bFGF mRNA Research Articles

Uterine infusion with bone marrow mesenchymal stem cells improves endometrium thickness in a rat model of thin endometrium.

Bone marrow mesenchymal stem cells (BMSCs) show multidirectional differentiation and possess immunoregulatory properties. Although transplantation of BMSCs has a therapeutic effect on many diseases, it is unclear whether BMSC transplantation can be used as a therapy for a thin endometrium. To explore whether transplantation of BMSCs directly into the uterine cavity can improve endometrium thickness, a thin endometrium rat model was established by infusing ethanol into the uterine cavity. In all, 48 rats with thin endometrium and 24 normal rats were divided into 3 groups: (1) normal group, (2) experimental group transplanted with BMSCs into uterine cavity, and (3) control group transplanted with saline into the uterine cavity. The morphology of the endometrium, the regeneration and receptivity of the endometrium, and the mechanisms involved in BMSC therapy were subsequently analyzed by hematoxylin and eosin staining, Western blot analysis, and reverse transcription-polymerase chain reaction throughout an observation period of 3 estrus cycles. The rats in the experimental group had a significantly thicker endometrial lining and exhibited higher expression of cytokeratin, vimentin, integrin αγβ3, and leukaemia inhibitor factor (LIF) than that of the control group (P < .05). Bromodeoxyuridine -positive cells were detected in the endometrium after BMSC transplantation. Some proinflammatory cytokines, such as tumor necrosis factor α messenger RNA (mRNA) and interleukin (IL)-1βmRNA, were significantly downregulated, and anti-inflammatory cytokines, such as basic fibroblast growth factor (bFGF) mRNA and IL-6mRNA, were significantly upregulated in the experimental group compared to the control group (P < .05). In conclusion, BMSCs improved endometrium thickness, probably via their migration and immunomodulatory properties. Uterine perfusion with BMSCs represents a promising new tool for the currently intractable problem of an inadequate, thin endometrium.

In situ normoxia enhances survival and proliferation rate of human adipose tissue-derived stromal cells without increasing the risk of tumourigenesis.

Adipose tissue-derived stromal cells (ASCs) natively reside in a relatively low-oxygen tension (i.e., hypoxic) microenvironment in human body. Low oxygen tension (i.e., in situ normoxia), has been known to enhance the growth and survival rate of ASCs, which, however, may lead to the risk of tumourigenesis. Here, we investigated the tumourigenic potential of ASCs under their physiological condition to ensure their safe use in regenerative therapy. Human ASCs isolated from subcutaneous fat were cultured in atmospheric O2 concentration (21% O2) or in situ normoxia (2% O2). We found that ASCs retained their surface markers, tri-lineage differentiation potential, and self-renewal properties under in situ normoxia without altering their morphology. In situ normoxia displayed a higher proliferation and viability of ASCs with less DNA damage as compared to atmospheric O2 concentration. Moreover, low oxygen tension significantly up-regulated VEGF and bFGF mRNA expression and protein secretion while reducing the expression level of tumour suppressor genes p16, p21, p53, and pRb. However, there were no significant differences in ASCs telomere length and their relative telomerase activity when cultured at different oxygen concentrations. Collectively, even with high proliferation and survival rate, ASCs have a low tendency of developing tumour under in situ normoxia. These results suggest 2% O2 as an ideal culture condition for expanding ASCs efficiently while maintaining their characteristics.

Role of endogenous basic fibroblast growth factor in stem cells isolated from human exfoliated deciduous teeth

ObjectiveThis study aimed to investigate the role of endogenous basic fibroblast growth factor (bFGF) in stem cells isolated from human exfoliated deciduous teeth. MethodsCells were isolated from dental pulp tissues of human exfoliated deciduous teeth. The expression of stem cell markers was determined using conventional semi-quantitative polymerase chain reaction (PCR) and flow cytometry. The multipotential differentiation ability was also examined. The lentiviral shRNA or fibroblast growth factor receptor (FGFR) inhibitor was employed to inhibit bFGF mRNA expression and signal transduction, respectively. The colony formation ability was determined by low-density cell seeding protocol. The mRNA expression was evaluated using real-time quantitative PCR. The osteogenic differentiation was examined using alkaline phosphatase enzymatic activity assay and alizarin red staining. ResultsThe results demonstrated that the cells isolated from human exfoliated deciduous teeth (SHEDs) exhibited stem cell characteristics, regarding marker expression and multipotential differentiation ability (osteogenic, adipogenic, and neurogenic lineage). The sh-bFGF transduced SHEDs had lower colony forming unit and higher mineralization than those of the control. Similarly, the decrease of colony number and the increase of mineral deposition were noted upon exposing cells to FGFR chemical inhibitor. ConclusionThese results imply that the endogenous bFGF may participate in the colony formation and osteogenic differentiation ability. In addition, the inhibition of bFGF signalling may be useful to enhance osteogenic differentiation of stem cells.

Estradiol enhances the proliferation and migration of Ishikawa cells by promotion of angiogenesis induced by activation of NF-κB via AKT pathway

The aim of this study was to explore whether estradiol induces the expression of VEGF and bFGF in the endometrial cancer Ishikawa cells by activation of NF-κB via AKT pathway, and its effect on cell proliferation. Western blot was used to detect the AKT protein expression in Ishikawa cells after stimulation with estradiol, and the effect of AKT inhibitor or ER inhibitor on the activation of AKT. TransAM kit was used to detect the NF-κB p65 activity. qPCR and Western blot were used to detect the expression of VEGF and bFGF mRNA and proteins in the Ishikawa cells after estradiol treatment (E2 group), and pretreated with AKT inhibitor (AKT group) or ER inhibitor (ER group) or NF-κB inhibitor (NF-κB group), following the estradiol treatment. Flow cytometry and CFSE (carboxyfluorescein diacetate, succinimidyl ester) staining were used to examine the cell proliferation. Transwell was used to detect the migration ability of Ishikawa cells. Expression of p-AKT protein in the Ishikawa cells was markedly higher than that in the control group (P < 0.05). Expressions of p-AKT protein in the AKT and ER groups were significantly decreased than that in the E2 group (P < 0.05). The NF-κB activity was highest after stimulation with 1×10(-6) mol/L estradiol for 30 min to 1 h. AKT inhibitor significantly reduced the NF-κB activity (P < 0.05). The expressions of VEGF and bFGF mRNA and proteins in the E2 group were significantly increased than that in the control group (P < 0.05), and their expression in the AKT, ER and NF-κB groups were significantly decreased than that in the E2 group (P < 0.05). The proliferation and migration abilities of the Ishikawa cells were significantly increased after estradiol stimulation. Estradiol induces the production of VEGF and bFGF through activating NF-κB via AKT pathway, and enhances the proliferation and migration ability of cancer cells.

Effects of basic fibroblast growth factor and cyclin D1 on cigarette smoke-induced pulmonary vascular remodeling in rats.

Cigarette smoking may contribute to pulmonary hypertension in chronic obstructive pulmonary disease by resulting in pulmonary vascular remodeling that involves pulmonary artery smooth muscle cell proliferation. This study investigated the effects of basic fibroblast growth factor (bFGF) and cyclin D1 on the pulmonary vascular remodeling in smoking-exposed rats. Twenty-four male Wistar rats were randomly divided into four groups. Three tobacco-exposed groups were exposed to the smoke produced by 20 cigarettes for 60 min, twice a day for two, four or eight weeks, and the control group were exposed to fresh air. The expression of bFGF and cyclin D1 in the pulmonary arterial smooth muscle cells were determined using immunohistochemistry. Quantitative polymerase chain reaction was conducted to determine the expression levels of bFGF and cyclin D1 mRNA. In addition, the expression of bFGF and cyclin D1 proteins was evaluated by western blotting. The expression of bFGF and cyclin D1 at the mRNA and protein levels was shown to increase with the duration of smoke exposure (P<0.05). The correlation analysis indicated the expression of bFGF and cyclin D1 was positively associated with the pulmonary vessel wall thickness. The expression of bFGF was positively associated with that of cyclin D1. Collectively, the data demonstrated that the upregulation of bFGF and cyclin D1 occurred in rats subjected to smoke exposure, which may be associated with the abnormal proliferation of the smooth muscle cells in the pulmonary arteries.

Multilayered adipose-derived regenerative cell sheets created by a novel magnetite tissue engineering method for myocardial infarction

BackgroundAdipose-derived regenerative cells (ADRCs) are a promising source of autologous stem cells for regeneration and repair of damaged tissue. Herein, we investigated the therapeutic potential of ADRC sheets created by a magnetite tissue engineering technology (Mag-TE) for myocardial infarction. Methods and resultsAdipose tissue was obtained from wild-type (WT) mice and ADRCs were isolated. ADRCs incubated with magnetic nanoparticle-containing liposomes (MCLs) were cultured. MCL-labeled ADRCs were mixed with a diluted extracellular matrix (ECM) precursor, and a magnet was placed on the reverse side. Magnetized ADRCs formed multilayered cell sheets after a 24-h incubation. WT mice were subjected to myocardial infarction by permanent ligation of the left anterior descending artery. We then transplanted the constructed ADRC sheet or a cell-free collagen gel sheet, as a control, onto the infarcted myocardium using an Alnico magnet before skin closure. Cardiac parameters were measured by echocardiogram, and angiogenesis was determined by tissue capillary density. ADRC sheet-treated mice showed significant improvements in systolic function, infarct wall thinning, and fibrotic length after myocardial infarction. ADRC sheet implantation also promoted angiogenesis in both the infarct area and the border zone in WT mice after myocardial infarction. The angiogenic effects of ADRC sheets were attributed to an increased expression of VEGF and bFGF mRNA in ischemic hearts. ConclusionsADRC sheets created by this Mag-TE method protect the heart against pathological cardiac remodeling. Our ADRC sheets have the potential to be a novel regenerative strategy for ischemic heart disease.

Expression of vascular endothelial growth factor and basic fibroblast growth factor in acute rejection reaction following rat orthotopic liver transplantation.

The aim of the present study was to investigate the expression levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in acute rejection reaction (ARR) following orthotopic liver transplantation in a rat model. Serum VEGF and bFGF levels were detected using ELISA, and their expression levels in liver and spleen tissues were determined using immunohistochemistry. The mRNA expression levels of VEGF and bFGF were detected by conducting a quantitative polymerase chain reaction during the ARR following orthotopic liver transplantation. The expression levels of VEGF and bFGF in the serum 3 days following liver transplantation were significantly higher compared with those in the other groups (1 and 7 days following transplantation; P<0.01). In addition, the numbers of cells in the liver tissue that were shown to be positive for the expression VEGF and bFGF using immunohistochemistry were significantly higher 3 days following transplantation than at the other time points (P<0.0001). Furthermore, the numbers of cells positive for VEGF and bFGF expression in the spleen detected 3 days following the transplantation surgery were also significantly higher compared with those at the other time points (P<0.01). VEGF and bFGF mRNA expression levels were also increased from 1 day following the surgery and reached a peak at day 3, prior to declining gradually and remaining at a relatively high level. VEGF and bFGF mRNA expression levels changed dynamically, by peaking and then declining, in ARR following orthotopic liver transplantation. These changes may have an important impact on angiogenesis and the inflammatory reaction, and the identification of these changes increases the current understanding of ARR following orthotopic liver transplantation.

Thyroid hormone induced angiogenesis through the integrin αvβ3/protein kinase D/histone deacetylase 5 signaling pathway

Thyroid hormone is reported to induce angiogenesis, which is mediated by the membrane receptor integrin αvβ3, but the precise signaling pathway is still not very clear. Recently, studies have shown that protein kinase D (PKD) regulates the recycling of integrin αvβ3, which is required for cell migration. Moreover, phosphorylated PKD stimulates histone deacetylase 5 (HDAC5) phosphorylation and nuclear export in endothelial cells. As a potent pro-angiogenic growth factor, basic fibroblast growth factor (bFGF (FGF2)) is a downstream target gene of HDAC5. Therefore, we examined the hypothesis that a novel signaling pathway through integrin αvβ3/PKD/HDAC5 might contribute to thyroxine (T4)-induced angiogenesis. We selected human umbilical vein endothelial cells (HUVECs) for treatment. Angiogenesis was assessed using wound-healing and tubulogenesis assays. Signaling molecules, including phosphorylated PKD and HDAC5, were measured by western blotting. bFGF mRNA was analyzed by real-time PCR. Our results showed that T4 (100 nmol/l) stimulated the migration and formation of tube-like structures of HUVECs, whereas tetraiodothyroacetic acid (Tetrac, 100 nmol/l) inhibited T4-induced cell migration. Importantly, T4 promoted the phosphorylation of PKD and HDAC5. These effects were inhibited respectively by Tetrac, PKC inhibitor (2.5 μmol/l) and PKD siRNA. Meanwhile, T4 could promote the cytoplasmic accumulation of phosphorylated HDAC5 in HUVECs. In addition, bFGF mRNA expression in HUVECs significantly increased within 2 h of T4 treatment, but was decreased by Tetrac. Our findings indicate that T4 increases the expression of bFGF mRNA via the integrin αvβ3/PKD/HDAC5 signaling pathway, which plays an important role in angiogenesis.

Wound healing effect of acupuncture through activating angiogenesis in mice

In East Asian countries, acupuncture is used to treat wounds; however, the therapeutic mechanism is not well established. In this study, the effects of acupuncture on wound healing and the therapeutic mechanism was investigated in C57BL/6 mice. A 4-mm biopsy punch was used to create four full-thickness wounds on the dorsum of the mice on BL48 and BL51. The wound healing effects were compared in three groups after wounding; an untreated group was used as the negative control, and acupuncture-treated (on BL49 and BL52) and Madecassol® ointment-treated groups were used as the positive controls. The wounds were digitally photographed and the wound size was measured daily until 10 days after wounding. The wound closure was quantified using CANVAS 7SE software. The wound tissues were collected at 24 h and 48 h after wounding for the PCR analysis of VEGF, TGF-β1, and bFGF expression. The wounds in the acupuncture-treated group closed more rapidly than in the other groups, and at 6 days after wounding, a statistically significant difference was observed. The expression of VEGF mRNA (F(3, 23) = 18.47, P < 0.0001) and TGF-β1 mRNA (F(3, 21) = 8.547, P = 0.0007) in the acupuncture-treated group was increased at 48 h after wounding. The expression of bFGF mRNA (F(3, 24) = 5.398, P = 0.0055) was increased in the acupuncture-treated group; however, compared with the untreated group, the difference was not statistically significant. Therefore, acupuncture accelerated the wound healing via the activation of angiogenic growth factors.

Rat bone marrow mesenchymal stem cells improve regeneration of thin endometrium in rat

To investigate whether bone marrow mesenchymal stem cell (BMSC) treatment could improve the regeneration of endometrium and improve the endometrial receptivity in an experimental model of thin endometrium. Randomized, control trial, animal research. National key laboratory. Sprague-Dawley rats. Bone marrow mesenchymal stem cell transplantation by tail vein IV injection. Endometrial thickness, the expression of mark proteins for endometrial cell, and endometrial receptivity. The endometrium was significantly thicker and the expression of cytokeratin, vimentin, integrin αγβ3, and leukemia inhibitor factor were significantly stronger compared with the control group. Some proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) messenger RNA (mRNA) and interleukin-1β mRNA, were significantly down-regulated, and anti-inflammatory cytokines, such as fibroblast growth factor-β (bFGF) mRNA and interleukin-6 mRNA, were significantly up-regulated in the experimental group compared with the control group. The BMSCs have beneficial effect on thin endometrium, and may play a role through migration and immunomodulatory of BMSCs.

Valproic acid inhibits tumor angiogenesis in mice transplanted with Kasumi-1 leukemia cells

Histone deacetylase (HDAC) inhibitors have been reported to inhibit tumor angiogenesis via the downregulation of angiogenic factors. Our previous in vitro studies demonstrated that valproic acid (VPA) exerted antitumor effects on Kasumi-1 cells, which are human acute myeloid leukemia cells with an 8;21 chromosome translocation. In the present study, the effects of VPA on tumor angiogenesis were investigated in mice transplanted with Kasumi-1 cells. Semi-quantitative reverse transcription-polymerase chain reaction, western blotting and immunohistochemistry were used to detect the expression of vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR2) and basic fibroblast growth factor (bFGF). The tumor microvessel density was measured following staining with an anti-CD34 antibody. Chromatin immunoprecipitation was used to study the effect of VPA-induced histone hyperacetylation on VEGF transcription. An intraperitoneal injection of VPA inhibited tumor growth and angiogenesis in mice transplanted with Kasumi-1 cells. The mRNA and protein expression of VEGF, VEGFR2 and bFGF were inhibited by VPA treatment. In addition, VPA downregulated HDAC, increased histone H3 acetylation and enhanced the accumulation of hyperacetylated histone H3 on the VEGF promoters. The findings of the present study indicate that VPA, an HDAC inhibitor, exerts an antileukemic effect through an anti-angiogenesis mechanism. In conclusion, the mechanism underlying VPA-induced anti-angiogenesis is associated with the suppression of angiogenic factors and their receptors. VPA may increase the accumulation of acetylated histones on the VEGF promoters, which possibly contributes to the regulation of angiogenic factors.

Cytomegalovirus Infection Accelerates the Process of Chronic Rejection in Rat Liver Transplantation

ObjectiveAnalysis of the pathogenesis of cytomegalovirus (CMV) infection for rat chronic liver rejection. MethodsBased on animal models that we previously developed, we divided control and a CMV infection group to analyze survival, histopathology and rejection activity. The level and mRNA expression of transforming growth factor-β1, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF) were measured in peripheral blood serum and liver tissue. ResultsObliterative arteriopathy and fibrosis was significantly greater in the CMV infection versus the control group (P < .05). PDGF mRNA expression in rat liver and in peripheral blood were significantly higher in the CMV infection versus the control group (P < .05). bFGF mRNA expression in the CMV infection group was also significantly higher than among the controls (P < .05). ConclusionsCMV infection aggravated foam cell obliterative arteriopathy and liver fibrosis in chronic liver rejection, processes that accelerate the immune response, which may correlate with early high PDGF and bFGF expression.

The Angiogenic Peptide Vascular Endothelial Growth Factor–Basic Fibroblast Growth Factor Signaling is Up‐Regulated in a Rat Pressure Ulcer Model

The purpose of this study is to investigate the mRNA and protein expression levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in pressure ulcers, and to elucidate the molecular mechanism by which VEGF and bFGF are involved in pressure ulcer formation. A rat model of ischemia-reperfusion pressure ulcer was established by magnetic disk circulating compression method. Real-time fluorescence quantitative PCR and Western blot assays were conducted to detect the mRNA and protein expression of VEGF and bFGF in the tissues of rat I-, II-, and III-degree pressure ulcers, the surrounding tissues, and normal skin. Our study confirmed that the mRNA and protein expression levels of VEGF and bFGF in the tissues of rat I-degree pressure ulcer were significantly higher than that in the II- and III-degree pressure ulcer tissues (P < 0.05). The expression of VEGF and bFGF in the tissues surrounding I- and II-degree pressure ulcers were higher than the rats with normal skin. The expression of VEGF and bFGF in the tissues of rat III-degree pressure ulcer was lower than that in the surrounding tissues and normal skin (P < 0.05). There was a significant positive correlation between change in the VEGF and bFGF. The results showed that with an increase in the degree of pressure ulcers, the expression of VEGF and bFGF in pressure ulcers tissue are decreased. This leads to a reduction in angiogenesis and may be a crucial factor in the formation of pressure ulcers.

Influence of monochromatic light exposure on the growth of Miiller cell and the expression of myopia-related cell factors in vitro

Background Research showed that exposure of 530 nm monochromatic light can induce myopia in animal,and retinal Muller cells participate in the formation of myopia.However,the effect and mechanism of retinal Muller cells during the formation of monochromatic light induced-myopia is below understood.Objective This study was to investigate biologic characteristics of rat retina Muller cells and the expression of cell factors in Muller cells after being illuminated by the 530 nm monochromatic light,and discuss the role of the retina Muller cells in myopia induced by monochromatic light.Methods Immortalized rat retinal Muller cells were cultured with DMEM containing 10％ fetal bovine serum in a self-made cell incubator with monochromatic light by adjusting luminance of 530 nm LED source.The cells were exposed to 125,250 and 500 lx luminance respectively for 6,12 and 24 hours,and the cells without light-irradiation were used as control.The growth of the cells under the different light time and different illuminations was described by MTT as the absorbance at the wavelength 570 nm (A570),and cell cycle analysis of Muller cells was performed by flow cytometry 48 hours after cultured,and the expression of transforming growth factor-beta 1 (TGF-β1),tyrosine hydroxylase(TH),inducible nitric oxide synthase(iNOS) and basic fibroblast growth factor(bFGF)in the cells were detected by reverse transcription PCR(RT-PCR),respectively.Results The Muller cells were uniform in size with polygonal shape and defined edges.No statistically significant difference was found in the A570 value in the cells of the 125 lx and 250 lx illuminated groups compared with the control group in various time points(P＞0.05).However,significant lowing was seen in the A570 value in the cells of the 500 lx illuminating for 12 hours and 24 hours in comparison with the control group (P =0.013,0.001).Compared with the control group,the ratio of the number between G2 and G1 phase was not significantly declined in 125 lx,250 lx illuminating for 48 hours (P =0.073,0.330),and the ratio in the 500 lx illuminating group was significantly lower than those in the 250 lx illuminated group and the control group (P =0.028,0.038).RT-PCR revealed that the expression of TGF-β1 mRNA in the cells was higher in the 250 lx illuminated group than that of the 500 lx illuminated group (P=0.006).The expression of iNOS mRNA was gradually upregulated in the 250 lx illuminated group compared with the control group (P =0.001),but that in the 500 lx illuminated group was downregulated (P =0.000).The expression of bFGF mRNA was raised in the 125 lx and 250 lx groups but reduced in the 500 lx group when compared with the control group(P=0.002,0.000,0.005).Also,the expression of TH mRNA was significantly increased in the 250 lx group(P=0.000),but decreased in the 500 lx group(P=0.000,P=0.001).Conclusions The monochromatic light of 530 nm can inhibit the growth of rat Muller cells and downregulate the expression of myopia-related cell factors and therefore exert effect in the formation of myopia. Key words: Monochromatic light; Muller cell; Myopia; Transforming growth factor-beta 1 ; Tyrosine hydroxylase; Inducible nitric oxide synthase; Basic fibroblast growth factor

Early growth response‐1 in the pathogenesis of cysteamineinduced duodenal ulcer

We showed earlier that the immediate‐early gene &amp; its zinc finger transcription factor early growth response (Egr) product were enhanced, including the expression of target genes (e.g., bFGF, PDGF, VEGF) in early stage of cysteamine (C)‐induced DU. Our recent studies demonstrated that deletion of egr‐1 gene led to 4.4‐fold increase in C‐induced DU in egr‐1 knockout (KO) vs. wild‐type (WT) mice. mRNA expression of bFGF, VEGF &amp; PDGF was still increased but protein expression of bFGF, VEGF &amp; PDGF was diminished in egr‐1 KO mice. We hypothesized that specific miRNAs in egr‐1 KO mice might be activated &amp; inhibit protein synthesis of egr‐1 target genes. Using miRXplore Microarray study we found that 11 miRNAs (e.g., mir‐696, mir‐467E, mir‐152 &amp; mir‐301B) were elevated by 1.8‐ to 1.9‐fold in egr‐1 KO mice vs. WT mice. Mir‐1892 decreased by 1.7‐fold in egr‐1 KO vs. WT mice. These miRNAs partially share validated targets relating to growth factor responses, e.g., FGFR3 &amp; VEGF. We also demonstrated that DU was significantly reduced in rats treated with adenovirus encoding egr‐1 on days 7 &amp; 14 (p &lt; 0.05) respectively. The mRNA &amp; protein expressions of egr‐1, bFGF, VEGF &amp; PDGF markedly increased in rats after egr‐1 gene therapy. We conclude that Egr‐1 plays a preventive role in the pathogenesis of DU &amp; further studies are needed to identify miRNA expression in egr‐1 KO &amp; WT mice with DU.(This work was supported by the Veterans Health Administration Merit Review Grant to S. Szabo).

Increase of Grb2 and Ras Proteins and Expression of Growth Factors in LPS Stimulated Odontoblast-like Dental Pulp Cells

Inflammatory cells express the inflammatory cytokines and growth factors induced by lipopolysaccharide (LPS). Odontoblasts are located at the pulp-dentin interface and extend their cell processes far into the dentin where they are the first cells to encounter microorganisms or their products. Therefore, this study examined the expression of some growth factors related to the signal pathway, such as growth factor receptor binding protein 2 (Grb2)-Ras in odontoblast-like dental pulp cells, after a treatment with LPS. After 60 minutes, the mRNA and protein expression levels of Grb2 and Ras were higher in the LPS-treated cells than in the control cells. The level of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) mRNA expression was increased significantly to a level similar to that of Grb2 and Ras at 60 minutes. The platelet-derived growth factor-AA (PDGF-AA) mRNA level was expressed strongly in the odontoblast like dental pulp cells without an association with LPS stimulation. Scanning electron microscopy revealed many extensions of the cytoplasmic processes and the number of processes increased gradually at 30, 60 and 90 minutes after LPS stimulation. From these results VEGF and bFGF expression might be induced through the Grb2-Ras signal transduction pathway in LPS treated odontoblasts.

Hypertrophy of the ligamentum flavum in lumbar spinal canal stenosis is associated with increased bFGF expression

A prospective study was undertaken to investigate basic fibroblast growth factor (bFGF) expression in hypertrophic ligamentum flavum (LF) from patients with lumbar spinal canal stenosis (LSCS) and to determine whether there was a correlation of bFGF expression with LF thickness. Twenty patients with lumbar spinal canal stenosis were enrolled in this study. bFGF mRNA and protein expressions in LF were analyzed using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA), respectively. The thickness of LF was measured by axial T1-weighted magnetic resonance imaging. Expression of bFGF was substantially higher in the hypertrophic LF group than in the control group (P < 0.001) as quantified by quantitative real-time PCR. In immunohistochemical study, bFGF was positively stained on the fibroblasts within hypertrophic LF compared to nonpathologic LF of controls. Subsequent ELISA analysis revealed that bFGF concentration in the hypertrophic LF group was remarkably higher than that in the control group (P = 0.003). The thickness of LF in the hypertrophic LF was significantly greater than that in the control group (P < 0.001). LSCS patients with greater severity of LF hypertrophy had significantly higher bFGF levels in the LF tissues (P < 0.001). Furthermore, the bFGF concentration exhibited a positive correlation with the LF thickness (r = 0.974, P < 0.001). These findings suggest that the increased expression of bFGF is associated with the hypertrophy of ligamentum flavum in patients with LSCS.

Smad7 gene transfer attenuates angiogenesis in peritoneal dialysis rats

Transforming growth factor-β (TGF-β) has been shown to play a role in peritoneal angiogenesis associated with peritoneal dialysis (PD). The present study investigated whether blockade of TGF-β signalling with Smad7 has a therapeutic effect on PD induced-peritoneal angiogenesis. A rat model of peritoneal dialysis was induced by a daily intraperitoneal injection of 4.25% Dianeal and lipopolysaccharides. PD rats were transfected with a doxycycline regulated, Smad7-expressing plasmid using an ultrasound-microbubble-mediated system on day 0 and day 14 after initiation of PD and an empty vector was used as control. Peritoneal microvessel density (MVD) in peritoneal tissue was assessed by anti-CD31 immunohistochemistry after 4 weeks of PD and peritoneal angiogenic growth factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) was also examined by immunofluorescence, western blot and reverse transcription-polymerase chain reaction. In contrast to the normal control group, at 4 weeks after PD, PD rats displayed peritoneal lesions, peritoneal angiogenesis and increased mRNA and protein expression of VEGF, bFGF and PDGF. Smad7 gene transfer significantly attenuated the peritoneal MVD and inhibited the upregulation of VEGF, bFGF and PDGF. Moreover, inhibition of peritoneal angiogenesis by overexpression of Smad7 was associated with inhibition of phosphorylation of Smad3 and downregulation of TGF-β expression. Smad7 gene transfer via an ultrasound-microbubble-mediated system is able to attenuate peritoneal angiogenesis in a rat model of PD. Those results suggest that blockade of the TGF-β/Smad signalling pathway may represent a novel therapeutic approach to prevent PD-induced peritoneal angiogenesis.

RNA interference-mediated silencing of VEGF and bFGF suppresses endostatin secretion in pancreatic carcinoma cells

Pro-angiogenic factors [vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)] and anti-angiogenic factors (endostatin) play important roles in the progression of pancreatic cancer. The purpose of the present study was to investigate the knockdown effect by either VEGF or bFGF siRNA on the expression and secretion of endostatin in pancreatic carcinoma cells. Pancreatic carcinoma cell lines (sw1990, Panc-1 and PCT-3) were treated with VEGF and bFGF siRNA. The expression of VEGF, bFGF and endostatin in pancreatic carcinoma cell lines was determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. Secretion of endostatin was measured by enzyme-linked immunosorbent assay (ELISA). bFGF and VEGF siRNA significantly reduced the expression of bFGF and VEGF mRNA, respectively, but did not affect mRNA and protein expression of endostatin in pancreatic carcinoma cell lines. However, secretion of endostatin in PCT-3, Panc-1 and sw1990 cells was significantly inhibited by bFGF and VEGF siRNA. This study demonstrated that pro-angiogenic factors (VEGF and bFGF) differentially modulate expression and secretion of anti-angiogenic factors (endostatin). This result may have important implications in the anti-angiogenesis therapy in pancreatic cancer.

Propofol Increases Expression of Basic Fibroblast Growth Factor After Transient Cerebral Ischemia in Rats

Anesthetics such as propofol can provide neuroprotective effects against cerebral ischemia. However, the underlying mechanism of this beneficial effect is not clear. Therefore, we subjected male Sprague-Dawley rats to 2h of middle cerebral artery occlusion and investigated how post-ischemic administration of propofol affected neurologic outcome and the expression of basic fibroblast growth factor (bFGF). After 2h of ischemia, just before reperfusion, the animals were randomly assigned to receive either propofol (20mgkg(-1)h(-1)) or vehicle (10% intralipid, 2mlkg(-1)h(-1)) intravenously for 4h. Neurologic scores, infarct volume, and brain water content were measured at different time points after reperfusion. mRNA level of bFGF was measured by real-time PCR, and the protein expression level of bFGF was analyzed by immunohistochemistry and Western blot. At 6, 24, 72h, and 7days of reperfusion, infarct volume was significantly reduced in the propofol-treated group compared to that in the vehicle-treated group (all P<0.05). Propofol post-treatment also attenuated brain water content at 24 and 72h and reduced neurologic deficit score at 72h and 7days of reperfusion (all P<0.05). Additionally, in the peri-infarct area, bFGF mRNA and protein expression were elevated at 6, 24, and 72h of reperfusion compared to that in the vehicle-treated group (all P<0.05). These results show that post-ischemic administration of propofol provides neural protection from cerebral ischemia-reperfusion injury. This protection may be related to an early increase in the expression of bFGF.