Expression Of Fibroblast Growth Factor Research Articles

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.

Investigation into the protective effects of hypaconitine and glycyrrhetinic acid against chronic heart failure of the rats

BackgroundThe present study aimed to determine the protective effects of hypaconitine (HA) and glycyrrhetinic acid (GA) against chronic heart failure (CHF) in the rats and to explore the underlying molecular mechanisms.MethodsThe CHF rat model was established by transverse-aortic constriction (TAC) operation. Transthoracic echocardiography and hematoxylin eosin (HE) staining were used to evaluate the pathophysiological and histopathological changes of CHF model. The total cholesterol (TCHO) and triglyceride (TG) levels were determined by ELISA assay. The protein expression of fibroblast growth factor 2 (FGF2), vascular endothelial growth factor A (VEGFA) and endothelial nitric oxide synthase (eNOS) in the rat ventricular tissues was determined by immunohistochemistry. The serum metabolites were determined by LC-MS/MS assay.ResultsAfter applied the HA + GA, the cardiac tissue and structure were obviously improved, and the HA + GA treatment also significantly reduced the plasma levels of TCHO and TG in the CHF rats. The expression of FGF2 and VEGFA protein was up-regulated and the expression of eNOS protein was down-regulated in the ventricular tissues of CHF rats, which was significantly restored after HA + GA treatment. HA + GA treatment down-regulated serum isonicotinic acid, phosphatidylcholine, cardiolipin, estrogen glucuronide, and glycocholic acid, up-regulated serum sphingosine and deoxycholic acid in the CHF rats.ConclusionsIn conclusion, HA + GA showed protective effects on CHF in the rats, and the HA + GA may exert protective effects by reducing lipid levels, up-regulating the expression of FGF2 and VEGFA proteins, attenuating eNOS protein expression, and modulating metabolic pathways. However, the molecular mechanisms underlying HA + GA-mediated effects still require further examination.

FGF2 Is Protective Towards Cisplatin-Induced KGN Cell Toxicity by Promoting FTO Expression and Autophagy.

It is widely known that chemotherapy-induced apoptosis of granulosa was the main reason for premature ovarian failure (POF). In addition, accumulating evidence has demonstrated that autophagy was involved in it. Studies before have reported that fibroblast growth factor-2 (FGF2) could attenuate cell death via regulating autophagy. In our previous study, FGF2 could decrease granulosa cell apoptosis in cisplatin-induced POF mice. Furthermore, obesity-associated protein [fat mass and obesity-associated protein (FTO)], which decreased significantly in POF mice, could inhibit cell apoptosis via activating autophagy. Moreover, downregulation of FTO could decrease the expression of paracrine factor FGF2. However, the relationship between FTO and FGF2 in granulosa cell autophagy is still unknown. In the present study, Cell Counting Kit-8 (CCK-8) and 5‐ethynyl‐2‐deoxyuridine (EdU) assays showed that exogenous addition of FGF2 could promote cisplatin-induced injured granulosa cell proliferation. Western blotting indicated that FGF2 could inhibit apoptosis of injured granulosa cells via autophagy. Inhibition of autophagy by chemicals suppressed the effect of FGF2 and promoted injured cell apoptosis. In addition, the expression of FTO was decreased in injured cells, and FGF2 addition could reverse it. Overexpression of FTO reduced injured cell apoptosis via activating the autophagy process. Our findings indicated that FGF2 activates autophagy by regulating the expression of FTO, thereby reducing the apoptosis of the injured cells.

Abstract 1674: FGF8 as a modulator of Wilms' tumor in vitro growth and longevity

Abstract Wilms’ tumor is the most common pediatric renal tumor accounting for 6-7% of all childhood cancers. Development of faithful Wilms’ tumor cell lines is needed to identify novel therapeutics. Currently, established Wilms’ tumor cell lines that are suitable for high throughput studies only represent anaplastic Wilms, a rare subtype. In contrast, cell lines representing the more common favorable histology Wilms’ tumor (FHWT) often have poor proliferative capacity and are unable to passage long-term (e.g. >20 passages). For this reason, there is a need to establish robust cell lines representing FHWT. We hypothesize that there are core factors missing from the cell culture medium which prevent proper growth signaling and proliferation. We first performed bulk RNA-sequencing from patients and associated cell lines at Aflac Cancer and Blood Disorders Center with an underlying diagnosis of FHWT. Transcriptomic analyses showed that the reduced growth and proliferation in these FHWT cell lines may be due to reduced presence of the mitogenic fibroblast growth factor 8 (FGF8) in the cell culture environment as compared to the tumor environment. Wilms’ tumor has notably high FGF8 expression levels compared to all other pediatric tumors, implicating FGF8 as a potential driving factor in tumor maintenance. To characterize the phenotypic effects of FGF8 reintroduction in short term FHWT cell lines, we both endogenously overexpressed FGF8 via a lentiviral vector and ectopically added recombinant FGF8 to the cell culture medium. Qualitatively, endogenous overexpression of FGF8 induced distinct morphological changes in cells, converting the enlarged and flattened cells into more focal cells resembling proliferative early passage cells. Quantitatively, overexpression of FGF8 in one favorable histology cell line led to an increased rate of cellular proliferation and extended the cellular longevity by seven passages (47% above baseline). RNA-sequencing analysis of these FGF8 overexpressing cells implicated the anti-apoptotic gene, NTSR1, and an extracellular matrix protein, SNED1, as potential downstream targets of FGF8 overexpression. However, ectopic addition of recombinant FGF8 protein to the cellular medium had less pronounced effects, with seemingly cell-line specific effects. These data demonstrate FGF8 as possibly being required for survival in certain Wilms’ tumor cell lines that can be modulated to influence in vitro proliferation and longevity. Citation Format: Garrett Cooper, Benjamin Lee, Nate Smyth, Karuna Mittal, Taylor Lawrence, Hunter Jonus, Jenny Shim, Kelly Goldsmith, Andrew Hong. FGF8 as a modulator of Wilms' tumor in vitro growth and longevity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1674.

Lentiviral interferon: A novel method for gene therapy in bladder cancer

Interferon alpha (IFNα) gene therapy is emerging as a new treatment option for patients with non-muscle invasive bladder cancer (NMIBC). Adenoviral vectors expressing IFNα have shown clinical efficacy treating bacillus Calmette-Guerin (BCG)-unresponsive bladder cancer (BLCA). However, transient transgene expression and adenoviral immunogenicity may limit therapeutic activity. Lentiviral vectors can achieve stable transgene expression and are less immunogenic. In this study, we evaluated lentiviral vectors expressing murine IFNα (LV-IFNα) and demonstrate IFNα expression by transduced murine BLCA cell lines, bladder urothelium, and within the urine following intravesical instillation. Murine BLCA cell lines (MB49 and UPPL1541) were sensitive to IFN-mediated cell death after LV-IFNα, whereas BBN975 was inherently resistant. Upregulation of interleukin-6 (IL-6) predicted sensitivity to IFN-mediated cell death mediated by caspase signaling, which when inhibited abrogated IFN-mediated cell killing. Intravesical therapy with LV-IFNα/Syn3 in a syngeneic BLCA model significantly improved survival, and molecular analysis of treated tumors revealed upregulation of apoptotic and immune-cell-mediated death pathways. In particular, biomarker discovery analysis identified three clinically actionable targets, PD-L1, epidermal growth factor receptor (EGFR), and ALDHA1A, in murine tumors treated with LV-IFNα/Syn3. Our findings warrant the comparison of adenoviral and LV-IFNα and the study of novel combination strategies with IFNα gene therapy for the BLCA treatment.

MicroRNA-146a attenuates isoproterenol-induced cardiac fibrosis by inhibiting FGF2.

Cardiac fibrosis is a key factor of heart failure. Increasing evidence suggests that microRNAs (miRNAs/miRs) serve vital roles in the pathogenesis of cardiac fibrosis. The present study aimed to investigate the role of miR-146a-5p in isoproterenol (ISO)-induced cardiac fibrosis. Reverse transcription-quantitative PCR analysis demonstrated that miR-146a-5p expression was downregulated in ISO-treated rat heart tissue and ISO-induced cardiac fibroblasts (CFs). Conversely, the expression levels of basic fibroblast growth factor 2 (FGF2), collagen I and smooth muscle α-actin (α-SMA) were upregulated in ISO-treated rat cardiac tissue and CFs. Furthermore, viability and differentiation were inhibited in ISO-induced CFs transfected with miR-146a-5p mimics. Dual-luciferase reporter assay confirmed that miR-146a-5p targeted FGF2. Notably, FGF2 expression was suppressed following overexpression of miR-146a-5p, while FGF2 expression increased following miR-146a-5p knockdown. In addition, FGF2 knockdown suppressed the expression levels of FGF2, collagen I and α-SMA levels in CFs. Taken together, the results of the present study suggested that the miR-146a-5p/FGF2 pathway may be a novel therapy for cardiac fibrosis.

Fibroblast growth factor-9 expression in airway epithelial cells amplifies the type I interferon response and alters influenza A virus pathogenesis.

Influenza A virus (IAV) preferentially infects conducting airway and alveolar epithelial cells in the lung. The outcome of these infections is impacted by the host response, including the production of various cytokines, chemokines, and growth factors. Fibroblast growth factor-9 (FGF9) is required for lung development, can display antiviral activity in vitro, and is upregulated in asymptomatic patients during early IAV infection. We therefore hypothesized that FGF9 would protect the lungs from respiratory virus infection and evaluated IAV pathogenesis in mice that overexpress FGF9 in club cells in the conducting airway epithelium (FGF9-OE mice). However, we found that FGF9-OE mice were highly susceptible to IAV and Sendai virus infection compared to control mice. FGF9-OE mice displayed elevated and persistent viral loads, increased expression of cytokines and chemokines, and increased numbers of infiltrating immune cells as early as 1 day post-infection (dpi). Gene expression analysis showed an elevated type I interferon (IFN) signature in the conducting airway epithelium and analysis of IAV tropism uncovered a dramatic shift in infection from the conducting airway epithelium to the alveolar epithelium in FGF9-OE lungs. These results demonstrate that FGF9 signaling primes the conducting airway epithelium to rapidly induce a localized IFN and proinflammatory cytokine response during viral infection. Although this response protects the airway epithelial cells from IAV infection, it allows for early and enhanced infection of the alveolar epithelium, ultimately leading to increased morbidity and mortality. Our study illuminates a novel role for FGF9 in regulating respiratory virus infection and pathogenesis.

Circ-HUWE1 Knockdown Alleviates Amyloid-β-Induced Neuronal Injury in SK-N-SH Cells via miR-433-3p Release-Mediated FGF7 Downregulation.

Alzheimer's disease (AD) is a progressive neurodegenerative disease, characterized by Amyloid-β accumulation-induced neuronal injury. Emerging evidence shows that circular RNA (circRNA) is involved in AD development. The aim of this study was to illustrate the role of circ-HUWE1 in Amyloid-β accumulation-induced neuronal injury. Quantitative real-time PCR (qPCR) or western blot was conducted for the expression analysis of circ-HUWE1, miR-433-3p, and fibroblast growth factor 7 (FGF7). In functional assays, cell viability was determined by CCK-8 assay, and cell apoptosis was examined by flow cytometry assay, the protein levels of apoptosis-related markers, and caspase1 or caspase3 activity. The release of pro-inflammatory factors was monitored by ELISA. The predicted binding relationship between miR-433-3p and circ-HUWE1 or FGF7 was validated by dual-luciferase reporter assay. We discovered that circ-HUWE1 absence alleviated Amyloid-β-induced cell viability degradation, cell apoptosis, and inflammatory responses in SK-N-SH cells. MiR-433-3p was a target of circ-HUWE1, and miR-433-3p inhibition reversed the effects of circ-HUWE1 knockdown. In addition, FGF7 was a downstream target of miR-433-3p whose function could be abolished by FGF7 reintroduction. Circ-HUWE1 positively regulated FGF7 expression via competitively targeting miR-433-3p. Moreover, circ-HUWE1 knockdown activated the WNT signaling pathway in Amyloid-β-treated SK-N-SH cells by targeting the miR-433-3p/FGF7 axis. In conclusion,circ-HUWE1 knockdown alleviates Amyloid-β-induced neuronal injury in SK-N-SH cells via miR-433-3p release-mediated FGF7 depletion.

Canonical Wnt signaling and the regulation of divergent mesenchymal Fgf8 expression in axolotl limb development and regeneration.

The expression of fibroblast growth factors (Fgf) ligands in a specialized epithelial compartment, the Apical Ectodermal Ridge (AER), is a conserved feature of limb development across vertebrate species. In vertebrates, Fgf 4, 8, 9, and 17 are all expressed in the AER. An exception to this paradigm is the salamander (axolotl) developing and regenerating limb, where key Fgf ligands are expressed in the mesenchyme. The mesenchymal expression of Amex.Fgf8 in axolotl has been suggested to be critical for regeneration. To date, there is little knowledge regarding what controls Amex.Fgf8 expression in the axolotl limb mesenchyme. A large body of mouse and chick studies have defined a set of transcription factors and canonical Wnt signaling as the main regulators of epidermal Fgf8 expression in these organisms. In this study, we address the hypothesis that alterations to one or more of these components during evolution has resulted in mesenchymal Amex.Fgf8 expression in the axolotl. To sensitively quantify gene expression with spatial precision, we combined optical clearing of whole-mount axolotl limb tissue with single molecule fluorescent in situ hybridization and a semiautomated quantification pipeline. Several candidate upstream components were found expressed in the axolotl ectoderm, indicating that they are not direct regulators of Amex.Fgf8 expression. We found that Amex.Wnt3a is expressed in axolotl limb epidermis, similar to chicken and mouse. However, unlike in amniotes, Wnt target genes are activated preferentially in limb mesenchyme rather than in epidermis. Inhibition and activation of Wnt signaling results in downregulation and upregulation of mesenchymal Amex.Fgf8 expression, respectively. These results implicate a shift in tissue responsiveness to canonical Wnt signaling from epidermis to mesenchyme as one step contributing to the unique mesenchymal Amex.Fgf8 expression seen in the axolotl.

Acrylamide induced glucose metabolism disorder in rats involves gut microbiota dysbiosis and changed bile acids metabolism

Acrylamide (AA) is a common food contaminant that causes glucose metabolism disorders (GMD). However, the underlying mechanism remains unclear. Female Sprague Dawley (SD) rats were treated with AA via gavage for 21 days, and the glucose and insulin levels, gut microbiota, intestinal barrier, and metabolism were analyzed. The results revealed that AA elevated serum glucose levels, reduced insulin levels and caused intestinal barrier injury. The 16S amplicon sequencing and non-targeted metabolomics showed that AA induced gut microbiota dysbiosis and bile acids (BAs) metabolism disorder. Specifically, AA decreased the abundance of Lactobacillus and Bacteroides in the cecal contents, and increased the cholic acid (CA) content in feces. Meanwhile, the expression of ileum apical sodium-dependent bile acid transporter (ASBT) responsible for CA reabsorption was suppressed. Further analysis indicated that BAs sensing nuclear receptor farnesoid X receptor (FXR) gene was activated and glucagon-like peptide-1 (GLP-1) which stimulates insulin secretion was downregulated. In addition, activation of FXR increased the expression of fibroblast growth factor 15 (FGF15), which resulted in the inhibition of hepatic BAs synthesis. Overall, this study demonstrated that AA-induced GMD is associated with the gut-microbiota-CA-FXR/GLP-1 axis. These findings add new knowledge to the AA-induced GMD and provide a basis for potential AA toxicity mitigation by manipulation of the gut microbiota.

Tubular cells produce FGF2 via autophagy after acute kidney injury leading to fibroblast activation and renal fibrosis

ABSTRACT Following acute kidney injury (AKI), renal tubular cells may stimulate fibroblasts in a paracrine fashion leading to interstitial fibrosis, but the paracrine factors and their regulation under this condition remain elusive. Here we identify a macroautophagy/autophagy-dependent FGF2 (fibroblast growth factor 2) production in tubular cells. Upon induction, FGF2 acts as a key paracrine factor to activate fibroblasts for renal fibrosis. After ischemic AKI in mice, autophagy activation persisted for weeks in renal tubular cells. In inducible, renal tubule-specific atg7 (autophagy related 7) knockout (iRT-atg7-KO) mice, autophagy deficiency induced after AKI suppressed the pro-fibrotic phenotype in tubular cells and reduced fibrosis. Among the major cytokines, tubular autophagy deficiency in iRT-atg7-KO mice specifically diminished FGF2. Autophagy inhibition also attenuated FGF2 expression in TGFB1/TGF-β1 (transforming growth factor, beta 1)-treated renal tubular cells. Consistent with a paracrine action, the culture medium of TGFB1-treated tubular cells stimulated renal fibroblasts, and this effect was suppressed by FGF2 neutralizing antibody and also by fgf2- or atg7-deletion in tubular cells. In human, compared with non-AKI, the renal biopsies from post-AKI patients had higher levels of autophagy and FGF2 in tubular cells, which showed significant correlations with renal fibrosis. These results indicate that persistent autophagy after AKI induces pro-fibrotic phenotype transformation in tubular cells leading to the expression and secretion of FGF2, which activates fibroblasts for renal fibrosis during maladaptive kidney repair. Abbreviations: 3-MA: 3-methyladnine; ACTA2/α-SMA: actin alpha 2, smooth muscle, aorta; ACTB/β-actin: actin, beta; AKI: acute kidney injury; ATG/Atg: autophagy related; BUN: blood urea nitrogen; CCN2/CTGF: cellular communication network factor 2; CDKN2A/p16: cyclin dependent kinase inhibitor 2A; CKD: chronic kidney disease; CM: conditioned medium; COL1A1: collagen, type I, alpha 1; COL4A1: collagen, type IV, alpha 1; CQ: chloroquine; ECM: extracellular matrix; eGFR: estimated glomerular filtration rate; ELISA: enzyme-linked immunosorbent assay; FGF2: fibroblast growth factor 2; FN1: fibronectin 1; FOXO3: forkhead box O3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HAVCR1/KIM-1: hepatitis A virus cellular receptor 1; IHC: immunohistochemistry; IRI: ischemia-reperfusion injury; ISH: in situ hybridization; LTL: lotus tetragonolobus lectin; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PDGFB: platelet derived growth factor, B polypeptide; PPIB/cyclophilin B: peptidylprolyl isomerase B; RT-qPCR: real time-quantitative PCR; SA-GLB1/β-gal: senescence-associated galactosidase, beta 1; SASP: senescence-associated secretory phenotype; sCr: serum creatinine; SQSTM1/p62: sequestosome 1; TASCC: TOR-autophagy spatial coupling compartment; TGFB1/TGF-β1: transforming growth factor, beta 1; VIM: vimentin

Does freeze dried amnion useful as scaffold for mesenchymal stem cell for repair of vesicovaginal fistula? An overview of proliferative and remodeling phase in the wound healing process

BackgroundSurgery is still the main solution for vesicovaginal fistula, but postoperative wound healing represents a challenge, and the recurrence rate remains high. There is a need to develop therapeutic methods to increase the success of therapy and women's quality of life. ObjectiveTo explore whether human freeze-dried amnion is useful as a mesenchymal stem cell scaffold for repair of vesicovaginal fistula through assessment of the proliferative and remodelling phases. MethodsThis experiment was undertaken using a New Zealand rabbit model. The research was divided into two stages: (1) an experiment to create a model of a vesicovaginal fistula; and (2) a laboratory experiment to close a vesicovaginal fistula as a result of the first stage. The second stage used a post-test-only control group design. The wound-healing process was assessed based on the expression of platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), occludin and claudin-4. Data were analysed descriptively and statistically. ResultsExpression of PDGF, VEGF, FGF, occludin and claudin-4 in vesicovaginal fistula models sutured with human freeze-dried amnion was higher compared with models without human freeze-dried amnion. Significant differences were found in average expression of PDGF, VEGF, FGF, occludin and claudin-4. ConclusionHuman freeze-dried amnion plays a role in the wound-healing process in vesicovaginal fistula repair models. It is hoped that this research will improve urogynaecological services.

Cardioprotection by selective SGLT-2 inhibitors in a non-diabetic mouse model of myocardial ischemia/reperfusion injury: a class or a drug effect?

Major clinical trials with sodium glucose co-transporter-2 inhibitors (SGLT-2i) exhibit protective effects against heart failure events, whereas inconsistencies regarding the cardiovascular death outcomes are observed. Therefore, we aimed to compare the selective SGLT-2i empagliflozin (EMPA), dapagliflozin (DAPA) and ertugliflozin (ERTU) in terms of infarct size (IS) reduction and to reveal the cardioprotective mechanism in healthy non-diabetic mice. C57BL/6 mice randomly received vehicle, EMPA (10mg/kg/day) and DAPA or ERTU orally at the stoichiometrically equivalent dose (SED) for 7days. 24h-glucose urinary excretion was determined to verify SGLT-2 inhibition. IS of the region at risk was measured after 30min ischemia (I), and 120min reperfusion (R). In a second series, the ischemic myocardium was collected (10th min of R) for shotgun proteomics and evaluation of the cardioprotective signaling. In a third series, we evaluated the oxidative phosphorylation capacity (OXPHOS) and the mitochondrial fatty acid oxidation capacity by measuring the respiratory rates. Finally, Stattic, the STAT-3 inhibitor and wortmannin were administered in both EMPA and DAPA groups to establish causal relationships in the mechanism of protection. EMPA, DAPA and ERTU at the SED led to similar SGLT-2 inhibition as inferred by the significant increase in glucose excretion. EMPA and DAPA but not ERTU reduced IS. EMPA preserved mitochondrial functionality in complex I&II linked oxidative phosphorylation. EMPA and DAPA treatment led to NF-kB, RISK, STAT-3 activation and the downstream apoptosis reduction coinciding with IS reduction. Stattic and wortmannin attenuated the cardioprotection afforded by EMPA and DAPA. Among several upstream mediators, fibroblast growth factor-2 (FGF-2) and caveolin-3 were increased by EMPA and DAPA treatment. ERTU reduced IS only when given at the double dose of the SED (20mg/kg/day). Short-term EMPA and DAPA, but not ERTU administration at the SED reduce IS in healthy non-diabetic mice. Cardioprotection is not correlated to SGLT-2 inhibition, is STAT-3 and PI3K dependent and associated with increased FGF-2 and Cav-3 expression.

Mesh-like electrospun membrane loaded with atorvastatin facilitates cutaneous wound healing by promoting the paracrine function of mesenchymal stem cells

BackgroundFunctional electrospun membranes are promising dressings for promoting wound healing. However, their microstructure and drug loading capacity need further improvements. It is the first time to design a novel mesh-like electrospun fiber loaded with atorvastatin (ATV) and investigated its effects on paracrine secretion by bone marrow-derived mesenchymal stem cells (BMSCs) and wound healing in vivo.MethodsWe fabricated a mesh-like electrospun membrane using a copper mesh receiver. The physical properties of the membranes were evaluated by SEM, FTIR spectroscopy, tensile strength analysis, and contrast angle test. Drug release was measured by plotting concentration as a function of time. We tested the effects of conditioned media (CM) derived from BMSCs on endothelial cell migration and angiogenesis. We used these BMSCs and performed RT-PCR and ELISA to evaluate the expressions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) genes and proteins, respectively. The involvement of FAK and AKT mechanotransduction pathways in the regulation of BMSC secretion by material surface topography was also investigated. Furthermore, we established a rat model of wound healing, applied ATV-loaded mesh-like membranes (PCL/MAT) seeded with BMSCs on wounds, and assessed their efficacy for promoting wound healing.ResultsFTIR spectroscopy revealed successful ATV loading in PCL/MAT. Compared with random electrospun fibers (PCL/R) and mesh-like electrospun fibers without drug load (PCL/M), PCL/MAT induced maximum promotion of human umbilical vein endothelial cell (HUVEC) migration. In the PCL/MAT group, the cell sheet scratches were nearly closed after 24 h. However, the cell sheet scratches remained open in other treatments at the same time point. The PCL/MAT promoted angiogenesis and led to the generation of longer tubes than the other treatments. Finally, the PCL/MAT induced maximum gene expression and protein secretion of VEGF and b-FGF. As for material surface topography effect on BMSCs, FAK and AKT signaling pathways were shown to participate in the modulation of MSC morphology and its paracrine function. In vivo, PCL/MAT seeded with BMSCs significantly accelerated healing and improved neovascularization and collagen reconstruction in the wound area compared to the other treatments.ConclusionsThe mesh-like topography of fibrous scaffolds combined with ATV release creates a unique microenvironment that promotes paracrine secretion of BMSCs, thereby accelerating wound healing. Hence, drug-loaded mesh-like electrospun membranes may be highly efficacious for wound healing and as artificial skin. It is a promising approach to solve the traumatic skin defect and accelerate recovery, which is essential to developing functional materials for future regenerative medicine.

The Role of Recombinant Secretory Leukocyte Protease Inhibitor to CD163, FGF-2, IL-1 and IL-6 Expression in Skin Wound Healing.

BackgroundThe wound healing process can be optimized through the addition of a biomaterial such as recombinant secretory leukocyte protease inhibitor (rSLPI). The SLPI is a non-glycosylated proteomic material that inhibits protease enzymes and has anti-inflammatory properties, thus accelerating wound healing. This study analyzed the administration of rSLPI doses 0.04 cc and 0.06 cc in skin wound healing on the CD163 expression of macrophages and cytokines such as interleukin 1 (IL-1), interleukin 6 (IL-6) and fibroblast growth factor 2 (FGF-2).Materials and MethodsrSLPI produced from Escherichia coli TOP10 as the cloning host, BL21 (DE3) strains as the expression host and pET30a plasmids were used for the expression system construction. The wound was created on Wistar rat dorsal skin, then rSLPI 0.04 cc and 0.06 cc was administered. In the next four days, the back skin was biopsied and stained by immunohistochemistry to analyze the CD163, FGF-2, IL-1 and IL-6 expression.ResultsThe administration of rSLPI increased CD163 and FGF-2 expression dependent on dose (p<0.05). On the other hand, administration of rSLPI decreased IL-1 and IL-6 expression depending on dose (p <0.05).ConclusionThe administration of rSLPI is able to accelerate the wound healing process by increasing the CD163 and FGF-2 expression. The cytokines such as IL-1 and IL-6 decreased depending on rSLPI doses.

Epicardial histone deacetylase 3 promotes myocardial growth through a novel microRNA pathway

Establishment of the myocardial wall requires proper growth cues from nonmyocardial tissues. During heart development, the epicardium and epicardium‐derived cells (EPDCs) instruct myocardial growth by secreting essential factors including fibroblast growth factor 9 (FGF9) and insulin‐like growth factor 2 (IGF2). However, it is poorly understood how the epicardial secreted factors are regulated, in particular by chromatin modifications for myocardial formation. To understand whether and how histone deacetylase 3 (HDAC3) in the developing epicardium regulates myocardial growth, we deleted Hdac3 in the developing murine epicardium. Mutant hearts showed ventricular myocardial wall hypoplasia with reduction of EPDCs. The cultured embryonic cardiomyocytes with supernatants from Hdac3 knockout (KO) mouse epicardial cells (MECs) also showed decreased proliferation. Genome‐wide transcriptomic analysis revealed that Fgf9 and Igf2 were significantly down‐regulated in Hdac3 KO MECs. We further found that Fgf9 and Igf2 expression is dependent on HDAC3 deacetylase activity. The supplementation of FGF9 or IGF2 can rescue the myocardial proliferation defects treated by Hdac3 KO supernatant. Mechanistically, we identified that microRNA (miR)‐322 and miR‐503 were upregulated in Hdac3 KO MECs and Hdac3 epicardial KO hearts. Overexpression of miR‐322 or miR‐503 repressed FGF9 and IGF2 expression, while knockdown of miR‐322 or miR‐503 restored FGF9 and IGF2 expression in Hdac3 KO MECs. Our findings reveal a critical signaling pathway in which epicardial HDAC3 promotes compact myocardial growth by stimulating FGF9 and IGF2 through repressing miR‐322/miR‐503, providing novel insights in elucidating etiology of congenital heart defects, and conceptual strategies to promote myocardial regeneration.

EFFECT OF THE ACTIVATION TIME OF PLATELET RICH PLASMA AND COLLAGEN ON THE LEVELS OF FIBROBLAST GROWTH FACTOR IN GINGIVAL CREVICULAR FLUID

Background: Gingival recession is a condition that occurs when the gingival margin is more apically than the cemento-enamel junction and causes the tooth root to open, so a root closure procedure is required. Platelet rich plasma (PRP) contains growth factors, including Fibroblast growth factor which plays a role in the process of angiogenesis and cell proliferation. The activation time is known to affect platelet count and FGF expression. The purpose of this study was to determine the effect of the activation time of platelet rich plasma and collagen on the expression of fibroblast growth factor in the gingival crevicular fluid Method: The research sample was gingival crevicular fluid which was taken using paper point #30 for 30 seconds as many as 18 samples. Samples were taken before surgery, day 5 and day 7 after surgery. The samples were divided into 2 groups based on the activation time, 1 hour and 24 hours PRP-collagen. Changes in FGF levels were examined using ELISA. Repeated Meassure ANOVA test was conducted to compare the differences in each time observation and post hoc was performed to compare the differences between groups for each time observation. Result: There was no significant difference in FGF levels in the gingival crevicular fluid at each time and between groups. Conclusion: There was no effect of the activation time of platelet rich plasma and collagen on the levels of fibroblast growth factor in gingival crevicular fluid

COMBINATION OF DECIDUAL DENTAL STEM CELL AND PLATELET RICH PLASMA IN FIBROBLAST FORMATION IN THE JAWBONE REGENERATION OF WISTAR RATS

Background: Orthodontic tooth movement is caused by mechanichal forces that cause pseudo-inflammation with the result that FGF (Fibroblast Growth Factors) was activated. The combination of SHED (Stem Cell From Human Exfoliated Deciduous Theeth) and PRP (Platelet Rich Plasma) hydrogels can affect the formation of fibroblast in the alveolar bone regeneration. The purpose of this study is the effect determination of SHED and PRP hydrogels for fibroblast formation in alveolar bone regeneration in wistar rats. Method: An experimental study was held using post-test only control group design. There were four groups, namely the PRP hydrogel group, the SHED hydrogel group, the SHED and PRP hydrogel group, and the povidone iodine group (n=8) treated with orthodontic tooth movement. Their mandibular tissue was made as histologycal slide, and the FGF expression were observed microscopically with 100x magnification. The data analyzed by ANNOVA and LSD test. Result: There was a significant effect of the combination of SHED and PRP hydrogel in ordering fibroblast in alveolar bone regeneration by ANNOVA test 0.001 (&lt;0.05). This is because SHED and PRP are able to stimulate FGF expression through external stimulation. Conclusion: It concluded that the combination of SHED and PRP hydrogel affected fibroblast formation on alveolar bone regeneration in wistar rats

Transcutaneous Auricular Vagus Nerve Stimulation Promotes White Matter Repair and Improves Dysphagia Symptoms in Cerebral Ischemia Model Rats.

BackgroundClinical and animal studies have shown that transcutaneous auricular vagus nerve stimulation (ta-VNS) exerts neuroprotection following cerebral ischemia. Studies have revealed that white matter damage after ischemia is related to swallowing defects, and the degree of white matter damage is related to the severity of dysphagia. However, the effect of ta-VNS on dysphagia symptoms and white matter damage in dysphagic animals after an ischemic stroke has not been investigated.MethodsMiddle cerebral artery occlusion (MCAO) rats were randomly divided into the sham, control and vagus nerve stimulation (VNS) group, which subsequently received ta-VNS for 3 weeks. The swallowing reflex was measured once weekly by electromyography (EMG). White matter remyelination, volume, angiogenesis and the inflammatory response in the white matter were assessed by electron microscopy, immunohistochemistry, stereology, enzyme-linked immunosorbent assay (ELISA) and Western blotting.Resultsta-VNS significantly increased the number of swallows within 20 s and reduced the onset latency to the first swallow. ta-VNS significantly improved remyelination but did not alleviate white matter shrinkage after MCAO. Stereology revealed that ta-VNS significantly increased the density of capillaries and increased vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF2) expression in the white matter. ta-VNS significantly alleviated the increase inTLR4, MyD88, phosphorylated MAPK and NF-κB protein levels and suppressed the expression of the proinflammatory factors IL-1β and TNF-α.ConclusionThese results indicated ta-VNS slightly improved dysphagia symptoms after ischemic stroke, possibly by increasing remyelination, inducing angiogenesis, and inhibiting the inflammatory response in the white matter of cerebral ischaemia model rats, implying that ta-VNS may be an effective therapeutic strategy for the treatment of dysphagia after ischemic stroke.

Decrease in LINC00963 attenuates the progression of pulmonary arterial hypertension via microRNA-328-3p/profilin 1 axis.

BackgroundPulmonary arterial hypertension (PAH) is a severe cardiopulmonary disease characterized by vascular hyperplasia and remodeling. Long noncoding RNA LINC00963 can regulate cell proliferation and metastasis in nonsmall cell lung cancer. However, the function of LINC00963 on PAH progression is rarely reported.MethodsQuantitative real‐time PCR was used to determine the expression levels of LINC00963, microRNA (miRNA)‐328‐3p, and profilin 1 (PFN1), as well as vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF‐2), and hypoxia‐inducible factor (HIF)‐α. The protein level of PFN1 was measured by western blotting. The viability and migration of hypoxia‐induced pulmonary arterial smooth muscle cells (PASMCs) were assessed by 3‐(4, 5‐dimethyl‐2‐thiazolyl)‐2, 5‐diphenyl‐2‐h‐tetrazolium bromide, and transwell assays, respectively. The target relationships between miR‐328‐3p and LINC00963/PFN1 were confirmed by dual‐luciferase reporter assay. A PAH mouse model was conducted to explore the effects of hypoxia on cardiopulmonary functions.ResultsIn hypoxia‐induced PASMCs and PAH mouse model, high expression levels of LINC00963 and PFN1, and low expression of miR‐328‐3p, were determined. The viability, migration of hypoxia‐induced PASMCs, the expression of VEGF, FGF‐2, and HIF‐α were significantly repressed by transfection of si‐LINC00963 or miR‐328‐3p mimics. The inhibitory effects of LINC00963 silencing on cell viability, migration, and the levels of VEGF, FGF‐2, and HIF‐α were partly eliminated by miR‐328‐3p inhibitor or increasing the expression of PFN1. Hypoxia treatment increased the levels of RVSP, mPAP, and RV/(LV+S), as well as the thickness of pulmonary artery wall.ConclusionsSilencing of LINC00963 ameliorates PAH via modulating miR‐328‐3p/PFN1.