FGF-2 Research Articles

LHX2 facilitates the progression of nasopharyngeal carcinoma via activation of the FGF1/FGFR axis

BackgroundDistant metastasis and recurrence remain the main obstacle to nasopharyngeal carcinoma (NPC) treatment. However, the molecular mechanisms underlying NPC growth and metastasis are poorly understood.MethodsLHX2 expression was examined in NPC cell lines and NPC tissues using quantitative reverse transcription-polymerase chain reaction, western blotting and Immunohistochemistry assay. NPC cells overexpressing or silencing LHX2 were used to perform CCK-8 assay, colony-formation assay, EdU assay, wound-healing and invasion assays in vitro. Xenograft tumour models and lung metastasis models were involved for the in vivo assays. The Gene Set Enrichment Analysis (GSEA), ELISA assay, western blot, chromatin immunoprecipitation (ChIP) assay and Luciferase reporter assay were applied for the downstream target mechanism investigation.ResultsLIM-homeodomain transcription factor 2 (LHX2) was upregulated in NPC tissues and cell lines. Elevated LHX2 was closely associated with poor survival in NPC patients. Ectopic LHX2 overexpression dramatically promoted the growth, migration and invasion of NPC cells both in vitro and in vivo. Mechanistically, LHX2 transcriptionally increased the fibroblast growth factor 1 (FGF1) expression, which in turn activated the phosphorylation of STAT3 (signal transducer and activator of transcription 3), ERK1/2 (extracellular regulated protein kinases 1/2) and AKT signalling pathways in an autocrine and paracrine manner, thereby promoting the growth and metastasis of NPC. Inhibition of FGF1 with siRNA or FGFR inhibitor blocked LHX2-induced nasopharyngeal carcinoma cell growth, migration and invasion.ConclusionsOur study identifies the LHX2-FGF1-FGFR axis plays a key role in NPC progression and provides a potential target for NPC therapy.

Neutrophil membrane-coated therapeutic liposomes for targeted treatment in acute lung injury

Acute lung injury (ALI) is one of the most common comorbidities associated with sepsis and can lead to acute respiratory distress syndrome. Intense inflammatory response due to excessive activation and uncontrolled infiltration of neutrophils are the central processes in the development of sepsis-induced ALI. In this study, a biomimetic nanoplatform that is a neutrophil membrane-coated liposome-loaded acidic fibroblast growth factor (aFGF@NMLs), which can selectively target the inflamed lung and effectively alleviate sepsis-induced ALI via inflammation suppression, was constructed. In vitro findings revealed that aFGF@NMLs has pro-inflammatory cytokine binding capabilities and can promote cellular uptake, substantially attenuate inflammatory responses, and enhance cellular antioxidant capacity. The in vivo results show that aFGF@NMLs can specifically accumulate in injured lungs in ALI mice after intravenous injection, thereby reducing the secretion of pro-inflammatory cytokines, inhibiting pulmonary cell apoptosis, and promoting lung function recovery. In conclusion, aFGF@NMLs demonstrated anti-inflammatory effects, mitigated the progression of ALI, and contributed to the disease prognosis. This research offers an innovative strategy and concept for the clinical treatment of diseases related to pulmonary inflammation.

Fibroblast growth factor signalling influences homologous recombination-mediated DNA damage repair to promote drug resistance in ovarian cancer

BackgroundOvarian cancer patients frequently develop chemotherapy resistance, limiting treatment options. We have previously shown that individuality in fibroblast growth factor 1 (FGF1) expression influences survival and chemotherapy response.MethodsWe used MTT assays to assess chemosensitivity to cisplatin and carboplatin following shRNA-mediated knockdown or heterologous over-expression of FGF1 (quantified by qRT-PCR and immunoblot analysis), and in combination with the FGFR inhibitors AZD4547 and SU5402, the ATM inhibitor KU55933 and DNA-PK inhibitor NU7026. Immunofluorescence microscopy was used to quantify the FGF1-dependent timecourse of replication protein A (RPA) and γH2AX foci formation.ResultsPharmacological inhibition of FGF signalling reversed drug resistance in immortalised cell lines and in primary cell lines from drug-resistant ovarian cancer patients, while FGF1 over-expression induced resistance. Ataxia telangiectasia mutated (ATM) phosphorylation, but not DNA adduct formation was FGF1 dependent, following cisplatin or carboplatin challenge. Combining platinum drugs with the ATM inhibitor KU55933, but not with the DNA-PK inhibitor NU7026 re-sensitised resistant cells. FGF1 expression influenced the timecourse of damage-induced RPA and γH2AX nuclear foci formation.ConclusionDrug resistance arises from FGF1-mediated differential activation of high-fidelity homologous recombination DNA damage repair. FGFR and ATM inhibitors reverse platinum drug resistance, highlighting novel combination chemotherapy approaches for future clinical trial evaluation.

The EGFR-STYK1-FGF1 axis sustains functional drug tolerance to EGFR inhibitors in EGFR-mutant non-small cell lung cancer

Non-small cell lung cancer (NSCLC) patients harboring activating mutations in epidermal growth factor receptor (EGFR) are sensitive to therapy with EGFR tyrosine kinase inhibitors (TKI). Despite remarkable clinical responses using EGFR TKI, surviving drug tolerant cells serve as a reservoir from which drug resistant tumors may emerge. This study addresses the need for improved efficacy of EGFR TKI by identifying targets involved in functional drug tolerance against them. To this aim, a high-throughput siRNA kinome screen was performed using two EGFR TKI-sensitive EGFR-mutant NSCLC cell lines in the presence/absence of the second-generation EGFR TKI afatinib. From the screen, Serine/Threonine/Tyrosine Kinase 1 (STYK1) was identified as a target that when downregulated potentiates the effects of EGFR inhibition in vitro. We found that chemical inhibition of EGFR combined with the siRNA-mediated knockdown of STYK1 led to a significant decrease in cancer cell viability and anchorage-independent cell growth. Further, we show that STYK1 selectively interacts with mutant EGFR and that the interaction is disrupted upon EGFR inhibition. Finally, we identified fibroblast growth factor 1 (FGF1) as a downstream effector of STYK1 in NSCLC cells. Accordingly, downregulation of STYK1 counteracted the afatinib-induced upregulation of FGF1. Altogether, we unveil STYK1 as a valuable target to repress the pool of surviving drug tolerant cells arising upon EGFR inhibition. Co-targeting of EGFR and STYK1 could lead to a better overall outcome for NSCLC patients.

P-383 Endometrial scratching: A strategy to stimulate the angiogenesis aspect of implantation in unexplained, repeated implantation failure

Abstract Study question Does endometrial scratching serve as an angiogenic stimulator during implantation in unexplained, repeated implantation failure (uRIF) patients? Summary answer Endometrial scratching can reimburse and regulate genes involved in both endometrial angiogenesis and receptivity. What is known already The vascular endothelial growth factor (VEGF), as an angiogenic factor, is well-known for its function in a variety of physiological and pathological aspects of female reproduction. Following the embryo transfer in the In-Vitro Fertilization cycles, the most challenging clinical obstacle to overcome is RIF. Endometrial scratching is a technique considered as an option for enhancing the embryo implantation of uRIF patients by modulating the different cellular and molecular components regulating endometrial receptivity before and during implantation. Study design, size, duration Twenty uRIF patients were enrolled based on a randomized controlled trial (RCT) to study the expression of genes involved in endometrial angiogenesis and receptivity functions (VEGFR1, VEGFR2, COL18A1, E-cadherin, FGF1) following ES. Ten uRIF patients were randomly assigned to the intervention group (twice endometrial sampling in the follicular and luteal phases) and ten in the control group (only luteal phase sampling) prior to the ovarian stimulation cycle. Participants/materials, setting, methods Women who failed to conceive following three or more IVF/ICSI cycles, high-quality embryo transfer, and at least one blastocyst embryo transfer cycle met the inclusion criteria. Post-biopsy endometrial samples were divided evenly. One component was placed in 10% formalin for histology dating and another in RNALater for genomic analysis and preserved at -80 °C for RNA extraction. Main results and the role of chance The expression levels of VEGFR1 and VEGFR2 (both as the receptors of VEGF) were significantly increased after ES (P <0.05). The mRNA expression level of COL18A1 was significantly decreased after ES. COL18A1 is an inhibitor of VEGF which can impede VEGF-mediated signaling and endothelial cell proliferation, via blocking of its receptor specifically VEGFR2. The E-cadherin expression level was significantly decreased (P <0.05) in the endometrium of the intervention group after scratching. E-cadherin is a cell-cell adhesion molecule that has a pivotal role in maintaining normal epithelial architecture, establishing cell polarity, glandular differentiation, and morphogenesis. Data showed that Fibroblast Growth Factor1 (FGF1) expression level was significantly increased in the intervention group (P <0.05). FGF1 promotes the formation of blood vessels which improves endometrial trophoblastic interaction and affects embryo implantation. Limitations, reasons for caution To further understand the function of these changes, it is preferable to compare the data with endometrium from a fertile group, which is ongoing. Wider implications of the findings On the timeline of embryo implantation, angiogenic factors (e.g., VEGFR1 and VEGFR2), their inhibitors (e.g., E-cadherin and COL18A1), and activators (e.g., FGF1) work together to establish a dynamic environment responsible for endometrial receptivity, which has been compromised in the endometrium of RIF patients.ES can reimburse and regulate these angiogenesis processes. Trial registration number IRCT20210316050723N1

1384-P: Serum Fibroblast Growth Factor 1 Levels and Its Association with ß-Cell Function and Insulin Sensitivity in Adults with Glucose Intolerance

Objectives: Beneficial role of Fibroblast growth factor 1 (FGF1) in the regulation of glucose metabolism and adipose tissue remodeling was suggested in rodents. We investigated an association between serum FGF1 levels and metabolic parameters in adults with glucose intolerance. Research Designs and Methods: Serum FGF1 levels were examined using enzyme-linked immunosorbent assay in 92 individuals with glucose intolerance. Associations between serum FGF1 levels and metabolic parameters including body mass index (BMI) , waist circumference (WC) , glycated hemoglobin (HbA1c) and 75g oral glucose tolerance test-derived parameters including insulinogenic index (IGI) , Matsuda insulin sensitivity index (ISI) , and disposition index (DI) were examined. Results: Mean age was 49.8 years, and mean BMI was 30.7 kg/m2 of 92 participants. Among them, 87% had type 2 diabetes. There was no significant association between serum FGF1 level and BMI or WC by using censored regression analysis. On the other hand, serum FGF1 level was higher in patients with higher HbA1c (β=0.369, p=0.014) . This association was significant after adjusting for age, sex, and BMI (p=0.005) . Notably, serum FGF1 level was negatively associated with IGI and DI (β=−0.462, p=0.017; β=−0.469, p=0.014, respectively) , but not with ISI. Mean FGF1 concentrations were significantly lower with higher quartiles of IGI and DI (p for trend=0.035 and 0.015 for IGI and DI, respectively) . Conclusion: Serum concentration of FGF1 was significantly higher in individuals with higher blood glucose and lower insulin secretion, which suggests a possible role of FGF1 in the glucose metabolism, especially in the regulation of β cell function in humans. Disclosure J.Kim: None. J.Choi: None. S.Kim: None. N.Kim: None.

Differential Responses of Human Dental Pulp Stromal Cells to Bioceramic Materials: A Comparative In Vitro Study

The aim of this study was to compare the effects of white MTA-Angelus (wMTA), Biodentine® (Biodentine) and TotalFill® BC Root Repair MaterialTM putty (TotalFill) on human dental pulp stromal cells (hDPSCs) in vitro. hDPSCs were isolated from third molars of healthy young adults. Material elutes at different concentrations were prepared. Cells were exposed to the eluates for 1, 3, and 7 days. Cell proliferation was evaluated using 3-(4,5-dimethyl-thiazoyl)-2, 5-diphenyl-tetrazolium bromide assay. The expression of alkaline phosphatase (ALP), osteoprotegerin (OPG), osteocalcin (OC), collagen1A (Col1A), runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-1 (FGF-1), interleukin 6 (IL6), tumor necrosis factor alpha (TNFα), and interleukin-1-beta (IL1β) was determined by reverse transcription-polymerase chain reaction (RT-PCR). VEGF-A protein levels and ALP activity were quantified in the culture supernatant. Data were analyzed by two-way analysis of variance (ANOVA). p values <0.05 were considered statistically significant. hDPSC proliferation was decreased in a dose-related manner for all materials on day 3. The same effect was observed with wMTA and TotalFill on day 7. RT-PCR showed that Biodentine increased the expression of the osteogenic markers ALP, OPG, and OC. TotalFill decreased the ALP expression and activity, enhanced the production of angiogenic VEGF-A, and downregulated the inflammatory IL6 on day 7. Although the tested materials are used interchangeably in vital pulp therapy, the findings showed varied hDPSC responses. Biodentine did not affect cell proliferation and increased the expression of osteo-/odontogenic markers compared to wMTA and TotalFill, whereas TotalFill decreased cell proliferation and exhibited enhanced angiogenic and anti-inflammatory effects over time. The clinical significance of the results needs further investigation in an attempt to provide recommendations on the selection of bioceramic pulp capping material under different scenarios of pulpal pathosis.

F1A‐CreERT2 Mice: Targeting and Tracing Cardiomyocytes with Fgf1 Expression in Adult Mouse

Fibroblast growth factor 1 (FGF1) regulates many biological and homeostatic processes, including obesity and metabolic syndrome. In mice, Fgf1 gene contains at least three upstream promoters and their respective adjacent untranslated exons that spliced to the first protein coding exon mutually exclusively, giving rise to different Fgf1 mRNA variants. Among them, the Fgf1A transcript is predominantly expressed in the heart. FGF1 can induce cardiomyocyte regeneration and cardiogenesis in vitro and in vivo. Here, we generated a novel mouse line Fgf1A promoter (F1A)‐driven inducible Cre recombinase (CreERT2). We firstly demonstrated that the highest mRNA expression of CreERT2 were detected in the heart specifically of F1A‐CreERT2 mice, similar to that of Fgf1A mRNA. The F1A‐CreERT2 mice were crossed with ROSA26 mice, and the F1 mice were analyzed. The LacZ‐positive signals were detected exclusively in the heart after tamoxifen administration. The CreERT2‐mediated recombination in the tissues is monitored through LacZ‐positive signals, indicating the in situ localization of F1A‐positive cells. Consistently, these F1A‐positive cells with RFP‐positive signals or LacZ‐positive blue signals were co‐localized with cardiomyocytes expressing cardiac troponin T, suggesting cardiomyocyte‐specific activation of Fgf1A promoter. Our data suggested that the F1A‐CreERT2 mouse line could be used for time‐dependent and lineage tracing of Fgf1A‐expressing cells in vivo. F1A‐CreERT2 line is a valuable tool for in vivo lineage tracing of Fgf1A‐expressing cells during heart development and will facilitate the elucidation of the physiological and pathological role of Fgf1A in heart failure and metabolic syndrome.

Polydopamine modified acellular dermal matrix sponge scaffold loaded with a-FGF: Promoting wound healing of autologous skin grafts.

Despite increasing potentials as a skin regeneration template (DRT) to guide tissue healing, acellular dermal matrix (ADM) is still challenged by issues (like dense architecture, low cellular adhesion and poor vascularization), contributing to necrosis and shedding of upper transplanted skins. Modified with polydopamine (PDA), a novel and porous DRT capable of drug delivery was designed using porcine-derived ADM (PADMS) gels, termed PDA-PADMS. However, it was unclear whether it could efficiently deliver human acidic fibroblast growth factor (a-FGF) and regenerate skin defects. Herein, after being fabricated and optimized with PADMS gels in different ratios (1:6, 1:7, 1:8), PDA-PADMS loading a-FGF (PDA-PADMS-FGF) was evaluated by the morphology, physical& chemical properties, drug release and in-vitro biological evaluations, followed by full-thickness skin defects implanted with PDA-PADMS-FGF covered by transplanted skins. Apart from containing abundant collagen and elastin, porous PADMS (with a loose and uniform structure) was demonstrated to possess controlled release of a-FGF and biocompatibility attributed to PDA coating. Consistent with augmented cellular migration and proliferation in vitro, PDA-PADMS-FGF also accelerated wound healing and reduced scarring, improving collagen arrangement and neovascularization. In conclusion, PDA-PADMS-FGF has a good potential and application prospect as a matrix material for wound repair.

SDS‐induced amyloid fibrillation of acidic fibroblast growth factor

Fibroblast growth factors and their receptors regulate a broad range of biological functions from developmental processes during embryogenesis to various physiological roles in the adult state, including regulation of angiogenesis, wound healing, and metabolism. hFGF‐1 also plays a key role in neuroprotection against neurotoxicity in Alzheimer’s disease (AD). The anionic surfactant sodium dodecyl sulfate (SDS) is widely used as a model for induction of amyloid‐like fibrils. In this context, we examined the effects of SDS on FGF1 using a variety of biophysical techniques including, Transmission Electron Microscopy (TEM). The secondary structure of hFGF1 was monitored by incubating hFGF1 with different concentrations of SDS utilizing the far‐UV CD (190‐250 nm) spectroscopy. We found that from 0.08‐0.15mM concentration of SDS, the negative peak at ~206 shifted gradually towards ~212nm while at 0.2mM concentration, far‐UV CD spectra of hFGF‐1 exhibit significant changes with striking shifting of negative peak ~212 nm to ~218 nm and disappearance of positive band at ~228nm which signifies substantial loss of native β‐barrel structure. The aggregation of hFGF‐1 is detected systematically by observing the absorbance at 350nm. The results indicate that at 0.2mM SDS, hFGF1 highly aggregates. In addition to far‐UV CD spectra and turbidity measurement, the influence of SDS on the tertiary structural changes of hFGF1 was studied by mentoring the intrinsic fluorescence of hFGF1 incubated with various concentrations of SDS. The study reveals that 0.2mM SDS is the concentration that affect mostly on the tertiary structure of hFGF1 because of the highest ratio of 305 to 350 at this concentration. Thioflavin‐T (ThT) assay for quantification of Amyloid‐like fibril formation was used to investigate the concentration of SDS that induce maximum hfGF1 fibrils. This study showed that 0.2mM SDS is the concentration that induced the optimum formation of fibrils hFGF1. Based on the results of these experiments, we wanted to do more investigation to achieve more evidence about the effect of SDS on the fibrils hFGF1. Therefore, morphological study by TEM was employed to examine the effect of 0.2mM SDS and 0.6mM SDS on the formation of fibrils hFGF1. At incubation for 1‐hour fibrillar aggregates of hFGF‐1 with average diameter at about 20‐25nm is observed in presence of 0.2mM SDS. All the previous results using biophysical techniques indicate that SDS induces formation of fibrils hFGF1 concentration which assist to better understanding about the causative of the fibrils hFGF1 which lead to find a remedy for the disease related with it.

Synergistic effects of mechanical stimulation and crimped topography to stimulate natural collagen development for tendon engineering

Suitable scaffold structures and mechanical loading are essential for functional tendon engineering. However, the bipolar fibril structure of native tendon collagen is yet to be recaptured in engineered tendons. This study compared the development of Achilles tendons of postnatal rats with and without (via surgical section) mechanical loading to define the mechanism of mechanical stimulation-mediated tendon development. The results demonstrated that the severed tendons weakened mechanically and exhibited disorganization without a bipolar fibril superstructure. Proteomic analysis revealed differentially expressed key regulatory molecules related to the collagen assembly process, including decreased fibromodulin, keratocan, fibroblast growth factor-1, and increased lumican and collagen5a1 in the severed tendons with immunohistochemical verification. Additionally, a complex regulatory network of mechanical stimulation-mediated collagen assembly in a spatiotemporal manner was also revealed using bioinformatics analysis, wherein PI3K–Akt and HDAC4 may be the predominant signaling pathways. A wavy microgrooved surface (Y = 5.47sin(0.015x)) that biomimics tendon topography was observed to enhance the expression of collagen assembly molecules under mechanical loading, and the aforementioned pathways are particularly involved and verified with their respective inhibitors of LY-294002 and LMK-235. Furthermore, an electrospun crimped nanofiber scaffold (approximately 2 μm fiber diameter and 0.12 crimpness) was fabricated to biomimic the tenogenic niche environment; this was observed to be more effective on enhancing collagen production and assembly under mechanical stimulation. In conclusion, the synergistic effect between topographical niche and mechanical stimulation was observed to be essential for collagen assembly and maturation and should be applied to functional tendon engineering in the future. Statement of significanceIn biomaterial-mediated tendon regeneration, mechanical stimulation is essential for tendon collagen assembly. However, the underlying mechanisms remain not fully defined, leading to the failure of the native-like collagen regeneration. In this study, a mechanical stimulation deprivation model of rat tendon was established to reveal the mechanisms in tendon development and define the key regulatory molecules including small leucine-rich proteoglycans, lysyl oxidase and collagen V. After ensuring the importance of biomimetic structure in tendon remodeling, crimped nanofibers were developed to verify these regulatory molecules, and demonstrated that mechanical stimulation significantly enhanced collagen assembly via PIK3 and HDAC4 pathways in biomaterial-regulated tendon regeneration. This study provides more insightful perspectives in the physiologically remodeling progression of tendon collagen and design of tendon scaffolds.

Adipose tissue derived stromal cells in a gelatin-based 3D matrix with exclusive ascorbic acid signalling emerged as a novel neural tissue engineering construct: an innovative prototype for soft tissue.

The current study investigated a triad, which comprises of adipose tissue derived stem cells isolated from infrapatellar fat pad and gelatin/polyvinyl alcohol (PVA)-based matrix with exclusive ascorbic acid signalling. Though, the bio-mechanical properties of the gelatin–PVA blended scaffolds in wet condition are equivalent to the ECM of soft tissues in general, in this study, the triad was tested as a model for neural tissue engineering. Apart from being cytocompatible and biocompatible, the porosity of the scaffold has been designed in such a manner that it facilitates the cell signalling and enables the exchange of nutrients and gases. The highly proliferative stem cells from Passage 2 were characterized using both, mesenchymal and embryonic stem cell markers. As an initial exploration the mesenchymal stem cells at Passage 4 were exposed to ascorbic acid and basic fibroblast growth factor signalling for neuronal differentiation in 2D environment independently. The MSCs successfully differentiated and acquired neuron specific markers related to cytoskeleton and synapses. Subsequently, three phases of experiments have been conducted on the 3D gelatin/PVA matrix to prove their efficacy, the growth of stem cells, growth of differentiated neurons and the in situ growth and differentiation of MSCs. The scaffold was conducive and directed MSCs to neuronal lineage under specific signalling. Overall, this organotypic model triad could open a new avenue in the field of soft tissue engineering as a simple and effective tissue construct.

FGF1 Signaling Modulates Biliary Injury and Liver Fibrosis in the Mdr2-/- Mouse Model of Primary Sclerosing Cholangitis.

Fibroblast growth factor 1 (FGF1) belongs to a family of growth factors involved in cellular growth and division. MicroRNA 16 (miR‐16) is a regulator of gene expression, which is dysregulated during liver injury and insult. However, the role of FGF1 in the progression of biliary proliferation, senescence, fibrosis, inflammation, angiogenesis, and its potential interaction with miR‐16, are unknown. In vivo studies were performed in male bile duct–ligated (BDL, 12‐week‐old) mice, multidrug resistance 2 knockout (Mdr2 −/−) mice (10‐week‐old), and their corresponding controls, treated with recombinant human FGF1 (rhFGF1), fibroblast growth factor receptor (FGFR) antagonist (AZD4547), or anti‐FGF1 monoclonal antibody (mAb). In vitro, the human cholangiocyte cell line (H69) and human hepatic stellate cells (HSCs) were used to determine the expression of proliferation, fibrosis, angiogenesis, and inflammatory genes following rhFGF1 treatment. PSC patient and control livers were used to evaluate FGF1 and miR‐16 expression. Intrahepatic bile duct mass (IBDM), along with hepatic fibrosis and inflammation, increased in BDL mice treated with rhFGF1, with a corresponding decrease in miR‐16, while treatment with AZD4547 or anti‐FGF1 mAb decreased hepatic fibrosis, IBDM, and inflammation in BDL and Mdr2 −/− mice. In vitro, H69 and HSCs treated with rhFGF1 had increased expression of proliferation, fibrosis, and inflammatory markers. PSC samples also showed increased FGF1 and FGFRs with corresponding decreases in miR‐16 compared with healthy controls. Conclusion: Our study demonstrates that suppression of FGF1 and miR‐16 signaling decreases the presence of hepatic fibrosis, biliary proliferation, inflammation, senescence, and angiogenesis. Targeting the FGF1 and miR‐16 axis may provide therapeutic options in treating cholangiopathies such as PSC.

Circular RNA circLMF1 regulates PDGF-BB-induced proliferation and migration of human aortic smooth muscle cells by regulating the miR-125a-3p/VEGFA or FGF1 axis.

Atherosclerosis is a major cause of cardiovascular disease, in which vascular smooth muscle cells (VSMCs) proliferation and migration play a vital role. Circular RNAs (circRNAs) have been reported to be correlated with the VSMCs function. Therefore, this study is designed to explore the role and mechanism of circRNA lipase maturation factor 1 (circLMF1) in Human aortic VSMCs (HASMCs). The microarray was used for detecting the expression of circLMF1 in proliferative and quiescent HASMCs. Levels of circLMF1, microRNA-125a-3p (miR-125a-3p), vascular endothelial growth factor A (VEGFA), and fibroblast growth factor 1 (FGF1) were determined by real-time quantitative polymerase chain reaction (RT-qPCR). Cell viability, cell cycle progression, and migration were assessed by Cell Counting Kit-8 (CCK-8), flow cytometry, wound healing, and transwell assays, respectively. Western blot assay determined proliferating cell nuclear antigen (PCNA), Cyclin D1, matrix metalloproteinase (MMP2), osteopontin (OPN), VEGFA, and FGF1 protein levels. The possible interactions between miR-125a-3p and circLMF1, and miR-125a-3p and VEGFA or FGF1 were predicted by circbank or targetscan, and then verified by a dual-luciferase reporter, RNA Immunoprecipitation (RIP), RNA pull-down assays. CircLMF1, VEGFA, and FGF1 were increased, and miR-125a-3p was decreased in platelet-derived growth factor-BB (PDGF-BB)-inducted HASMCs. Functionally, circLMF1 knockdown hindered cell viability, cell cycle progression, and migration in PDGF-BB-treated HASMCs. Mechanically, circLMF1 could regulate VEGFA or FGF1 expression through sponging miR-125a-3p. Our findings revealed that circLMF1 deficiency could inhibit cell viability, cell cycle progression, and migration of PDGF-BB stimulated atherosclerosis model partly through the miR-125a-3p/VEGFA or FGF1 axis, suggesting that targeting circLMF1 can be a feasible therapeutic strategy for atherosclerosis.

MiR-143-3p Increases the Radiosensitivity of Breast Cancer Cells Through FGF1.

Breast cancer is a common malignant tumor in women. At present, the main treatment for breast cancer is radiotherapy. Due to the difference in radiosensitivity between individuals or tumor cells, the effect of radiotherapy is not good. Therefore, in radiotherapy, how to use various auxiliary means to reduce the radiation resistance of tumor, Therefore, it has become an important research topic to improve the radiosensitivity of the tumor. Fibroblast growth factor-1 (FGF1) plays an important role in tumor migration. Therefore, the study of miR-143-3p increasing the radiosensitivity of breast cancer cells through FGF1 is proposed in this paper. In this study, a control group experiment was set up to study. During the experiment, the relative expression of miR-143-3p was detected by fluorescent quantitative PCR of miRNA, and the cell irradiation experiment was created to analyze the radiosensitivity of breast cancer cells by comparing their survival fraction. The results of this study showed that when the radiation dose was 0, the survival scores of the three groups were all 1. The survival fraction of the experimental group decreased from 0.26 ± 0.045 to 0.068 ± 0.008 when the dose was added to 4Gy. The survival fraction of the experimental group was always greater than that of the two control groups. The results of this study show that miR-143-3p can increase the radiosensitivity of breast cancer cells through FGF1.

Nuclear Localization Sequence of FGF1 Is Not Required for Its Intracellular Anti-Apoptotic Activity in Differentiated Cells.

Fibroblast growth factor 1 (FGF1) is considered primarily as a ligand for FGF surface receptors (FGFRs) through which it activates a number of cellular responses. In addition to its canonical mode of action, FGF1 can act intracellularly, before secretion or after internalization and translocation from the cell exterior. The role of FGF1 inside the cell is to provide additional protection against apoptosis and promote cell survival. The FGF1 protein contains a specific N-terminal nuclear localization sequence (NLS) that is essential for its efficient transport to the nucleus. Here, we investigated the role of this sequence in the anti-apoptotic response of FGF1. To this end, we produced recombinant FGF1 variants with mutated or deleted NLS and added them to apoptosis-induced cells in which FGFR1 was inactive, either as a result of chemical inhibition or kinase-dead mutation. After internalization, all FGF1 variants were able to protect the differentiated cells from serum starvation-induced apoptosis. To verify the results obtained for NLS mutants, we knocked down LRRC59, a protein that mediates the nuclear transport of FGF1. Upon LRRC59 silencing, we still observed a decrease in caspase 3/7 activity in cells treated exogenously with wild-type FGF1. In the next step, FGF1 variants with mutated or deleted NLS were expressed in U2OS cells, in which apoptosis was then induced by various factors (e.g., starvation, etoposide, staurosporine, anisomycin and actinomycin D). Experiments were performed in the presence of specific FGFR inhibitors to eliminate FGFR-induced signaling, potentially activated by FGF1 proteins released from damaged cells. Again, we found that the presence of NLS in FGF1 is not required for its anti-apoptotic activity. All NLS variants tested were able to act as wild type FGF1, increasing the cell viability and mitochondrial membrane potential and reducing the caspase 3/7 activity and PARP cleavage in cells undergoing apoptosis, both transiently and stably transfected. Our results indicate that the nuclear localization of FGF1 is not required for its intracellular anti-apoptotic activity in differentiated cells and suggest that the mechanism of the stress response differs according to the level of cell differentiation.

FGF1 and insulin control lipolysis by convergent pathways

Inexorable increases in insulin resistance, lipolysis, and hepatic glucose production (HGP) are hallmarks of type 2 diabetes. Previously, we showed that peripheral delivery of exogenous fibroblast growth factor 1 (FGF1) has robust anti-diabetic effects mediated by the adipose FGF receptor (FGFR) 1. However, its mechanism of action is not known. Here, we report that FGF1 acutely lowers HGP by suppressing adipose lipolysis. On a molecular level, FGF1 inhibits the cAMP-protein kinase A axis by activating phosphodiesterase 4D (PDE4D), which separates it mechanistically from the inhibitory actions of insulin via PDE3B. We identify Ser44 as an FGF1-induced regulatory phosphorylation site in PDE4D that is modulated by the feed-fast cycle. These findings establish the FGF1/PDE4 pathway as an alternate regulator of the adipose-HGP axis and identify FGF1 as an unrecognized regulator of fatty acid homeostasis.

Establishing F1A-CreERT2 Mice to Trace Fgf1 Expression in Adult Mouse Cardiomyocytes.

Fibroblast growth factor 1 (FGF1) regulates many biological and physiological processes. In mice, Fgf1 gene contains at least three upstream promoters and are alternatively spliced to the first protein coding exon, giving rise to different Fgf1 mRNA variants (1A, 1B and 1G). Among them, the Fgf1A transcript is predominantly expressed in the heart. FGF1 can induce cardiomyocyte regeneration and cardiogenesis in vitro and in vivo. Here, we generated a novel mouse line using the Fgf1A promoter (F1A) driving the expression of the inducible Cre recombinase (CreERT2). We firstly demonstrated that the highest mRNA expression of CreERT2 were detected in the heart specifically of F1A-CreERT2 mice, similar to that of Fgf1A mRNA. The F1A-CreERT2 mice were crossed with ROSA26 mice, and the F1 mice were analyzed. The LacZ-positive signals were detected exclusively in the heart after tamoxifen administration. The CreERT2-mediated recombination in the tissues is monitored through LacZ-positive signals, indicating the in situ localization of F1A-positive cells. Consistently, these F1A-positive cells with RFP-positive signals or LacZ-positive blue signals were co-localized with cardiomyocytes expressing cardiac troponin T, suggesting cardiomyocyte-specific activation of Fgf1A promoter. Our data suggested that the F1A-CreERT2 mouse line could be used for time-dependent and lineage tracing of Fgf1A-expressing cells in vivo.

Methacrylated gelatin shape-memorable cryogel subcutaneously delivers EPCs and aFGF for improved pressure ulcer repair in diabetic rat model

Pressure ulcer (PU) in patients with diabetes mellitus (DM) is still a clinical intractable issue due to the complicated physiological characteristics by the prolonged high glucose level and impaired angiogenesis. The PU treatment includes surgical debridement, stem cell therapy and growth factors, leading to high cost and repeated professional involvement. Developing effective wound dressing combining the therapeutic cells and growth factors has become highly demanded. Herein, we reported the direct subcutaneous administration of endothelial progenitor cells (EPCs) and acid fibroblast growth factor (aFGF) with a shape-memorable methacrylated gelatin cryogel (EPCs/aFGF@GelMA) for the therapy of PU in rats with DM. This EPCs/aFGF@GelMA cryogel system presented microporous structure, elastic mechanical strength and enhanced cell migration property with controlled release of aFGF. Moreover, compared with EPCs/aFGF and GelMA alone, in vivo results showed that this EPCs/aFGF@GelMA system exhibited accelerated wound closure rate, enhanced granulation formation, collagen deposition as well as re-epithelization. Importantly, we found that the excellent positive performance of EPCs/aFGF@GelMA is due to its up-regulation of HIF-ɑ upon the wound site, modulating the microenvironment of wound site to initiate the impaired local angiogenesis. Collectively, this hybrid gelatin cryogels show great promise for biomedical applications, especially in tissue engineering and regenerative medicine.

Oxidative stress-induced growth inhibitor 1 in alcohol-induced liver cirrhosis.

Human oxidative stress-induced growth inhibitor 1 (OSGIN1) is a protein identified in 2001 which belongs to the OKL38 protein family. The aim of the study was to investigate the levels of this protein depending on the severity of alcohol-induced liver cirrhosis. The study group consisted of 60 patients: 30 patients with cirrhosis in the P-Ch A and B stage and 30 in the P-Ch C stage. The control group consisted of 18 healthy individuals without liver diseases, who did not abuse alcohol. Oxidative stress induced growth inhibitor 1 (OSGIN1), fibroblast growth factor 1 (FGF1) and fibroblast growth factor 21 (FGF21) were determined in blood serum using enzyme-linked immunosorbent assay (ELISA) kits. All absorbance readings were conducted using an Epoch Microplate Spectrophotometer (BioTek Instrumentals, Inc., Winooski, VT, USA). OSGIN1, FGF1 and FGF21 concentrations were determined using Sandwich enzyme immunoassay kits (by Cloud Clone Corp., Katy, TX, USA). Statistica 13.3 (TIBCO Software, Inc.) was used for data analysis. The concentration of OSGIN1 was 0.028 ± 0.017 in the control group which increased with the advancement of liver cirrhosis (stage of Pugh-Child): 0.075 ± 0.098 in the P-Ch A + B group and 0.121 ± 0.134 in the P-Ch C stage. Multiple comparison tests confirmed statistically significant differences in OSGIN1 concentration between the control group and P-Ch C (p <0.02). Significant correlations were noted between OSGIN1 and FGF1 (r = 0.39; p = 0.004) and between OSGIN1 and FGF21 (r = 0.53; p <0.0001). The study revealed that the level of OSGIN1 increased significantly in the P-Ch C stage of liver cirrhosis. It is possible that OSGIN1 may be used for the non-invasive diagnosis of ALD, but its possible diagnostic value is still very uncertain.