithelium as compared to group 2. Treatment with BMSCs was associated with significant improvement in the morphology and number of lingual epithelial cells and taste buds in the tongues of diabetic rats as compared to BV-treated rats, which was due to the local upregulation of TGF-β1 and VEGF gene expression.

Wearable Magnetoelectric Stimulation for Chronic Wound Healing by Electrospun CoFe2O4@CTAB/PVDF Dressings.

Magnetoelectric stimulation is a promising therapy for various disorders due to its high efficacy and safety. To explore its potential in chronic skin wound treatment, we developed a magnetoelectric dressing, CFO@CTAB/PVDF (CCP), by electrospinning cetyltrimethylammonium bromide-modified CoFe2O4 (CFO) particles with polyvinylidene fluoride. Cetyltrimethylammonium bromide (CTAB) serves as a dispersion surfactant for CFO, with its quaternary ammonium cations imparting antibacterial and hydrophilic properties to the dressing. Electrospinning polarizes polyvinylidene fluoride (PVDF) molecules and forms a fibrous membrane with flexibility and breathability. With a wearable electromagnetic induction device, a dynamic magnetic field is established to induce magnetostrictive deformation of CFO nanoparticles. Consequently, a piezoelectric potential is generated on the surface of PVDF nanofibers to enhance the endogenous electrical field in the wound, achieving a cascade coupling of electric-magnetic-mechanical-electric effects. Bacteria and cell cultures show that 2% CTAB effectively balances antibacterial property and fibroblast activity. Under dynamic magnetoelectric stimulation, the CCP dressing demonstrates significant upregulation of TGF-β, FGF, and VEGF, promoting L929 cell adhesion and proliferation. Moreover, it facilitates the healing of diabetic rat skin wounds infected with Staphylococcus aureus within 2 weeks. Histological and molecular biology evaluations confirm the anti-inflammatory effect of CTAB and the accelerated formation of collagen and vessel by electrical stimulation. This work provides insights into the application of magnetoelectric stimulation in the healing of chronic wounds.