CircPDE7B/miR-661 axis accelerates the progression of human keloid fibroblasts by upregulating fibroblast growth factor 2 (FGF2).

Circular RNAs (circRNAs) are implicated in keloidogenesis and development. We aimed to investigate the role of a new identified phosphodiesterase 7B-derived circRNA (hsa_circ_0002198; henceforth named as PDE7B) in human keloid fibroblasts (HKFs) and to further confirm its mechanism via competing endogenous RNA (ceRNA) network. Transcriptional and translational levels of circPDE7B, microRNA (miR)-661, fibroblast growth factor 2 (FGF2), cleaved caspase3, B-cell lymphoma (bcl)-2, and bcl-2-associated X protein (bax) were detected by real-time quantitative PCR and western blotting. Relationship among circPDE7B, miR-661, and FGF2 was confirmed by bioinformatics algorithm, dual-luciferase reporter assay, RNA immunoprecipitation, RNA pull-down assay, and Spearman's rank correlation analysis. Cell progression was measured by cell counting kit-8 assay, 5-ethynyl-2-deoxyuridine assay, transwell assays, and flow cytometry. Expression of circPDE7B was upregulated in human keloid tissues and HKFs, accompanied with miR-661 downregulation and FGF2 upregulation. High circPDE7B accelerated proliferation, migration, and invasion, and inhibited apoptosis. These effects were paralleled with increased bcl-2 and decreased cleaved caspase3 and bax. Moreover, low circPDE7B played opposite effects to high circPDE7B. Restoring miR-661 could suppress HKFs progression, while blocking miR-661 could facilitate that. Notably, miR-661 was directly sponged by circPDE7B and then directly governed FGF2 gene expression. Deleting miR-661 and re-expressing FGF2 both abrogated the suppression of circPDE7B knockdown in HKFs progression. In conclusion, circPDE7B might contribute to HKFs progression via functioning as ceRNA for miR-661, suggesting a novel circPDE7B/miR-661/FGF2 pathway underlying keloid formation and treatment.

Clinical significance of miR-139-5p and FGF2 in ovarian cancer.

PURPOSE: To estimate the expression and clinical significance of miR-139-5p and fibroblast growth factor 2 (FGF2) in ovarian cancer (OC). METHODS: Of the 198 female patients undergoing surgical treatment in our hospital, 101 patients with ovarian tumor resection were allocated in a study group and 97 with ovarian resection for benign lesions were allocated in a control group. MiR-139-5p and FGF2 expression was quantified, and associations between miR-139-5p and FGF2 and clinicopathological features of OC were analyzed, as well as their diagnostic performances (receiver operating characteristic (ROC) curve). RESULTS: The study group presented lower miR-139-5p level and higher FGF2 level (both p<0.05). Significant associations of miR-139-5p and FGF2 with tumor differentiation and clinical stage were noted in OC (p<0.05). MiR-139-5p was reversely associated with clinical stage and positively associated with tumor differentiation (p<0.05), FGF2 was positively correlated with clinical stage and negatively correlated with tumor differentiation (p<0.05). The overall survival in the study group was 70.41%. The survival in high miR-139-5p expression group and low FGF2 expression group improved remarkably (p<0.05). The area under the curve (AUC) of combined detection (0.91) was higher than that of single detection. CONCLUSION: MiR-139-5p shows a decreased expression and FGF-2 shows an increased expression in OC, and they are associated with clinical stage and tumor differentiation. Combined detection of miR-139-5p and FGF-2 contributes to the diagnosis and treatment of OC, and is an available biomarker for the diagnosis and prognosis of patients.

Upregulation of fibroblast growth factor 2 contributes to endometriosis through SPRYs/DUSP6/ERK signaling pathway.

Previous studies report that fibroblast growth factor 2 (FGF2) modulates Sproutys (SPRYs)/dual specificity phosphatase 6 (DUSP6)/extracellular signal-regulated kinase (ERK) signaling pathway in endometrial glandular epithelial cells. However, its role in endometriosis remains unclear. The expression patterns and localization of related proteins in endometrium patients' samples were determined using quantitative reverse transcription PCR, Western blotting, and immunohistochemistry, respectively. Human endometrial stromal cells (HESCs) were isolated and transfected with small interfering RNA (siRNA) targeting FGF2 (FGF2-siRNA). Cell viability was determined using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. It was found that FGF2 mRNA and protein levels were increased in the ectopic endometrium, whilst the mRNA and protein levels of SPRYs/DUSP6/ERK signaling pathway related-genes were dysregulated. Spearman's rank correlation analysis revealed a negative correlation between FGF2 and SPRYs/DUSP6 signaling pathway-related proteins. In vitro study demonstrated that FGF2 silencing suppressed cell proliferation. Our results suggest that FGF2 upregulation might contribute to endometriosis via the regulation of the SPRYs/DUSP6/ERK signaling pathway.

AAV2-mediated and hypoxia response element-directed expression of bFGF in neural stem cells showed therapeutic effects on spinal cord injury in rats.

Neural stem cell (NSCs) transplantation has been one of the hot topics in the repair of spinal cord injury (SCI). Fibroblast growth factor (FGF) is considered a promising nerve injury therapy after SCI. However, owing to a hostile hypoxia condition in SCI, there remains a challenging issue in implementing these tactics to repair SCI. In this report, we used adeno-associated virus 2 (AAV2), a prototype AAV used in clinical trials for human neuron disorders, basic FGF (bFGF) gene under the regulation of hypoxia response element (HRE) was constructed and transduced into NSCs to yield AAV2-5HRE-bFGF-NSCs. Our results showed that its treatment yielded temporally increased expression of bFGF in SCI, and improved scores of functional recovery after SCI compared to vehicle control (AAV2-5HRE-NSCs) based on the analyses of the inclined plane test, Basso-Beattie-Bresnahan (BBB) scale and footprint analysis. Mechanistic studies showed that AAV2-5HRE-bFGF-NSCs treatment increased the expression of neuron-specific neuronal nuclei protein (NeuN), neuromodulin GAP43, and neurofilament protein NF200 while decreased the expression of glial fibrillary acidic protein (GFAP) as compared to the control group. Further, the expressions of autophagy-associated proteins LC3-II and Beclin 1 were decreased, whereas the expression of P62 protein was increased in AAV2-5HRE-bFGF-NSCs treatment group. Taken together, our data indicate that AAV2-5HRE-bFGF-NSCs treatment improved the recovery of SCI rats, which is accompanied by evidence of nerve regeneration, and inhibition of SCI-induced glial scar formation and cell autophagy. Thus, this study represents a step forward towards the potential use of AAV2-5HRE-bFGF-NSCs for future clinical trials of SCI repair.

HOXD-AS1 facilitates cell migration and invasion as an oncogenic lncRNA by competitively binding to miR-877-3p and upregulating FGF2 in human cervical cancer.

BACKGROUND: Long non-coding RNAs (LncRNAs) are dysregulated in multiple human cancers and they are highly involved in tumor progression. Previous studies have identified the oncogenic lncRNA HOXD cluster antisense RNA 1 (HOXD-AS1) in human cancers, while its roles in cervical cancer (CC) remain unclear. Herein we intended to characterize the implication of HOXD-AS1 in CC. METHODS: qRT-PCR was applied to examine the relative expression of HOXD-AS1 in CC tissues, cell lines and transfected cells. Wound healing and transwell assays were applied to detect cell migration and invasion alteration. The targeting relationship between miRNA and mRNA/lncRNA was determined by dual luciferase reporter, qRT-PCR and western blot assays. RESULTS: HOXD-AS1 was overexpressed in CC tissues and cell lines. Its higher level predicted worse prognosis of CC patients. SiRNA mediated knockdown of HOXD-AS1 repressed CC cell migration and invasion, and its overexpression did the opposite. Mechanistically, HOXD-AS1 acted as a competing endogenous RNA (ceRNA) to sponge miR-877-3p and led to upregulation of FGF2, a target of miR-877-3p. Importantly, either miR-877-3p overexpression or FGF2 inhibition could abolish the migration and invasion promotion induced by HOXD-AS1. CONCLUSION: HOXD-AS1 functions as a tumor-promoting lncRNA via the miR-877-3p/FGF2 axis in CC. HOXD-AS1 might be a promising therapeutic target as well as a novel prognostic biomarker for CC.

MicroRNA-936 promotes proliferation and invasion of gastric cancer cells by down-regulating FGF2 expression and activating P13K/Akt signaling pathway.

OBJECTIVE: This study was designed to investigate whether microRNA-936 can be involved in the development of gastric cancer (GCa) by down-regulating FGF2 expression and activating the phosphatidylinositol 3-kinase/protein kinase B (P13K/Akt) signaling pathway. PATIENTS AND METHODS: Quantitative polymerase chain reaction (qPCR) was carried out to examine microRNA-936 and FGF2 levels in GCa tissue samples and adjacent normal ones, and further in GCa cell lines. After transfection of microRNA-936 inhibitor in GCa cell lines BGC and SGC, cell invasion, and proliferation abilities were evaluated by transwell and cell counting kit-8 (CCK-8) assays, respectively. In addition, the Dual-Luciferase reporting assay was conducted to verify the binding relationship between microRNA-936 and FGF2. After simultaneous transfection of microRNA-936 inhibitor and si-FGF2 in GCa cells, we detected the expression of FGF2/P13K/Akt by performing qPCR and Western blot experiments to further verify the regulation of microRNA-936 on FGF2 and PI3K/AKT pathway. RESULTS: QPCR detection revealed that microRNA-936 was remarkably up-regulated while FGF2 was conversely down-regulated in GCa tissue samples, indicating a negative correlation between the two. In addition, compared with normal gastric mucosal cells GES, microRNA-936 showed a significant increased expression in GCa cell lines. Meanwhile, down-regulation of microRNA-936 caused a marked reduction in invasive and proliferation ability of GCa cells. Dual-Luciferase reporting assay demonstrated a direct binding of microRNA-936 to FGF2. QPCR and Western blot showed that microRNA-936 can inhibit FGF2 expression and activate the PI3K/AKT pathway at the same time. Further studies found that silencing FGF2 induced an enhancement in cell proliferation and invasiveness, which could be reversed by simultaneous downregulation of microRNA-936. The above observations suggested that microRNA-936 may accelerate the progression of GCa by inhibiting FGF2 expression and activating the PI3K/AKT pathway. CONCLUSIONS: Overexpression of microRNA-936 can be conducive to the development of GCa, mainly through the down-regulation of FGF2 and activation of the P13K/Akt signaling pathway.

Regulation of MYO18B mRNA by a network of C19MC miRNA-520G, IFN-γ, CEBPB, p53 and bFGF in hepatocellular carcinoma.

MYO18B has been proposed to contribute to the progression of hepatocellular carcinoma (HCC). However, the signals that govern MYO18B transcription are not known. Here we show that, a network of C19MC miRNA-520G, IFN-γ, CEBPB and p53 transcriptional-defects promote MYO18B mRNA expression in HCCs. IFN-γ by itself suppresses MYO18B transcription, but promotes it when miRNA-520G is stably overexpressed. Similarly, CEBPB-liver-enriched activator protein (LAP) isoform overexpression suppresses MYO18B transcription but promotes transcription when the cells are treated with IFN-γ. Furthermore, miR-520G together with mutant-p53 promotes MYO18B transcription. Conversely, bFGF suppresses MYO18B mRNA irrespective of CEBPB, miR-520G overexpression or IFN-γ treatment. Finally high MYO18B expression reflects poor prognosis while high MYL5 or MYO1B expression reflects better survival of HCC patients. Thus, we identified a network of positive and negative regulators of MYO18B mRNA expression which reflects the survival of HCC patients.

Fermentation, purification and immunogenicity of a recombinant tumor multi-epitope vaccine, VBP3.

The recombinant multi-epitope vaccine called VBP3 is designed to suppress tumor growth and angiogenesis through targeting both basic fibroblast growth factor (bFGF) and vascular endothelial growth factor A (VEGFA). We are aiming to produce VBP3 vaccine in a large scale and provide sufficient protein for pre-clinical study. High cost and potential toxicity are severe limitations of IPTG and we investigated whether lactose can mediate VBP3 induction. Firstly, we identified the biological characteristics and established a culture bank of VBP3 strains. The best-performing strains were selected and the fermentation mode of medium, bacterial growth and protein expression were optimized in shake flasks. We scaled up the VBP3 production in 10 L bioreactor using lactose as inducer and the protein yield was comparable with IPTG induction. Next, the target protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, with a SDS-PAGE purity over 90%. Further, the purified VBP3 vaccine was subcutaneously injected in BALB/c mice and elicited high-titer anti-bFGF (1:32,000) and anti-VEGFA (1:4000) antibodies. Take together, lactose was an applicable inducer for VBP3 production and the eligible product of VBP3 was harvested in the large-scale fermentation, supporting the industrial production and pre-clinical study in the future. The VBP3 vaccine with superior immunogenicity might be used as a potential therapeutic vaccine for tumor treatment.

Early growth response gene mediates in VEGF and FGF signaling as dissected by CRISPR in corpus luteum of water buffalo.

The EGR family comprises of EGR 1, EGR 2, EGR 3 and EGR 4 which are involved in the transactivation of several genes. A broad range of extracellular stimuli by growth factors is capable of activating EGR mediated transactivation of genes involved in angiogenesis and cell proliferation. However, their role in controlling VEGF A and FGF 2 signaling in the CL of water buffalo is not known. The present study was conducted to understand the role of EGR mediated regulation of VEGF A and FGF 2 signaling in buffalo luteal cells. Towards this goal, luteal cells were cultured and treated with VEGF A and FGF 2 and the mRNA expression pattern of EGR family members were documented. The EGR 1 message was found to be up-regulated in luteal cells of buffalo at 72 hours of culture. The functional validation of EGR 1 gene was accomplished by knocking out (KO) of EGR 1 in cultured luteal cells by CRISPR/Cas9 mediated gene editing technology. The EGR 1 KO cells were then cultured and stimulated with VEGF A and FGF 2. It was observed that VEGF A and FGF 2 induced angiogenesis, cell proliferation and steroidogenesis in wild type luteal cells, whereas the response of the growth factors was attenuated in the EGR 1 KO cells. Taken together our study provides evidence convincingly that both VEGF and FGF mediate their biological action through a common intermediate, EGR 1, to regulate corpus luteum function of buffalo.

LncRNA PCGEM1 promotes renal carcinoma progression by targeting miR-433-3p to regulate FGF2 expression.

Long non-coding RNAs (lncRNAs) are implicated in the development of carcinomas, containing renal carcinoma. The competing endogenous RNA (ceRNA) network is well-known in modulating the pathological and physiological processes of tumors. Still and all, the function role of oncogenic lncRNA PCGEM1 prostate-specific transcript (PCGEM1) in renal carcinoma was undefined till now. This paper aimed to figure out the role and mechanism of PCGEM1 in renal carcinoma. In this study, PCGEM1 was observed to be lifted in renal carcinoma cells. Loss-of-function experiments displayed that silencing of PCGEM1 repressed cell proliferation and migration, and activated apoptosis in renal carcinoma. FISH assay and subcellular fractionation assay indicated that PCGEM1 was largely located in the cytoplasm. As demonstrated, PCGEM1 interacted with microRNA433-3p (miR-433-3p). Subsequently, luciferase reporter and RIP experiments together with qRT-PCR certified that PCGEM1 and fibroblast growth factor 2 (FGF2) functioned as ceRNA for miR-433-3p, leading to the upregulation of FGF2 expression. Finally, rescue assays exhibited that FGF2 overexpression rescued the inhibited cell progression caused by PCGEM1 downregulation. MiR-433-3p inhibitor could reverse the cell growth and migration caused by PCGEM1 downregulation. The present research investigated the molecular mechanism underlying PCGEM1 in renal carcinoma, exposing a PCGEM1-mediated therapy for the treatment of patients with renal carcinoma.

Osteoblasts grown on microroughened titanium surfaces regulate angiogenic growth factor production through specific integrin receptors.

Cellular attachment and response to biomaterials are mediated by integrin receptor binding to extracellular matrix proteins adsorbed onto the material surface. Osteoblasts interact with their substrates via several integrin complexes including fibronectin-binding α5ß1 and collagen-binding α1ß1 and α2ß1. Knockdown of α2 or ß1 integrin subunits inhibits the production of factors that promote an osteogenic microenvironment, including osteocalcin, osteoprotegerin, and TGFß1. Osteoblasts also secrete several angiogenic growth factors such as VEGF-A (VEGF165), FGF-2, and angiopoietin 1, which are regulated by titanium surface topography and surface energy. Here, we examined whether signaling through integrin receptor complexes regulates production and secretion of angiogenic factors during osteoblast differentiation on microtextured Ti surfaces. To do this, integrin subunits α1, α2, α5, and ß1 were stably silenced in MG63 osteoblast-like cells cultured on grit-blasted/acid-etched hydrophobic Ti (SLA) or on hydrophilic SLA (modSLA). VEGF-A production increased in response to Ti surface topography and energy in integrin α2, α5, and ß1 silenced cells but decreased in α1-silenced cells. FGF-2 decreased on modSLA substrates in both α1 and α2-silenced cells but was unchanged in response to silencing of either α5 or ß1. In integrin α1, α2, and ß1-silenced cells, Ang-1 increased on modSLA but α5-silencing did not affect Ang-1 production during surface mediated differentiation. These results suggest that signaling through specific integrin receptor complexes during osteoblast differentiation on microstructured Ti substrates, regulates the production of angiogenic factors by those cells, and this is differentially regulated by surface hydrophilicity. STATEMENT OF SIGNIFICANCE: Successful implantation of synthetic biomaterials into bone depends on the biological process known as osseointegration. Osseointegration is a highly regulated communication of cells that orchestrates the migration of progenitor cells towards the implant site and promotes the deposition and mineralization of extracellular matrix proteins within the implant microenvironment, to tightly join the implant to native bone. In this process, angiogenesis functions as the initiation site of progenitor cell migration and is necessary for matrix deposition by providing the necessary nutrients for bone formation. In the present study, we show a novel regulation of specific angiogenic growth factors by integrin receptor complexes. This research is important to develop biomaterials that promote and maintain osseointegration through proper vascularization and prevent implant failure in patients lacking sufficient angiogenesis.

bFGF overexpression adipose derived mesenchymal stem cells improved the survival of pulmonary arterial endothelial cells via PI3k/Akt signaling pathway.

Pulmonary arterial hypertension (PAH) is characterized as pulmonary arterial endothelial dysfunction and endothelial cells over proliferation, therefore, the repair of pulmonary arterial endothelial cells has been a common goal in treating PAH. In the present study, human adipose derived mesenchymal stem cells (ASCs) were transfected with bFGF lentiviral vector and co-cultured with monocrotaline pyrrole treated human pulmonary arterial endothelial cells (HPAECs). The results showed that bFGF-ASCs improved the proliferation, viability and decreased the apoptosis of HPAECs, besides, improved PAH was observed in PAH rat models. Western blot analysis showed that the PI3k and p-Akt protein expression level increased in HPAECs, suggesting the activation of the PI3k/Akt signaling pathway. With the administration of LY294002, the bFGF induced HPAECs survival and PI3k/Akt signaling activation were successfully blocked. The present study demonstrated that bFGF transfected ASCs improved the survival of HPAECs by activating the PI3k/Akt pathway.

circRAD18 sponges miR-208a/3164 to promote triple-negative breast cancer progression through regulating IGF1 and FGF2 expression.

As a new rising star of non-coding RNA, circular RNAs (circRNAs) emerged as vital regulators with biological functions in diverse of cancers. However, the function and precise mechanism of the vast majority of circRNAs in triple-negative breast cancer (TNBC) occurrence and progression have not been clearly elucidated. In the current study, we identified and further investigated hsa_circ_0002453 (circRAD18) by analyzing our previous microarray profiling. Expression of circRAD18 was found significantly upregulated in TNBC compared with normal mammary tissues and cell lines. circRAD18 was positively correlated with T stage, clinical stage and pathological grade and was an independent risk factor for TNBC patients. We performed proliferation, colony formation, cell migration, apoptosis and mouse xenograft assays to verify the functions of circRAD18. Knockdown of circRAD18 significantly suppressed cell proliferation and migration, promoted cell apoptosis and inhibited tumor growth in functional and xenograft experiments. Through luciferase reporter assays, we confirmed that circRAD18 acts as a sponge of miR-208a and miR-3164 and promotes TNBC progression through upregulating IGF1 and FGF2 expression. Altogether, our research revealed the pivotal role of circRAD18-miR-208a/3164-IGF1/FGF2 axis in TNBC tumorigenesis and metastasis though the mechanism of competing endogenous RNAs. Thus, circRAD18 may serve as a novel prognostic biomarker and potential target for TNBC treatment in the future.

MicroRNA-23c inhibits articular cartilage damage recovery by regulating MSCs differentiation to chondrocytes via reducing FGF2.

OBJECTIVE: The aim of the study was to explore the role of microRNA-23c in the differentiation of marrow stromal cells (MSCs) to chondrocytes and its potential mechanism. MATERIALS AND METHODS: MSCs were first isolated from rat bone marrow for cell culture. Surface antigens of MSCs (CD29 and CD34) were identified by flow cytometry. MSCs were induced for chondrogenic differentiation in MCDM (Mesenchymal Stem Cell Chondrogenic Differentiation Medium) for 0, 3, and 7 days, respectively, followed by detection of RUNX2, microRNA-23c and FGF2 expressions by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Alcian blue staining was performed to access proteoglycan deposition in MSCs transfected with microRNA-23c mimics or inhibitor. Western blot was conducted to detect the protein expressions of ACAN and COL2A1 in MSCs. The binding condition between microRNA-23c and FGF2 was verified by dual-luciferase reporter gene assay. Finally, MSCs were co-transfected with microRNA-23c mimics and FGF2 overexpression plasmid for rescue experiments. RESULTS: On the fourth day of MSCs isolation, MSCs were in an elongated shape. Flow cytometry results showed positive expression of CD29 and negative expression of CD34, which were consistent with MSCs phenotype. QRT-PCR data elucidated that the mRNA levels of RUNX2 and FGF2 gradually increased, whereas microRNA-23c expression decreased with the prolongation of chondrogenic differentiation. Transfection of microRNA-23c mimics in MSCs remarkably elevated microRNA-23c expression. Alcian blue staining showed that microRNA-23c overexpression results in less proteoglycan deposition in MSCs than that of controls. Both mRNA and protein expressions of ACAN and COL2A1 decreased after microRNA-23c overexpression. Dual-luciferase reporter gene assay confirmed that FGF2 binds to microRNA-23c. Further Western blot results demonstrated that FGF2 expression is negatively regulated by microRNA-23c. FGF2 overexpression reversed the inhibitory effects of microRNA-23c on proteoglycan deposition, as well as expressions of ACAN and COL2A1. CONCLUSIONS: MicroRNA-23c expression decreases during chondrogenic differentiation of MSCs, which inhibits MSCs differentiation to chondrocytes by inhibiting FGF2.

Effects of transplantation of FGF-2-transfected MSCs and XACB on TNF-α expression with avascular necrosis of the femoral head in rabbits.

Objective: The present study aimed to investigate the effect of the transplantation of basic fibroblast growth factor (FGF-2) gene-transfected mesenchymal stem cells (MSCs) and xenogeneic antigen-cancellous bone (XACB) on tumor necrosis factor-α (TNF-α) expression with avascular necrosis of the femoral head (ANFH) in rabbits. Methods: The models of steroid-induced osteonecrosis in rabbits were randomly divided into five groups: A (model), B (XACB), C (XACB + MSCs), D (XACB + MSCs + LV), and E (XACB + MSCs + LV-FGF-2) groups. The therapeutic effect was evaluated by Hematoxylin and Eosin (H&E) staining. Immunohistochemical and RT-PCR assays were used to detect the protein and mRNA expression of TNF-α in the femoral head, respectively. Results: At 12 weeks after the operation, the defect in rabbits in group E was completely repaired, while defects in rabbits in the other groups were not completely repaired, and the area of new bone formation was higher, when compared with the other groups (P<0.05). Furthermore, the protein and mRNA expression TNF-α was lower at 3, 6, and 12 weeks after surgery, when compared with the other groups, and the difference was statistically significant (P<0.05). Conclusion: FGF-2/MSCs/XACB could promote the repair of ANFH, and may be correlated to the inhibition of TNF-α expression.

Chemically Modified Messenger RNA: Modified RNA Application for Treatment of Achilles Tendon Defects.

Different regenerative medicine approaches for tendon healing exist. Recently, especially gene therapy gained popularity. However, potential mutagenic and immunologic effects might prevent its translation to clinical research. Chemically modified mRNA (cmRNA) might bypass these limitations of gene therapy. Therefore, the purpose of this study was to evaluate the early healing properties of Achilles tendon defects in rats treated with basic fibroblast growth factor (bFGF) cmRNA. Forty male Lewis rats were used for the study and randomly assigned to two study groups: (1) treatment with cmRNA coding for bFGF and (2) noncoding cmRNA control. Protein expression was measured using in vivo bioluminescence imaging at 24, 48, and 72 h, as well as 14 days. Animals were euthanized 2 weeks following surgery. Biomechanical, histological, and immunohistological analyses were performed with the significance level set at p < 0.05. Protein expression was evident for 3 days. At 14 days, bioluminescence imaging revealed only little protein expression. Biomechanically, tendons treated with bFGF cmRNA showed a construct stiffness closer to the healthy contralateral side when compared with the control group (p = 0.034), without any significant differences in terms of load to failure. Hematoxylin and eosin staining detected no side effects of the treatment, as signs of inflammation, or necrosis. Furthermore, it revealed the shape of the nuclei to be more oval in the bFGF group in the tendon midsubstance (p = 0.043) with a reduced cell count (p = 0.035). Immunohistological staining for type I, II, III, and IV collagen did not differ significantly between the two groups. In conclusion, this pilot study demonstrates the feasibility of a novel messenger RNA (mRNA)-based therapy for Achilles tendon defects using chemically modified mRNA coding for bFGF.

VEGF-A/VEGFR-2 and FGF-2/FGFR-1 but not PDGF-BB/PDGFR-ß play important roles in promoting immature and inflammatory intraplaque angiogenesis.

Various angiogenic factors have been shown to play important roles in intraplaque angiogenesis, while little is known about the dynamic expression change and interplay between various angiogenic factors and intraplaque angiogenesis under high cholesterol conditions. New Zealand rabbits underwent balloon injury of the abdominal artery and then were assigned to a control group (n = 15, normal chow) or high cholesterol group (n = 25, 1% high cholesterol diet). At weeks 4, 6, 8, 10, and 12 after acclimation, rabbits (high cholesterol group, n = 5; control group, n = 3) were euthanized. No lesions were observed in the control group. From week 4 to week 12, the expression of vascular endothelial growth factor A (VEGF-A), VEGF receptor 2 (VEGFR-2), fibroblast growth factor 2 (FGF-2), FGF receptor 1 (FGFR-1), platelet-derived growth factor-BB (PDGF-BB), and tumor necrosis factor alpha (TNF-α), the vulnerability index (VI) and the microvessel density (MVD) were significantly elevated in the high cholesterol group; however, PDGF receptor ß (PDGFR-ß) expression showed little change. Analysis by double-label immunofluorescence (CD31 and Ng2) and FITC-dextran indicated that the neovessels within the plaque were leaky due to a lack of pericytes. As indicated by Pearson's correlation analysis, there was a highly positive correlation between the VI, MVD, macrophage content, and TNF-α level, and the levels of VEGF-A/VEGFR-2 and FGF-2/FGFR-1. However, no correlations were observed between PDGFR-ß levels and the VI or MVD. High expression of VEGF-A/VEGFR-2 and FGF-2/FGFR-1 but not of PDGF-BB/PDGFR-ß may contribute to immature and inflammatory intraplaque angiogenesis and plaque instability in a rabbit model of atherosclerosis.

[The Novel Short Isoform of Securin Stimulates the Expression of Cyclin D3 and Angiogenesis Factors VEGFA and FGF2, but Does Not Affect the Expression of MYC Transcription Factor].

Pituitary tumor-transforming gene-1 (PTTG1) encodes securin, a multifunctional protein involved in development of various types of cancer. Securin participates in the regulation of sister chromatids separation and the expression of multiple genes involved in the control of the cell cycle, metabolism, and angiogenesis. In several human cell lines, we have found a novel short isoform of securin mRNA, which does not contain exons 3 and 4. After the translation of this new mRNA, a shortened protein is produced that, like the full-size form, is able to activate the transcription of cyclin D3 gene (CCND3), which controls the G1/S transition and angiogenesis factors VEGFA (vascular endothelial growth factor), and FGF2 (fibroblast growth factor 2) in HEK293 cells. However, unlike the full-size protein, the short isoform of PTTG1 does not affect the MYC gene expression because it lacks the DNA-binding domain, which is needed for its interactions with the MYC promoter. Furthermore, the short form of securin does not influence the expression of MYC transcriptional targets, such as TP53 and IL-8. Thus, we found a novel isoform of securin which is able to activate a more restricted repertoire of genes compared to the full-size protein.

In-office tooth bleaching with 38% hydrogen peroxide promotes moderate/severe pulp inflammation and production of ll-1ß, TNF-ß, GPX, FGF-2 and osteocalcin in rats.

OBJECTIVES: To study the intensity of inflammatory infiltrate and production of interleukin-1ß (ll-1ß), tumor necrosis factor-ß (TNF-ß), fibroblast growth factor-2 (FGF-2), glutathione peroxidase (GPX), and osteocalcin in response to in-office tooth bleaching in rats. MATERIAL AND METHODS: Twenty male Wistar rats were randomized into four groups (n=5) according to the received treatment (tooth bleaching or no treatment - control) and the period of euthanasia after treatment (24 h or 10 days). We performed tooth bleaching using a 38% hydrogen peroxide gel on maxillary and mandibular incisors. After euthanasia, incisors (20 per group) were processed for histological analysis, immunohistochemistry staining of ll-1ß, TNF-ß, FGF-2 and GPX and osteocalcin by immunofluorescence. We analyzed data using the Mann-Whitney and Kruskal-Wallis/Dunn tests (p<0.05). RESULTS: The bleached groups presented statistically significant differences regarding the pulp inflammation stage compared with the control groups. Bleached teeth showed moderate/severe inflammatory infiltrate and control groups presented absent inflammatory cells or a negligible number of mononuclear cells (p<0.001) at two times (24 h and 10 days). There was strong staining for ll-1ß, TNF-ß, and GPX in bleached groups at 24 h and strong staining for ll-1ß, TNF-ß, GPX and FGF-2 at 10 days. After 10 days of tooth bleaching, the bleached group showed a statistically superior amount of osteocalcin than the other groups (p<0.01). CONCLUSIONS: Tooth bleaching with 38% hydrogen peroxide causes severe pulp inflammation, but characteristics of tissue repair after 10 days.

Effects of fibroblast growth factors 2 and low intensity pulsed ultrasound on the repair of knee articular cartilage in rabbits.

OBJECTIVE: To investigate the effect of low intensity ultrasound irradiation combine with fibroblast growth factors (FGF2) on the repair of the knee articular cartilage and to explore its mechanism in rabbit. MATERIALS AND METHODS: The model of the rabbit knee joint injury was established. 40 rabbits were divided into four groups, including control group, model group, FGF2 group and FGF2 + low intensity pulsed ultrasound group (FGF2 + LIPU). The knee joints of rabbits were taken at 4 and 8 weeks, respectively. Histopathological changes were detected by Hematoxylin and Eosin stain (HE) and evaluated by Wakitani score. The expression of FGF2 mRNA was detected by Real-time polymerase chain reaction (RT-PCR) and the levels of Collagen I and Collagen II protein were analyzed by Western blotting. RESULTS: In FGF2 group and FGF2 + LIPU group, it was found that the tissues of knee joint were gradually repaired following the change of time. Further, the recovery was better in FGF2 + LIPU group. Cartilage defect areas were filled with cartilage-like cells and the repair surface was fused with surrounding cartilage in FGF2 and FGF2 + LIPU groups. Wakitani scores were consistent with HE results. The expressions of FGF2 mRNA were higher in FGF2 and FGF2 + LIPU group than the model group. Western blotting results showed that the levels of Collagen I and Collagen II protein in FGF2 and FGF2 + LIPU groups were significantly increased compared with that in model group. CONCLUSIONS: FGF2 and LIPU combined application on the rabbit knees joint repair is better than FGF2 alone. FGF2 and LIPU combination can promote the synthesis and secretion of collagen in chondrocytes, promote the differentiation and maturation of chondrocytes during the repair of cartilage defects.