Expression Of bFGF Research Articles

Significance of Basic Fibroblast Growth Factor (BFGF) in The Development of Postresuscitation Changes in Population of Cerebellar Purkinje Cells

Objective: to evaluate the relationship of postresuscitation changes in the level of bFGF protein expression and morphological patterns of the cerebellar Purkinje cells. Materials and methods. Albino adult male rats were subjected to 10 minutes of systemic circulatory arrest caused by cardiac vascular fascicle ligation. The status of a hypoxiasensitive neuronal population of cerebellar Purkinje cells were investigated in different postresuscitation periods. Total numbers of Purkinje cells per mm of their layer length were estimated by a histological analysis of the specimens stained with cresyl violet after the Nissl procedure. An immunocytochemical analysis was performed to determine the number of bFGFpositive (weakly and strongly stained) and GDNFnegative neurons per mm of their layer length and the total population density. Results. Dynamics of the process of neuronal death and changes of bFGF expression level in the population of the Purkinje cells were determined postresuscitation. Next day after the resuscitation the level of expression of bFGF in the neuronal population significantly increased. At the same time point, the neuronal death did not occur. However, on day 4 the level of bFGF expression decreased and overall density of the population declined. Immunohistochemical study revealed that bFGFnegative cells undergone death. In the later stages of the process postresuscitation (days 7 and 14) the bFGF expression level increased again. At the same time deepening and/or enhancing of the pathological changes in the neuronal population were not observed. Conclusion. The data indicate that ischemiareperfusion significantly affect the expression of bFGF protein, inducing its elevation within the Purkinje cell population of resuscitated animals. The initial rise in the level of bFGF protein within the neuronal population might prevent the development of a nerve cell death process. The subsequent reduction in the bFGF level is accompanied by the neuronal loss. Therefore, the ability to produce bFGF is an important factor affecting the resistance of neurons to postresuscitation damage. Moreover, the bFGF is considered as promising candidate molecule for developing alternative therapeutic strategies to prevent and/or treatment posthypoxic encephalopathies.

Elevation of Microglial Basic Fibroblast Growth Factor Contributes to Development of Neuropathic Pain after Spinal Nerve Ligation in Rats.

Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistological analysis of spinal cord and pain behavior analysis in a rat neuropathic pain model were conducted to examine the function of microglial basic fibroblast growth factor (bFGF) in the development of neuropathic pain. To investigate the role of bFGF in spinal microglia during the development of allodynia following spinal nerve ligation in rats. Evidence suggests that the production of bFGF by spinal cord glial cells is increased in response to peripheral nerve injury. Although an association between bFGF and astrocytes has been widely reported, the relationship between bFGF and microglia, particularly with respect to the development of neuropathic pain, remains poorly understood. Spinal nerve ligation rats were used. After surgery, bFGF expression in the spinal cord was investigated using RT-PCR and immunohistochemistry. Neutralizing antibodies to bFGF were injected intrathecally into rats after spinal nerve ligaton. Spinal cords were used for RT-PCR analysis and pain behavior was analyzed using the von Frey test. bFGF mRNA expression was significantly increased in the spinal cord 6 hours after spinal nerve ligation compared with untreated rats. Immunohistochemical analysis revealed that bFGF co-localized with ionized calcium-binding adaptor molecule 1, a microglial marker, and myeloperoxidase. Neutralizing antibodies to bFGF attenuated mechanical allodynia and myeloperoxidase mRNA expression. bFGF increased in spinal microglia during the development allodynia after spinal nerve ligation. Thus, controlling bFGF release from microglia during the acute stage of peripheral nerve injury may suppress the progression of allodynia. N/A.

Using basic fibroblast growth factor nanoliposome combined with ultrasound-introduced technology to early intervene the diabetic cardiomyopathy.

Basic fibroblast growth factor (bFGF)-loaded liposome (bFGF-lip) combined with ultrasound-targeted microbubble destruction (UTMD) technique was investigated to prevent diabetic cardiomyopathy (DCM). Cardiac function and myocardial ultrastructure were assessed. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining, immunohistochemistry staining, and Western blot assay were used to investigate the signal pathway underlying the expression of bFGF in DCM treatment. From Mason staining and TUNEL staining, bFGF-lip + UTMD group showed significant differences from the diabetes group and other groups treated with bFGF or bFGF-lip. The diabetes group showed similar results (myocardial capillary density, collagen volume fraction, and cardiac myocyte apoptosis index) to other bFGF treatment groups. Indexes from transthoracic echocardiography and hemodynamic evaluation also proved the same conclusion. These results confirmed that the abnormalities including diastolic dysfunctions, myocardial fibrosis, and metabolic disturbances could be suppressed by the different extents of twice-weekly bFGF treatments for 12 consecutive weeks (free bFGF or bFGF-lip +/− UTMD), with the strongest improvements observed in the bFGF-lip + UTMD group. The group combining bFGF-lip with UTMD demonstrated the highest level of bFGF expression among all the groups. The bFGF activated the PI3K/AKT signal pathway, causing the reduction of myocardial cell apoptosis and increase of microvascular density. This strategy using bFGF-lip and UTMD is a potential strategy in early intervention of DCM in diabetes.

Arginine ADP-ribosyltransferase 1 promotes angiogenesis in colorectal cancer via the PI3K/Akt pathway.

Arginine adenosine diphosphate (ADP)-ribosyltransferase 1 (ART1) is known to play an important role in many physiological and pathological processes. Previous studies have demonstrated that ART1 promotes proliferation, invasion and metastasis in colon carcinoma. However, it was unclear whether ART1 is involved in angiogenesis in cases of colorectal cancer (CRC). In the present study, lentiviral vector-mediated ART1-cDNA or ART1-shRNA were transfected into LoVo cells, and the LoVo cells transfected with ART1-cDNA or ART1-shRNA were co-cultured with human umbilical vein endothelial cells (HUVECs) to determine the influence of ART1 on HUVECs. The proliferation, migration and angiogenesis of HUVECs were monitored using a cell counting kit-8 assay, a Transwell migration assay and immunohistochemical analysis in intrasplenic allograft tumors, respectively. Hypoxia-inducible factor 1-α (HIF-1α), total (t-) Akt, phosphorylated (p-)Akt, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) expression levels were detected via western blot analysis. Our results revealed that HUVECs which were co-cultured with ART1-cDNA LoVo cells showed higher proliferation, migration and angiogenic abilities, but a reduction was noted in those cultured with ART1-shRNA LoVo cells; p-Akt, HIF-1α, VEGF and bFGF expression was increased in HUVECs cultured with ART1-cDNA-transfected LoVo cells, but reduced in ART1-shRNA-transfected LoVo cells. In a mouse xenograft model, we noted that the tumor microvessel density (MVD) was significantly increased in intrasplenic transplanted ART1-cDNA CT26 tumors but decreased in intrasplenic transplanted ART1-shRNA tumors. These data suggest that ART1 promoted the expression of HIF-1α via the Akt pathway in tumor cells. It also upregulated VEGF and bFGF and enhanced angiogenesis in HUVECs. Thus, we suggest that ART1 plays an important role in the invasion of CRC cells and the metastasis of CRC.

Prognostic Value of Basic Fibroblast Growth Factor (bFGF) in Lung Cancer: A Systematic Review with Meta-Analysis.

BackgroundBasic fibroblast growth factor (bFGF) is known to stimulate angiogenesis and thus to influence the proliferation, migration and survival of tumor cells. Many studies examined the relationship between human bFGF overexpression and survival in lung cancer patients, but the results have been mixed. To systematically summarize the clinical prognostic function of bFGF in lung cancer, we performed this systematic review with meta-analysis.MethodStudies were identified by an electronic search of PubMed, EMBASE, China National Knowledge Infrastructure and Wanfang databases, including publications prior toAugust 2014. Pooled hazard ratios (HR) for overall survival (OS) were aggregated and quantitatively analyzed by meta-analysis.ResultsTwenty-two studies (n = 2154) were evaluated in the meta-analysis. Combined HR suggested that bFGF overexpression had an adverse impact on survival of patients with lung cancer(HR = 1.202,95%CI, 1.022–1.382). Our subgroup analysis revealed that the combined HR evaluating bFGF expression on OS in operable non-small cell lung cancer (NSCLC) was 1.553 (95%CI, 1.120–1.986); the combined HR in small cell lung cancer (SCLC) was 1.667 (95%CI, 1.035–2.299). There was no significant impact of bFGF expression on survival in advanced NSCLC.ConclusionThis meta-analysis showed that bFGF overexpression is a potential indicator of worse prognosis for patients with operable NSCLC and SCLC, but is not associated with outcome in advanced NSCLC. The data suggests that high bFGF expression is highly related to poor prognosis. Nevertheless,more high-quality studies should be performed in order to provide additional evidence for the prognostic value of bFGF in lung cancer.

Quercetin-3-O-glucoside suppresses pancreatic cancer cell migration induced by tumor-deteriorated growth factors in vitro.

Analysis using Universal exPress Codes (UPCs) with the public microarray database GEO indicates significantly higher mRNA expressions of VEGF-A, bFGF, and bFGFR2 in pancreatic cancers than those in normal pancreatic tissues. Human pancreatic cancer cell line CFPAC-1 and SNU-213 had relatively differential sensitivity to exogenous VEGF-A, bFGF, and TGF-β1 in migration property. Treatment of quercetin-3-O-glucoside suppressed the migratory activity induced by TGF-β and VEGF-A even at relatively low dosages in CFPAC-1, but not in bFGF-activated SNU-213 cells. However, high dosages of quercetin-3-O-glucoside sufficiently suppressed the migratory activity induced by bFGF in SNU-213 cells. Furthermore, co-treatment with low dose of gemcitabine plus quercetin-3-O-glucoside showed synergistic inhibition effects on the infiltrate activity induced by bFGF in CFPAC-1 and SNU-213 cells. These results collectively suggested that quercetin-3-O glucoside could act as an inhibitor of local metastasis induced by various growth factors in pancreatic cancers and be an effective adjuvant to boost chemotherapeutic efficacy of gemcitabine, currently used in pancreatic cancers.

Osmotic Induction of Angiogenic Growth Factor Expression in Human Retinal Pigment Epithelial Cells

BackgroundAlthough systemic hypertension is a risk factor of age-related macular degeneration, antihypertensive medications do not affect the risk of the disease. One condition that induces hypertension is high intake of dietary salt resulting in increased blood osmolarity. In order to prove the assumption that, in addition to hypertension, high osmolarity may aggravate neovascular retinal diseases, we determined the effect of extracellular hyperosmolarity on the expression of angiogenic cytokines in cultured human retinal pigment epithelial (RPE) cells.Methodology/Principal FindingsHyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Hypoxia and oxidative stress were induced by the addition of the hypoxia mimetic CoCl2 and H2O2, respectively. Alterations in gene expression were determined with real-time RT-PCR. Secretion of bFGF was evaluated by ELISA. Cell viability was determined by trypan blue exclusion. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. Hyperosmolarity induced transcriptional activation of bFGF, HB-EGF, and VEGF genes, while the expression of other cytokines such as EGF, PDGF-A, TGF-β1, HGF, and PEDF was not or moderately altered. Hypoxia induced increased expression of the HB-EGF, EGF, PDGF-A, TGF-β1, and VEGF genes, but not of the bFGF gene. Oxidative stress induced gene expression of HB-EGF, but not of bFGF. The hyperosmotic expression of the bFGF gene was dependent on the activation of p38α/β MAPK, JNK, PI3K, and the transcriptional activity of NFAT5. The hyperosmotic expression of the HB-EGF gene was dependent on the activation of p38α/β MAPK, ERK1/2, and JNK. The hyperosmotic expression of bFGF, HB-EGF, and VEGF genes was reduced by inhibitors of TGF-β1 superfamily activin receptor-like kinase receptors and the FGF receptor kinase, respectively. Hyperosmolarity induced secretion of bFGF that was reduced by inhibition of autocrine/paracrine TGF-β1 signaling and by NFAT5 siRNA, respectively. Hyperosmolarity decreased the viability of the cells; this effect was not altered by exogenous bFGF and HB-EGF. Various vegetable polyphenols (luteolin, quercetin, apigenin) inhibited the hyperosmotic expression of bFGF, HB-EGF, and NFAT5 genes.ConclusionHyperosmolarity induces transcription of bFGF and HB-EGF genes, and secretion of bFGF from RPE cells. This is in part mediated by autocrine/paracrine TGF-β1 and FGF signaling. It is suggested that high intake of dietary salt resulting in osmotic stress may aggravate neovascular retinal diseases via stimulation of the production of angiogenic factors in RPE cells, independent of hypertension.

Relationship between interleukin-8 levels in expressed prostatic secretion and expressions of bFGF and Bcl-2 in benign prostatic hyperplasia

To investigate the relationship between interleukin-8 levels in expressed prostatic secretion and expressions of basic fibroblast growth factor (bFGF) and Bcl-2 in benign prostatic hyperplasia (BPH). A series of 50 consecutive patients diagnosed with BPH and scheduled for transurethral resection of the prostate (TURP) were included. Patients were divided into two groups, simple BPH and BPH with chronic prostatitis (CP). The grade of inflammatory changes in the prostate was then determined blindly by two experienced pathologists, according to the classification system. Expressed prostatic secretion (EPS) was collected right before TURP for IL-8 detection using ELISA kits. The resected prostatic tissue was harvested for immunohistochemistry to measure the expression of Bcl-2 and bFGF. A total of 30 (60%) patients were confirmed to have prostatic histologic inflammation. The volume of the prostate in BPH with CP was obviously larger than that in simple BPH (P=0.022). The NIH chronic prostatitis symptom index (NIH-CPSI) and international prostate symptom score (IPSS) of BPH with CP were both higher than those of simple BPH (P<0.05). Significantly increased levels of IL-8 were observed in EPS obtained from BPH patients with CP (mean level, 43.29 ng/L), compared with patients with simple BPH (mean level, 36.90 ng/L) (P=0.003). The expression of Bcl-2 in BPH with CP (mean level, 6.17) increased significantly, compared with those in simple BPH (mean level, 2.45) (P=0.013). The expression of bFGF in BPH with CP (mean level, 7.57) also obviously elevated, compared with those in simple BPH (mean level, 5.05) (P=0.008). Correlation coefficients of IL-8 levels in EPS and expression of Bcl-2 or bFGF indicated that IL-8 had strong correlation with Bcl-2 and bFGF respectively both in simple BPH and BPH with CP (0<R<1, P<0.05). IL-8, Bcl-2 and bFGF are significantly up-regulated in BPH with CP patients, compared with simple BPH patients. Inflammation might play an important role in the outcome and development of benign prostatic hyperplasia and makes it more severe.

雌激素、胰岛素诱导子宫内膜癌细胞中VEGF和bFGF的表达及临床意义

初步探讨雌二醇、胰岛素诱导子宫内膜癌细胞系Ishikawa细胞中血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)的产生及临床意义。采用荧光定量PCR技术分别检测雌二醇、胰岛素、雌二醇 + 胰岛素作用后Ishikawa细胞中VEGF、bFGF基因表达的情况。结果显示相同浓度雌二醇(1 × 10−8 mol/L)，胰岛素(1 × 10−8 mol/L)，雌二醇 + 胰岛素(1 × 10−8 mol/L雌二醇 + 1 × 10−8 mol/L胰岛素)对Ishikawa细胞中VEGF、bFGF均有不同程度促进表达作用；VEGF、bFGF基因在雌二醇+胰岛素组中表达与雌二醇、胰岛素组比较无明显增高，差异没有统计学意义(p > 0.05)。因此，雌激素、胰岛素诱导子宫内膜癌细胞系Ishikawa细胞产生VEGF、bFGF，促进了子宫内膜癌发生、发展。 The aim of this article is to explore the expression and clinical significance of VEGF and bFGF in endometrial carcinoma. PCR technique was used to detect the variation of expression of VEGF and bFGF in estradiol, insulin and both adding group between Ishikawa cells. Estradiol, insulin, estradiol insulin have different degrees of promoting the expression of VEGF and bFGF at the same concentration of E2, Insulin, E2 + Insulin on Ishikawa cells; the expression of VEGF and bFGF in E2 + Insulin cell group in the same time point shows no significant increase than E2, Insulin cell group. The difference was not statistically significant (p > 0.05). VEGF and bFGF are important angiogenesis factors in endometrial carcinoma. Estrogen and insulin induced Ishikawa cells produce VEGF and bFGF and promote the occurrence and development of endometrial carcinoma.

(125)I-labeled anti-bFGF monoclonal antibody inhibits growth of hepatocellular carcinoma.

To investigate the inhibitory efficacy of (125)I-labeled anti-basic fibroblast growth factor (bFGF) monoclonal antibody (mAb) in hepatocellular carcinoma (HCC). bFGF mAb was prepared by using the 1G9B9 hybridoma cell line with hybridization technology and extracted from ascites fluid through a Protein G Sepharose affinity column. After labeling with (125)I through the chloramine-T method, bFGF mAb was further purified by a Sephadex G-25 column. Gamma radiation counter GC-1200 detected radioactivity of (125)I-bFGF mAb. The murine H22 HCC xenograft model was established and randomized to interventions with control (phosphate-buffered saline), (125)I-bFGF mAb, (125)I plus bFGF mAb, bFGF mAb, or (125)I. The ratios of tumor inhibition were then calculated. Expression of bFGF, fibroblast growth factor receptor (FGFR), platelet-derived growth factor, and vascular endothelial growth factor (VEGF) mRNA was determined by quantitative reverse transcriptase real-time polymerase chain reaction. The purified bFGF mAb solution was 8.145 mg/mL with a titer of 1:2560000 and was stored at -20 °C. After coupling, (125)I-bFGF mAb was used at a 1: 1280000 dilution, stored at 4 °C, and its specific radioactivity was 37 MBq/mg. The corresponding tumor weight in the control, (125)I, bFGF mAb, (125)I plus bFGF mAb, and (125)I-bFGF mAb groups was 1.88 ± 0.25, 1.625 ± 0.21, 1.5 ± 0.18, 1.41 ± 0.16, and 0.98 ± 0.11 g, respectively. The tumor inhibition ratio in the (125)I, bFGF mAb, (125)I plus bFGF mAb, and (125)I-bFGF mAb groups was 13.6%, 20.2%, 25.1%, and 47.9%, respectively. Growth of HCC xenografts was inhibited significantly more in the (125)I-bFGF mAb group than in the other groups (P < 0.05). Expression of bFGF and FGFR mRNA in the (125)I-bFGF mAb group was significantly decreased in comparison with other groups (P < 0.05). Groups under interventions revealed increased expression of VEGF mRNA (except for (125)I group) compared with the control group. (125)I-bFGF mAb inhibits growth of HCC xenografts. The coupling effect of (125)I-bFGF mAb is more effective than the concomitant use of (125)I and bFGF mAb.

Molecular Cloning and Expression of Novel Fibroblast Growth Factor-2 Conjugated with Immunodominant Domains of Pseudomonas exotoxin

Angiogenesis is very important in cancer growth and metastasis. Basic fibroblast growth factor (bFGF) as one of the most important angiogenesis factors is an attractive target for cancer vaccine. Due to low immunogenicity, it cannot stimulate an effective immune response. Theoretically, pseudomonas exotoxin (PE) as a potent immunogenic carrier protein when fused to low immunogenic antigens such as bFGF significantly increased immunogenicity of it. In this study, we tried to molecular cloning and expression of bFGF conjugated with immunodominant domains of pseudomonas exotoxin. The coding sequence of fusion protein composed of bFGF linked to PE domains 1b and 2 using EAAAK poly linker. The KDEL sequence was also used in C-terminal coding sequence. It was synthesized and expressed using recombinant DNA technology in the bacterial expression system. Expression of recombinant protein verified using SDS-PAGE and western blot analyses. Finally, it purified using Ni-affinity chromatography. The band close to 37 kDa in SDS-PAGE and western blot analyses was aligned completely to designed sequence. Purified recombinant protein also showed as a clear single band near to 37 kDa.

Stable Expression of Basic Fibroblast Growth Factor in Chloroplasts of Tobacco.

Basic fibroblast growth factor (bFGF) is a multifunctional factor in acceleration of cell proliferation, differentiation and transference, and therefore widely used in clinical applications. In this study, expression vector pWX-Nt03 harboring a codon-optimized bFGF gene was constructed and introduced into the tobacco chloroplasts by particle bombardment. After four rounds of selection, bFGF was proved to integrate into the chloroplast genome of regenerated plants and two of four transgenic plants were confirmed to be homoplastomic by PCR and Southern hybridization. ELISA assay indicated that bFGF represented approximately 0.1% of total soluble protein in the leaves of transplastomic tobacco plants. This is the first report of bFGF expression via chloroplast transformation in model plant, providing an additional option for the production of chloroplast-produced therapeutic proteins.

Spatiotemporal release of BMP-2 and VEGF enhances osteogenic and vasculogenic differentiation of human mesenchymal stem cells and endothelial colony-forming cells co-encapsulated in a patterned hydrogel

Reconstruction of large bone defects is limited by insufficient vascularization and slow bone regeneration. The objective of this work was to investigate the effect of spatial and temporal release of recombinant human bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor (VEGF) on the extent of osteogenic and vasculogenic differentiation of human mesenchymal stem cells (hMSCs) and endothelial colony-forming cells (ECFCs) encapsulated in a patterned hydrogel. Nanogels (NGs) based on polyethylene glycol (PEG) macromers chain-extended with short lactide (L) and glycolide (G) segments were used for grafting and timed-release of BMP2 and VEGF. NGs with 12kDa PEG molecular weight (MW), 24 LG segment length, and 60/40L/G ratio (P12-II, NG(10)) released the grafted VEGF in 10days. NGs with 8kDa PEG MW, 26 LG segment length, and 60/40L/G ratio (P8-I, NG(21)) released the grafted BMP2 in 21days. hMSCs and NG-BMP2 were encapsulated in a patterned matrix based on acrylate-functionalized lactide-chain-extended star polyethylene glycol (SPELA) hydrogel and microchannel patterns filled with a suspension of hMSCs+ECFCs and NG-VEGF in a crosslinked gelatin methacryloyl (GelMA) hydrogel. Groups included patterned constructs without BMP2/VEGF (None), with directly added BMP2/VEGF, and NG-BMP2/NG-VEGF. Based on the results, timed-release of VEGF in the microchannels in 10days from NG(10) and BMP2 in the matrix in 21days from NG(21) resulted in highest extent of osteogenic and vasculogenic differentiation of the encapsulated hMSCs and ECFCs compared to direct addition of VEGF and BMP2. Further, timed-release of VEGF from NG(10) in hMSC+ECFC encapsulating microchannels and BMP2 from NG(21) in hMSC encapsulating matrix sharply increased bFGF expression in the patterned constructs. The results suggest that mineralization and vascularization are coupled by localized secretion of paracrine signaling factors by the differentiating hMSCs and ECFCs.

Possible involvement of basic FGF in the upregulation of PDGFRβ in pericytes after ischemic stroke

Central nervous system (CNS) pericytes have been recognized as an indispensable component of the neurovascular unit. The expression of platelet-derived growth factor receptor β (PDGFRβ) is markedly increased in CNS pericytes after brain ischemia. It has been elucidated that PDGFRβ, expressed in pericytes and pericyte-derived fibroblast-like cells, plays important roles in the maintenance of the blood–brain barrier (BBB) and in the repair process in infarct areas. The aim of this study was to uncover how the PDGFRβ expression is regulated in pericytes after brain ischemia. We found that basic fibroblast growth factor (bFGF), but neither hypoxia at 1% O2 nor acidification at pH 6.5, significantly upregulated the PDGFRβ expression in human cultured CNS pericytes. SU5402, an inhibitor of FGF receptor (FGFR), and inhibitors of its downstream effectors Akt and Erk abolished the bFGF-induced upregulation of PDGFRβ. On the other hand, acidification significantly upregulated the expression of bFGF, while hypoxia upregulated the expression of FGFR1 in the pericytes. The expression of bFGF and FGFR1 was markedly induced in the ischemic hemisphere after ischemic insult in a middle cerebral artery occlusion stroke model. Immunofluorescent double labeling demonstrated that the expression of bFGF and FGFR1 was co-localized with PDGFRβ-positive cells in peri-infarct areas. Moreover, treatment with bFGF enhanced cell growth and the PDGF-BB-induced migratory activity of cultured pericytes, which were significantly suppressed by SU5402 or Sunitinib, an inhibitor of PDGFR. These data suggested that increased bFGF upregulates the expression of PDGFRβ and may enhance PDGFRβ-mediated pericyte functions after brain ischemia.

Abstract 15623: Transplantation of Adipose-derived Stem Cells Sheet for Accelerated Neovascularization and Engraftment in Acute Myocardial Infarction Rats

Background: Transplantation of adipose-derived stem cell (ADSCs) has been suggested to improve cardiac function after acute myocardial infarction (AMI). Poor retention rate and differentiation ability of the implanted ADSCs limited their therapeutic potential. This study investigated the application of cell sheet technology for ADSC to improve engraftment and functional recovery in rat AMI model. Methods: Mouse ADSC (mADSC) sheets were successfully obtained by using the thermo-responsive dishes. Three groups of sham (n=10), suspended mADSC myocardial injection (n=10) and mADSC sheet transplantation (n=10) were compared in rat AMI model of left anterior descending coronary artery ligation. Final analyses were performed at day 28 after cell implantation. Results: In rat AMI model, mADSC sheet showed higher cellular engraftment in the infarcted heart compared to suspended mADSCs and sham control (signal unit; 227.3±18.3, 2.0±2.0 and 0; p&lt;0.05). Cytokine analysis of the cell-implanted myocardial tissues showed that higher expression of IGF-1, bFGF, TGF-β1, and EGF in mADSC sheet compared to suspended mADSCs and sham control (p&lt;0.05). Microvascular density of the peri-infarct zone was also higher in mADSC sheet compared to suspended mADSCs and sham. Interestingly, the transplanted cell-derived microvessels determined by mouse specific anti-vWF antibody were detected only in mADSC sheet. Echocardiography showed the significant improvement of ejection fraction (EF) in both suspended mADSCs and mADSC sheet compared to sham control (ΔEF; 15.60±0.44%, 15.24±0.62% and 3.53±%0.18; p&lt;0.05). Conclusions: Although the functional recovery was comparable between ADSC sheet transplantation and suspended ADSC injection, ADSC sheet transplantation improved the cellular engraftment, paracrine effect of growth factor and cytokine secretion, and in-vivo neovascularization compared to suspended ADSC injection.

Abstract 14104: Replacement of Infarct Myocardium by Large Scale-Expanded Human Induced Pluripotent Stem Cell-Derived Cardiac Cell-Sheet in a Porcine Chronic Myocardial Infarction Model

Introduction: It has been shown that transplanted induced pluripotent stem cell (iPSC)-derived cardiac cells in the myocardial infarction (MI) heart synchronously contract with native myocardium to mechanically contribute to functional recovery in rodent models. We herein hypothesized that large scale cardiac cell-sheets generated by human iPSCs may induce a greater functional recovery than small scale ones after transplantation in chronic MI heart. Methods: Bioreactor-based three-dimensional suspension culture system was used for generating large scale-expanded human iPSC-derived cardiomyocytes, of which cardiac troponin T positivity was constantly 75-85%. Scaffold-free cell-sheets containing several cell number (1.0х10^6, 10^7, 10^8) were transplanted over the cardiac surface in porcine chronic MI heart (n=5 each). Tacrolimus and prednisolone were daily given in all pigs against xeno-transplantation-inducing immune reaction. Results: Echocardiographically, left ventricular systolic and diastolic dimensions were significantly decreasing and ejection fraction was significantly increasing in the 10^8 cell group. In addition, global myocardial structure was better preserved in the 10^8 cell group with presence of the graft in the infarct area macroscopically (Figure). Moreover, there were significantly less accumulation of interstitial fibrosis in the infarct-remote area and greater vascular density and expression of VEGF, bFGF, and SDF-1 in the infarct-border area in the 10^8 cell group than the other groups at 3 months after the transplantation. Conclusions: Large scale human iPSC-derived cardiac cells were engrafted in the infarct myocardium, showing substantial functional recovery in a porcine chronic MI heart, indicating that artificial cell-based myocardial replacement therapy may be achieved. In contrast, small scale cardiac cells induced modest functional recovery, suggesting paracrine mechanisms of this treatment.

Effects of 530 nm monochromatic light on basic fibroblast growth factor and transforming growth factor-β1 expression in Müller cells.

To expose rat retinal Müller cells to 530 nm monochromatic light and investigate the influence of varying light illumination times on basic fibroblast growth factor (bFGF) and transforming growth factor-β1 (TGF-β1) expression. Three groups of rat retinal Müller cells cultured in vitro under a 530 nm monochromatic light were divided into 6, 12 and 24h experimental groups, while cells incubated under dark conditions served as the control group. The bFGF and TGF-β1 mRNA expression, protein levels and fluorescence intensity of the Müller cells were analyzed. The bFGF mRNA expression and protein levels were significantly upregulated in Müller cells in all three experimental groups compared with the control group (P<0.05), while that of TGF-β1 was downregulated (P<0.05). Also, bFGF expression was positively correlated, but TGF-β1 expression was negatively correlated with illumination time. The largest changes for both cytokines were seen in the 24h group. The changes in bFGF and TGF-β1 fluorescence intensity were highest in the 24h group, and significant differences were observed among the experimental groups (P<0.05). The expressions of bFGF and TGF-β1 changed in a time-dependent manner in Müller cells exposed to 530 nm monochromatic light with 250 lx illumination intensity. Müller cells might play a role in the development of myopia by increasing bFGF expression or decreasing TGF-β1 expression. Changes in cytokine expression in retinal Müller cells may affect monochromatic light-induced myopia.

CD73 on B16F10 melanoma cells in CD73-deficient mice promotes tumor growth, angiogenesis, neovascularization, macrophage infiltration and metastasis

Ecto-5′-nucleotidase (CD73), an enzyme providing interstitial adenosine, mediates diverse physiological and pathological responses. In tumor progression, it has primarily an immunosuppressive role but is also thought to regulate neovascularization. However, the latter role is still in debate. When B16F10 melanoma was subcutaneously injected into CD73 knockout mice, changes in the tumor vasculature were not always observed. However, we demonstrated earlier that the growth and vascularization of B16F10 melanoma in CD73 knockout mice depend on the low presence of CD73 on tumor cells. To further analyze the role of CD73 on tumor growth and vascularization, we compared the changes in B16F10 melanoma subcutaneously injected into right flank of wild-type mice, CD73 knockout mice lacking host CD73 only, and CD73 knockout mice with tumor cell CD73 either inhibited with AOPCP (adenosine α,β-methylene 5′-diphosphate) or permanently knocked down through genetic modification. We report here that both inhibition and knockdown of tumor CD73 further inhibited tumor growth compared to host CD73 knockout alone. MAP-kinase signaling pathway activation also decreased more strongly in the stable knockdown. There was a significant reduction in the angiogenic activation of blood microvessels as observed by decreased anti-VEGFR2 staining. Stable CD73 knockdown also reduced endothelial cell proliferation as measured by anti-CD105 staining. However, only chemical inhibition with AOPCP significantly augmented the reduction in intratumoral microvessel density induced by host CD73 knockout. Such reduction was not observed when tumor CD73 was knocked down due to the much slower tumor growth and decreased oxygen demand as indicated by the low expression of Bad, a hypoxia marker. Decreased CD73 activity also led to the decreased expression of angiogenic factors, including VEGF and bFGF that was only partially reversed by hypoxia in tumors treated with AOPCP. Both inhibition and knockdown of tumor CD73 significantly decreased tumor macrophage infiltration and induced microenvironment changes, thereby influencing MI or MII macrophage polarization. Additionally, tumor cell CD73 is important in metastasis formation through adenosine-independent attachment to endothelium. We conclude that even low tumor cell CD73 expression has an undeniable role in melanoma progression, including the regulation of many aspects of angiogenesis. CD73 is thus a viable target in anti-angiogenic melanoma therapy.

Expressions of MMP-7 and bFGF and their correlations with tumor interstitial microvascular density and cancer metastasis in NSCLC

Objective To investigate the expressions of matrix metallopro-teinase-7 (MMP-7), basic fibroblast growth factor (bFGF) in NSCLC, and to elucidate the correlations among MMP-7, bFGF and MVD, as well as their relationships with cancer metastasis in NSCLC. Methods The expressions of MMP-7, bFGF and MVD were detected by immunohistochemical SP method in 80 cases of NSCLC and 40 cases of normal lung tissue, compare the expression of MMP-7 and bFGF in NSCLC and normal lung tissue, and then invest the relationship between their expression and lymph node metastasis in NSCLC. The MVD was investigated within the CD34 positive slides, check the relationship between MVD and the expression of MMP-7 , bFGF in NSCLC, and also its relationship with histology type, differentiation degree, lymph node metastasis and TNM stage. Results The positive rates of MMP-7 and bFGF were significantly higher in NSCLC than those in normal lung tissue (70.0% vs 12.5%, χ2=35.276, P=0.000; 65.0% vs 10.0%, χ2=32.411, P=0.000). The positive rates of MMP-7 and bFGF were also significantly higher in group with lymph node metastasis compared to those group without lymph node metastasis (83.0% vs 51.5%, χ2=9.139, P=0.003; 80.9% vs 42.4%, χ2=12.584, P=0.000). The numbers of MVD were significantly higher in cases with MMP-7 and bFGF positive expressions than those in cases with MMP-7 and bFGF negative expressions (46.78±11.11 vs 31.12±7.62, t=5.831, P=0.000; 45.05±10.97 vs 34.77±8.56, t=5.364, P=0.000). The number of MVD in group with lymph node metastasis was also significantly higher than that in group without lymph node metastasis (61.32±21.61 vs 40.52±18.31, t=3.865, P=0.008). The number of MVD in staging Ⅲ-Ⅳ group was significantly higher than that in stagingⅠ-Ⅱgroup ( 55.28±15.64 vs 41.73±16.48, t=2.531, P=0.013). Conclusion The expression levels of MMP-7 and bFGF in NSCLC are high, and the positive rates of MMP-7 and bFGF are correlated with the lymphnode metastasis. The numbers of MVD are significantly higher in cases with MMP-7 and bFGF positive expressions than those in cases with MMP-7 and bFGF negative expre-ssions. MVD is correlated with lymph node metastasis and the TNM stage. It suggests that the expressions of MMP-7, bFGF and MVD may involve in the invasion and metastasis of NSCLC. Key words: Carcinoma, non-small-cell lung; Immunohistochemistry; Matrix metalloproteinase-7; Basic fibroblast growth factor; Microvascular density

The effect of ultrashortwave therapy on the expression of fibroblast growth factor in the wounds of diabetics

Objective To observe the effect of ultrashortwave irradiation on the expression of basic fibroblast growth factor (bFGF) and the growth of new capillaries and pathological changes in wounds affecting diabetics. Methods Ninety healthy, adult, male Sprague-Dawley rats were randomly divided into a control group, a diabetic group and an experimental group, each of 30. A model of diabetes was induced in each rat of the diabetic and experimental groups by intraperitoneal injection of 55 mg/kg of streptozotocin. Skin ulcers were then established by scraping. The experimental group was given ultrashortwave treatment, while the other groups were given no intervention. Three, 7, 14 and 21 days after the injury, tissues at the edge and in the central part of the ulcers were collected and given hematoxylin-eosin staining, and microvessels were counted. Any pathological changes in the ulcers were observed under an optical microscope, and the expression of bFGF was detected using immunohistochemistry. Results In the control group, the 50% and 100% healing times were (8.94±0.87)d and (16.56±1.04)d respectively. The 50% and 100% healing times of the experimental group were (12.78±1.26)d and (23.25±1.54)d-significantly shorter than those of the diabetic group. The average microvessel count of the experimental group rats increased from (12.5±1.52) on day 3 to (17.83±1.94) on day 7 and then to (23.5±1.876) on day 14 and finally to (29.33±2.736) on day 21. Those counts were significantly greater than those of the diabetic group at the same time points. The average index of bFGF expression increased from (11.83±1.17) on day 3, to (23.33±1.51) on day 7 and further to (55.50±4.09) on day 21, and then decreased to (22.50±1.87) on day 21, but always remaining significantly higher than in the diabetic group at the same time points. Conclusion Ultrashortwave therapy can increase the expression of bFGF, promote capillary angiogenesis and quicken the healing of diabetics' wounds. Key words: Diabetes; Skin ulcers; Basic fibroblast growth factor; Ultrashortwave therapy