Expression Levels Of Fibroblast Growth Factor 2 Research Articles

MiR-129-5p/FGF2 Axis is Associated with Homocysteine-induced Human Umbilical Vein Endothelial Cell Injury.

Homocysteine (Hcy)-induced endothelial cell injury is a key event in atherosclerosis pathogenesis. In this study, we aimed to explore the mechanisms underlying Hcy-induced endothelial injury by assessing the effects of Hcy on endothelial cell proliferation and the microRNA (miR)-129-5p/fibroblast growth factor 2 (FGF2) axis. Human umbilical vein endothelial cells (HUVECs) were treated with Hcy to construct an endothelial cell injury model. Expression levels of FGF2 in Hcy-induced HUVECs were determined using quantitative real-time polymerase chain reaction and western blotting. An FGF2 overexpression lentiviral vector was constructed to upregulate FGF2 expression in HUVECs via lentivirus transduction. A cell counting kit-8 assay was used to explore the effects of FGF2 overexpression on HUVEC proliferation. An upstream regulatory miRNA was predicted, and its targetbinding relationship with FGF2 was evaluated using a dual-luciferase reporter assay. We found that FGF2 expression in HUVECs was inhibited by Hcy treatment. Lentivirus transduction led to the overexpression of FGF2 in HUVECs, which significantly reversed the effect of Hcy on endothelial cell proliferation. miR-129-5p was experimentally validated as an upstream regulator of FGF2, and its decreased levels in HUVECs led to increased FGF2 expression. In addition, HUVEC proliferation was enhanced by the knockdown of miR-129-5p, and this effect was reversed by Hcy treatment. Taken together, the results of this study revealed that Hcy inhibits FGF2 expression in HUVECs, and FGF2 is regulated by upstream miR-129-5p to improve the effect of Hcy on endothelial cell proliferation.

FGFR blockade inhibits targeted therapy-tolerant persister in basal FGFR1- and FGF2-high cancers with driver oncogenes

Cancer cell resistance arises when tyrosine kinase inhibitor (TKI)-targeted therapies induce a drug-tolerant persister (DTP) state with growth via genetic aberrations, making DTP cells potential therapeutic targets. We screened an anti-cancer compound library and identified fibroblast growth factor receptor 1 (FGFR1) promoting alectinib-induced anaplastic lymphoma kinase (ALK) fusion-positive DTP cell’s survival. FGFR1 signaling promoted DTP cell survival generated from basal FGFR1- and fibroblast growth factor 2 (FGF2)-high protein expressing cells, following alectinib treatment, which is blocked by FGFR inhibition. The hazard ratio for progression-free survival of ALK-TKIs increased in patients with ALK fusion-positive non-small cell lung cancer with FGFR1- and FGF2-high mRNA expression at baseline. The combination of FGFR and targeted TKIs enhanced cell growth inhibition and apoptosis induction in basal FGFR1- and FGF2-high protein expressing cells with ALK-rearranged and epidermal growth factor receptor (EGFR)-mutated NSCLC, human epidermal growth factor receptor 2 (HER2)-amplified breast cancer, or v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-mutated melanoma by preventing compensatory extracellular signal-regulated kinase (ERK) reactivation. These results suggest that a targeted TKI-induced DTP state results from an oncogenic switch from activated oncogenic driver signaling to the FGFR1 pathway in basal FGFR1- and FGF2-high expressing cancers and initial dual blockade of FGFR and driver oncogenes based on FGFR1 and FGF2 expression levels at baseline is a potent treatment strategy to prevent acquired drug resistance to targeted TKIs through DTP cells regardless of types of driver oncogenes.

Effect of acupuncture on the opening time of implantation window and endometrial receptivity in controlled ovarian hyperstimulation rats during peri-implantation period.

To investigate the effect of acupuncture for improving the pregnancy rate of COH rats from the viewpoint of regulating the opening time of the implantation window and endometrial receptivity. Experimental rats were randomly divided into normal group (N), model group (M) and acupuncture group(A), and samples were collected on Day 4, 5 and 6 after mating. COH rats were treated with acupuncture at SP6, LR3, and ST36 once a day for 7 times. The pinopodes were observed under a scanning electron microscope. Serum estrogen and progesterone levels were measured via ELISA. The protein and mRNA levels of estrogen receptor (ER), progesterone receptor (PR), leukemia inhibitory factor (LIF), integrin β3, vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF-2) in the endometrium were evaluated via West-blot, immunohistochemistry, and PCR. Compared with group N, the pregnancy rate of group M was significantly decreased (P<0.05), and the abnormal serum hormone levels and implantation window advancement were observed. Compared with group M, the pregnancy rate of group A was significantly increased (P<0.05), the supraphysiological serum progesterone levels were restored to normalcy (P<0.05), and the advanced implantation window was restored to a certain extent. Further, the abnormal ER, PR, LIF, integrin β3, VEGF, and FGF-2 expression levels of the endometrium got recovered to varying degrees. Acupuncture may restore the estrogen and progesterone balance in COH rats and the forward shift of the implantation window to a certain extent, improving the endometrial receptivity and finally improving the pregnancy rate of COH rats.

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.

Investigation of the potential therapeutic effect of cationic lipoplex mediated fibroblast growth factor-2 encoding plasmid DNA delivery on wound healing.

Developing an alternative and efficient therapy for wound healing has been an important research topic for pharmaceutical sciences. A straightforward but effective system for delivering fibroblast growth factor-2 (FGF-2) encoding plasmid DNA (pFGF-2) for wound healing therapy was aimed to develop in this study. In order to provide the delivery of pFGF-2, a delivery vector, namely, cationic lipid nanoparticle (cLN) was developed by the melt-emulsification process, complexed with pFGF-2 to form a lipoplex system and further characterized. The pFGF-2 binding and protecting ability of lipoplexes were evaluated. The cytotoxicity and transfection efficiency of the lipoplexes, FGF-2 expression levels, and in vitro wound healing ability have been investigated on the L929 fibroblast cell line. The obtained lipoplex system has a particle size of 88.53nm with a low PDI (0.185), and zeta potential values of 27.8mV with a spherical shape. The ability of cLNs to bind pFGF-2 and protect against nucleases was demonstrated by gel retardation assay. Furthermore, the developed FGF-2 carrying lipoplexes system showed significant transfection and FGF-2 expression ability comparing naked plasmid. Finally, scratch assay revealed that the developed system is able to promote in vitro cell proliferation/migration in 48h. Promising results have been achieved with the use of lipoplexes carrying pFGF-2, and this approach could be considered as a potentially applicable concept for the future gene-based wound healing therapies.

WITHDRAWN: Lnc-H19 enhances anaerobic glycolysis of keloid fibroblasts by targeting the miR-214-5p/FGF2 axis

Long non-coding RNA (lncRNA) H19 has been demonstrated as vital regulator in tumors. However, whether lnc-H19 mediated the development of keloid fibroblasts (KD) was unknown, this study was aimed to clarify the role and molecular mechanisms of lnc-H19 in KD. We have investigated the expression levels of lnc-H19, miR-214-5p and fibroblast growth factor 2 (FGF2) in KD skin samples and normal skin tissues as well as matched cells by real-time quantitative polymerase chain reaction (RT-qPCR) assay. The glycolysis ability of keloid fibroblasts was assessed by measuring glucose consumption, lactate production, and ATP level. The western blot assay was used to assay the expression levels of FGF2 and hexokinase 2 (HK2). Migration and invasion were analyzed by transwell in keloid fibroblasts. The bioinformatics database and dual-luciferase reporter assay were used to search and identify the target of miR-214-5p and lnc-H19. Lnc-H19 was overexpressed in KD tissues and keloid fibroblasts than normal skin tissues and normal fibroblasts, respectively. Small interfering RNA of lnc-H19 treatment markedly inhibited glycolysis, migration and invasion of keloid fibroblasts exposed to hypoxia, which was reserved by silencing of miR-214-5p or upregulation of FGF2. Mechanistically, lnc-H19 regulated KD development by regulation of miR-214-5p/FGF2 axis. In summary, lnc-H19 may exert regulatory functions in KD by targeting miR-214-5p/FGF2 axis, further regulated glycolysis, migration and invasion in keloid fibroblasts exposed to hypoxia, which might be a potential marker of KD diagnosis or progression.

MiR-124-3p Regulates FGF2-EGFR Pathway to Overcome Pemetrexed Resistance in Lung Adenocarcinoma Cells by Targeting MGAT5.

ObjectiveTo investigate whether miR-124-3p regulates the fibroblast growth factor 2 (FGF2)–epidermal growth factor receptor (EGFR) pathway by targeting MGAT5 to affect the pemetrexed resistance in lung adenocarcinoma cells.MethodsPC9-MTA and H1993-MTA anti-pemetrexed lung adenocarcinoma cell lines were constructed. The cell viability of anti-pemetrexed and parent lung adenocarcinoma cells was analyzed using MTS assay and reverse transcription PCR to determine the expression of miR-124-3p. CCK8 assay, colony formation assay, and flow cytometry were used to determine cells’ proliferation and apoptosis. FGF2–EGFR signaling pathway-related proteins and MGAT5 protein expression were quantified by Western blotting. The target relationship between miR-124-3p and MGAT5 was verified by double luciferase assay. A nude mouse model with a transplanted tumor was established using the anti-pemetrexed lung adenocarcinoma cells. Tumor volume and weight were determined, and the apoptosis of tumor cells was observed.ResultsThe half-maximal inhibitory concentration of pemetrexed in anti-pemetrexed lung adenocarcinoma cells was higher than that in parent lung adenocarcinoma cells, and the expression of miR-124-3p in the anti-pemetrexed cells was lower than that of the parent cells. In the miR-124-3p overexpression group, MGAT5 silencing group, and miR-124-3p+MGAT5 overexpression group, compared with the control group, the proliferation ability of cells and tumors was markedly reduced; their apoptosis rates were increased significantly; expression levels of FGF2 and p-EGFR/EGFR were decreased; and the growth rate and tumor volume and mass were reduced; however, the opposite results were obtained in the miR-124-3p silencing group (p<0.05).ConclusionmiR-124-3p may inhibit the FGF2–EGFR pathway by targeting MGAT5 to decrease pemetrexed resistance in lung adenocarcinoma cells.

Sonic Hedgehog, a Novel Endogenous Damage Signal, Activates Multiple Beneficial Functions of Human Endometrial Stem Cells.

Local endometrial stem cells play an important role in regulating endometrial thickness, which is an essential factor for successful embryo implantation and pregnancy outcomes. Importantly, defects in endometrial stem cell function can be responsible for thin endometrium and subsequent recurrent pregnancy losses. Therefore, many researchers have directed their efforts toward finding a novel stimulatory factor that can enhance the regenerative capacity of endometrial stem cells. Sonic hedgehog (SHH) is a morphogen that plays a key role in regulating pattern formation throughout embryonic limb development. In addition to this canonical function, we identified for the first time that SHH is actively secreted as a stem cell-activating factor in response to tissue injury and subsequently stimulates tissue regeneration by promoting various beneficial functions of endometrial stem cells. Our results also showed that SHH exerts stimulatory effects on endometrial stem cells via the FAK/ERK1/2 and/or phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways. More importantly, we also observed that endometrial stem cells stimulated with SHH showed markedly enhanced differentiation and migratory capacities and subsequent invivo therapeutic effects in an endometrial ablation animal model.

Function of fibroblast growth factor 2 in gastric cancer occurrence and prognosis.

The present study aimed to explore the role of fibroblast growth factor 2 (FGF2) in the development and prognosis of gastric cancer (GC). The relationship between FGF2 mRNA expression levels and the clinical characteristics of GC was investigated using microarray data from four GC cohorts involving 726 patients obtained from the Gene Expression Omnibus. The results of the present study indicated that FGF2 expression levels were an independent factor affecting the prognosis of GC. The primary functions of FGF2 were related to cell adhesion and angiogenesis, and patients with high levels of FGF2 expression had poorer TNM staging and prognosis; these differences were statistically significant. In terms of immune infiltration, a higher extent of M2 macrophage intrusion was observed in patients with higher levels of FGF2. However, the degree of infiltration by dendritic and CD4+ T cells was lower, and this difference was statistically significant. Multivariate Cox proportional hazards model analysis revealed that age, TNM staging and FGF2 expression levels were independent prognostic factors for GC. In summary, FGF2 expression was demonstrated to be an independent prognostic factor in GC, and higher levels of FGF2 may promote the progression of this malignancy.

MicroRNA-936 targets FGF2 to inhibit epithelial ovarian cancer aggressiveness by deactivating the PI3K/Akt pathway.

PurposeMicroRNA-936 (miR-936) was previously reported to be dysregulated and involved in the development of non-small cell lung cancer and glioma. However, the functional roles of miR-936 in epithelial ovarian cancer (EOC) remain unclear. In this study, we aimed to evaluate miR-936 expression in EOC and investigate its regulatory role in EOC cell behavior.MethodsThe expression of miR-936 in EOC was measured by RT-qPCR. Cell proliferation, apoptosis, migration, and invasion in vitro, as well as tumor growth in vivo, were determined by CCK-8, flow cytometry, migration and invasion assays, and xenograft models in nude mice, respectively. Bioinformatics analysis, luciferase reporter assays, RT-qPCR, and Western blot analysis were performed to investigate the relationship between miR-936 and fibroblast growth factor 2 (FGF2).ResultsmiR-936 expression was significantly downregulated in EOC tissues and cell lines. Low miR-936 expression was found to be correlated with the tumor size, FIGO stage, and lymphatic metastasis in EOC patients. Functional experiments indicated that ectopic miR-936 expression suppressed EOC cell proliferation, migration, and invasion; promoted cell apoptosis; and decreased tumor growth in vivo. In addition, the FGF2 gene was verified to be a direct target of miR-936 in EOC cells. FGF2 expression levels were upregulated in EOC tissues and were inversely correlated with miR-936 expression. Furthermore, effects of FGF2 silencing were similar to those of miR-936 overexpression in EOC cells. Recovered FGF2 expression rescued the miR-936-induced inhibitory effects in EOC cells. Notably, miR-936 was able to deactivate the PI3K/Akt signaling pathway in EOC cells by regulating FGF2 both in vitro and in vivo.ConclusionAltogether, our findings provided initial evidence that miR-936 inhibits the aggressiveness of EOC cells in vitro and in vivo, at least partially, by targeting FGF2-mediated suppression of the PI3K/Akt pathway. Therefore, the miR-936/FGF2/PI3K/Akt pathway is a promising therapeutic target for the treatment of EOC patients.

Gabapentin regulates expression of FGF2 and FGFR1 in dorsal root ganglia via microRNA-15a in the arthritis rat model.

Arthritis is an inflammatory disease with a prevalence rate of approximately 10% in China, which commonly manifests as pain. The aim of the current study was to investigate the function of gabapentin in the dorsal root ganglion in an arthritis rat model, and assess the effect of gabapentin on the expression of fibroblast growth factor 2 (FGF2) and FGF receptor 1 (FGFR1). A total of 30 healthy male Sprague-Dawley rats were randomly divided into the following three groups: Untreated group, control group and gabapentin group. Rats in the control and the gabapentin groups were injected with Freund's complete adjuvant to induce arthritis. A total of 7 days subsequent to model establishment, the gabapentin group was administered intraperitoneally gabapentin for 8 days. The alterations in thickness of paw pad and paw withdrawal mechanical threshold (PWMT) were detected, which indicated that the rats in the control and gabapentin groups presented with the symptoms of arthritis. In the control group, the PWMT value was significantly reduced (P<0.05), whereas the PWMT value was significantly increased in the gabapentin group. Immunohistochemistry demonstrated that the expression levels of FGF2 and FGFR1 were increased in the control group compared with the untreated group, while the expression levels of FGF2 and FGFR1 were reduced in the gabapentin group. Moreover, the FGF2 antagonist PD173074 partially improved the plantar thickness and PWMT of the arthritic rats. Bioinformatics analysis predicted microRNA-15a binding sites in the 3'untranslated regions (UTR) of FGF2 and FGFR1. Furthermore, the expression of microRNA-15a was reduced in the control group compared with untreated rats, whereas microRNA-15a in the gabapentin group was upregulated compared with the control. Additionally, the luciferase reporter assay confirmed that microRNA-15a could inhibit the protein expression through pairing with the 3'UTR of FGF2 and FGFR1 mRNAs. Gabapentin may relieve arthritis pain and reduce the expression of FGF2 and FGFR1 in dorsal root ganglia. Furthermore, microRNA-15a may be involved in the regulatory process.

Thermal Preconditioning May Prevent Tendon Adhesion by Up-Regulating HSP72 in Rats

The study aims to determine the effects of thermal preconditioning on tendon adhesion by regulating the expression of heat shock protein 72 (HSP72) in rat models. Sixty male Wistar rats were collected and randomly assigned into the thermal preconditioning and control groups. During the 4th and 8th weeks following surgery, 15 rats were sacrificed in each period respectively, and their tendon adhesion was observed and evaluated. Biomechanical testing was performed to measure the tensile strength and gliding distance of tendons. Hematoxylin-eosin (HE) was used to observe the morphological structure of the tendons. Immunohistochemical staining, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the HSP72, fibroblast growth factor-2 (FGF-2), fibroblast growth factor receptor-1 (FGFR-1), β-catenin, epithelial cell adhesion molecule (EPCAM), Tenomodulin and scleraxis protein expressions. Pearson correlation analysis was applied to analyze the correlation between HSP72 expression and tendon adhesion. At the 4th week after surgery, we found no differences in the tendon adhesion scores or mRNA and protein expressions of HSP72 between the thermal preconditioning and control groups. However, after the 8th week after surgery, the thermal preconditioning group had a lower tendon adhesion score and higher mRNA and protein expressions of HSP72 than the control group. During the same period, we found longer gliding distance and higher expression levels of FGF-2, FGFR-1, β-catenin, Tenomodulin and scleraxis, but lower EPCAM expression in the thermal preconditioning group. Pearson correlation analysis indicated that HSP72 mRNA and protein expression levels were negatively correlated with tendon adhesion. These findings provide evidence that thermal preconditioning may alleviate tendon adhesions via upregulation of HSP72 expression.

Abstract 730: The roles of FGF-2-NCAM/FGFR1 interplay in esophageal squamous cell carcinomas and its microenvironment including tumor-associated macrophages

Abstract Tumor-associated macrophages (TAMs) have important roles in the angiogenesis, tumor immunosuppression and tumor infiltration of various cancers. Previously, we reported that a large number of infiltrated tumor promoting CD204+ TAMs within esophageal squamous cell carcinomas (ESCCs) contributed to the significant association of clinicopathological malignancy and poor disease free survival. However, the relevant malignant progression mechanisms have not been studied explicitly yet. To elucidate roles of TAMs in ESCCs, we established an in vitro macrophage model using human peripheral blood acquired by healthy volunteer donors. Peripheral blood monocytes were treated with 25 ng/mL recombinant human M-CSF for 6 days to induce macrophage-like differentiation (macrophage-like cells), then exposed to the conditioned media of TE series ESCC cell lines (TECM) for 2 days to induce TAM-like cells. From the cDNA microarray data between macrophages-like cells treated with and without TECM, we interested in upregulated genes. Among highly expressed genes in TAM-like cells, we focused on neural cell adhesion molecule (NCAM) which was a possibility to act on the regulation between tumor and its surrounding stroma. Gene knockdown of NCAM by siRNA in TAM-like cells revealed significant suppression of cell survival and migration via decrease of PI3K/Akt signaling and downregulation of fibroblast growth factor receptor 1 (FGFR1) expression. Interestingly, fibroblast growth factor-2 (FGF-2) derived from ESCCs promotes FGF2/FGFR1 autocrine loop, and activates FGFR1 signaling in TAM-like cells. Furthermore, we analyzed 70 ESCC tissue samples by immunohistochemistry whether the expression levels of FGF-2 were associated with clinicopathological background factors of ESCC patients. Relationships of the expression levels of FGF-2 in human ESCC stromal cells including macrophages had a correlation with the depth of invasion, blood vessel invasion, TNM classification UICC cancer staging and high number of infiltrating CD163 positive and CD204 positive macrophages as a marker for the M2 macrophage phenotype. These findings may indicate novel roles of FGF-2-NCAM/FGFR1 interplay in ESCCs and its microenvironment including TAMs. Citation Format: NOBUHISA TAKASE, Yuichiro Koma, Noriaki Arai, Himiko Kodaira, Masayoshi Hosono, Yumi Ichihara, Mari Nishio, Manabu Shigeoka, Hiroshi Yokozaki. The roles of FGF-2-NCAM/FGFR1 interplay in esophageal squamous cell carcinomas and its microenvironment including tumor-associated macrophages. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 730.

Monoamine neurotransmitters and fibroblast growth factor-2 in the brains of rats with post-stroke depression.

The aim of the present study was to investigate the changes in the levels of serotonin (5-HT), dopamine (DA), norepinephrine (NE) and fibroblast growth factor-2 (FGF-2) in the brains of rats with post-stroke depression (PSD). A rat model of stroke was established by middle cerebral artery occlusion and the rats were randomly divided into two groups: Control and modification groups. The rats in the modification group had PSD, while the rats in the control group had experienced a stroke only. The PSD model was established by applying chronic mild stress to the individually housed rats. High-performance liquid chromatography was used to detect the levels of 5-HT, DA and NE, while western blotting was used to detect the FGF-2 protein expression levels in the frontal lobe and hippocampus. Quantitative polymerase chain reaction was also used to determine the mRNA expression levels of FGF-2 in the frontal lobes of the two groups. The levels of 5-HT, DA and NE in the frontal lobe and hippocampus of the rats in the PSD group were significantly lower than the levels observed in the rats in the stroke group (P<0.01). In addition, protein expression levels of FGF-2 in the frontal lobe of the rats in the PSD group were significantly lower when compared with the control group rats (P<0.01), however, the protein expression levels of FGF-2 in the hippocampus did not exhibit a statistically significant difference (P>0.05). The mRNA expression levels of FGF-2 in the frontal lobe of the rats in the modification group were significantly lower than the levels in the control group rats (P<0.01). Therefore, reduced levels of monoamine neurotransmitters and FGF-2 expression in the brains of rats with PSD are associated with the incidence of PSD.

Protective effects of silymarin on fumonisin B1-induced hepatotoxicity in mice

The present study was conducted to investigate the effect of silymarin on experimental liver toxication induced by Fumonisin B1 (FB1) in BALB/c mice. The mice were divided into six groups (n = 15). Group 1 served as the control. Group 2 was the silymarin control (100 mg/kg by gavage). Groups 3 and 4 were treated with FB1 (Group 3, 1.5 mg/kg FB1, intraperitoneally; and Group 4, 4.5 mg/kg FB1). Group 5 received FB1 (1.5 mg/kg) and silymarin (100 mg/kg), and Group 6 was given a higher dose of FB1 (4.5 mg/kg FB1) with silymarin (100 mg/kg). Silymarin treatment significantly decreased (p < 0.0001) the apoptotic rate. FB1 administration significantly increased (p < 0.0001) proliferating cell nuclear antigen and Ki-67 expression. Furthermore, FB1 elevated the levels of caspase-8 and tumor necrosis factor-alpha mediators while silymarin significantly reduced (p < 0.0001) the expression of these factors. Vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) expressions were significantly elevated in Group 4 (p < 0.0001). Silymarin administration alleviated increased VEGF and FGF-2 expression levels (p < 0.0001). In conclusion, silymarin ameliorated toxic liver damage caused by FB1 in BALB/c mice.

Short-Hairpin RNA Silencing of Endogenous Fibroblast Growth Factor 2 in Rat Hippocampus Increases Anxiety Behavior

The fibroblast growth factor system has been implicated in the pathophysiology of mood disorders in humans and in affective behavior in animal models. However, the studies have been either correlative or involved exogenous administration of fibroblast growth factor 2 (FGF2). None of them have directly linked endogenous FGF2 to changes in emotional responses. Therefore, we began a series of studies to knockdown FGF2 by RNA interference to examine the role of brain FGF2 in emotional responsiveness. We assessed the efficacy of short-hairpin RNA (shRNA) sequences targeted to FGF2 in COS7 cells transfected with a plasmid vector containing the full-length FGF2 sequence. We then sought to assess the effects of knocking down FGF2 gene expression in vivo on behavior. We microinjected a lentiviral vector containing either a shRNA targeting FGF2 or a nonsilencing sequence bilaterally into the dentate gyrus of the rat. In a reporter assay system, three different shRNA sequences resulted in significant FGF2 knockdown in vitro. Five weeks following a single microinjection of one of those sequences in vivo, we observed a significant decrease in FGF2 gene expression by messenger RNA in situ hybridization in the hippocampus. The FGF2 knockdown increased the time spent in the closed arms of the elevated-plus maze, a test of anxiety behavior. The FGF2 knockdown in the hippocampus resulted in an anxiogenic effect. Together with our findings of an inverse correlation between anxiety and FGF2 expression levels, these results implicate FGF2 in the genesis and expression of anxiety disorders.

Epithelium is required for maintaining FGFR-2 expression levels in facial mesenchyme of the developing chick embryo.

In the developing chick embryo, fibroblast growth factor-2 (FGFR-2) expression patterns correlate with outgrowth of facial prominences. Frontonasal mass prominences that form the pre-nasal cartilage and upper beak express high levels of FGFR-2 receptor, whereas maxillary prominences that form the flattened corners of the beak and palatal shelves express low FGFR-2 transcript levels. Facial epithelium is an abundant source of FGFs and is required to support outgrowth of mesenchymal tissue, including cartilage rod formation. Because FGFR-2 is highly expressed in regions of facial outgrowth and because epithelium is required for outgrowth of facial prominences, epithelium could be required to maintain FGFR-2 transcripts in facial mesenchyme. To test this hypothesis, we removed epithelium to inhibit outgrowth of regions of the embryonic face, grafted frontonasal mass and maxillary prominences into a host limb bud, and then examined changes in FGFR-2 expression using in situ hybridization. We also hybridized adjacent sections with collagen II probe to identify regions undergoing chondrogenesis. Our results indicate that removal of epithelium from frontonasal mass led to a decrease in FGFR-2 and collagen II expression 24 hr after grafting to host and that neither FGFR-2 nor collagen II expression increased to expected levels at 48 hr. These results suggest that there are signals in the epithelium required for increasing FGFR-2 and collagen II gene transcription, and the expression of these genes are linked to outgrowth of facial prominences.