Basic Fibroblast Growth Factor Protein Research Articles

Production and characterization of basic fibroblast growth factor protein in rice suspension cultures

The basic fibroblast growth factor (bFGF) plays a major role in cell growth, survival, and other mitogenic activities. General applications of this protein involve tissue repair, morphogenesis, embryonic development, cellular growth, etc. Due to its importance and high demand, research has been done on producing recombinant bFGF in different expression platforms. We have produced bFGF in transgenic rice (Oryza sativa) cells growing in liquid suspension culture. The gene construct consists of the rice alpha-amylase (RAmy) 3D sugar-inducible promotor and rice glutelin secretion signal peptide for controlled production and release of the bFGF protein by manipulating the sugar concentration for batch or semi-continuous bioreactor runs. A thermal shift assay was performed on the purified rice cell produced bFGF (rbFGF) and the results were compared with E. coli derived bFGF. The purified rbFGF protein from rice cells was tested for propagating human-induced pluripotent stem cells (hiPSCs) up to eight passages. Furthermore, the biological activity measured in both crude lysate and purified rbFGF showed a similar pattern of the extracellular signal-regulated kinase (ERK) activity compared to E. coli derived bFGF (control), indicating that rbFGF is active before purification and the protein activity is not inhibited by other components of the lysate. From the current results, the rice-produced rbFGF protein is biologically active and could serve as a viable product for various applications.

An interesting case of papillary thyroid carcinoma with heterotrophic ossification

Calcification in thyroid gland is associated with both benign and malignant lesion. However calcification is most frequently seen in malignant lesions. Calcification can give rise to metaplasia and bone containing marrow tissue. Papillary carcinoma with heterotrophic ossification has been associated with high incidence extrathyroidal invasion, multifocality, lymphnode metastsasis and older age group than in those without heterotrophic ossification. It has not been described as a specific entity in WHO classification because of its rarity. Expression of both basic fibroblast growth factor and bone morphogenetic protein was highest in papillary carcinoma with intratumoral heterotrophic ossification. In this case report we described a 71-year-old female patient who sought medical care for swelling in the neck for the past 4 years. The cervical ultrasound showed a 2 well defined nodules measuring 2x2 cms and 4mm in the right lobe with larger lesion showing peripheral rim of calcification and left lobe and isthumus shows well defined lesions measuring 5mm and 4 mm each. The clinical diagnosis was multinodular goitre. Patient subsequently underwent thyroidectomy. On histopathological investigation right lobe showed follicular adenoma and papillary carcinoma thyroid with extensive heterotrophic ossification and calcification within the tumor. The margins were free of neoplasia and there was no extrathyroidal extension. No preineural/ vascular invasion noted. One lymphnode was identified which was free of tumor. pTNM staging was pT1bNxMx.

Aflibercept Suppression of angiopoietin-2 in a Rabbit Retinal Vascular Hyperpermeability Model.

Anti-vascular endothelial growth factor (anti-VEGF) therapies, which attenuate the capacity of VEGF to bind to VEGF receptors, are standard-of-care options for various retinal disorders that are characterized by pathologic retinal angiogenesis and vascular permeability. Multiple receptors and ligands have also been reported as being involved in these pathways, including angiopoietin-1 (ANG1) and angiopoietin-2 (ANG2). Electrochemiluminescence immunoassays were used to detect human VEGF (hVEGF), as well as rabbit ANG2 and basic fibroblast growth factor protein levels in vitreous samples derived from a study evaluating the efficacy of the anti-VEGF agents ranibizumab, aflibercept, and brolucizumab in an hVEGF165-induced rabbit retinal vascular hyperpermeability model. hVEGF was completely suppressed in rabbit vitreous after anti-VEGF treatment for 28 days. ANG2 protein in vitreous and ANGPT2 mRNA in retina tissue were similarly suppressed, although the anti-VEGF agents do not directly bind to ANG2. Aflibercept demonstrated the greatest inhibitory effect in ANG2 levels in vitreous, which correlated with strong, durable suppression of intraocular hVEGF levels. This study explored the effects of anti-VEGF therapies beyond direct binding of VEGF by evaluating protein levels and the expression of target genes involved in angiogenesis and associated molecular mechanisms in the rabbit retina and choroid. In vivo data suggest that anti-VEGF agents currently used for the treatment of retinal diseases could provide beneficial effects beyond direct binding of VEGF, including suppression of ANG2 protein and ANGPT2 mRNA.

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

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

Acupuncture intervention prolongates thrombolysis time window by regulating cerebral cortex angiogenesis-related genes and proteins in cerebral infarction rats

To observe the effect of acupuncture on the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and endostatin (Endostatin, ES) mRNAs and proteins (angiogenesis related factors) in the ischemic penumbra region in rats with cerebral infarction (CI), so as to explore its underlying mechanisms in prolonging the time window of thrombolysis therapy for CI. A total of 48 male SD rats were randomly divided into sham operation, model, medication (6 h thrombolysis) and acupuncture (Acupunct)+medication groups (n=12 in each group). The CI model was established using modified auto-thrombus method. Six hours after thrombolysis, recombinant human tissue plasminogen activator (rt-PA,10 mg/kg) was given to rats of the thrombolysis group through tail vein. Acupuncture was applied at "Shuigou"(CV26) and bilateral "Neiguan" (PC6) 2 h after successful modeling, and the needles were retained for 30 minutes. Cerebral blood flow (CBF) was monitored during modeling in each group, and the neurological deficit score (0-7 points) was given 2 h and 24 h after successful modeling according to Bederson's methods. The cerebral infarction volume was observed after triphenyltetrazole chloride (TTC) staining. The protein and mRNA expression levels of VEGF, bFGF and ES in the ischemic penumbra region of the right cerebral cortex were detected by Western blot and real-time PCR, separately. The neurological deficit score at both 2 h and 24 h after modeling, percentage of cerebral infarction volume, and the expression levels of VEGF, bFGF and ES proteins and mRNAs in the model group were significantly higher than those of the sham operation group (P<0.01, P < 0.05). Compared with the model group, the neurological deficit score 24 h (not at 2 h) after modeling and percentage of cerebral infarction volume, and the expression levels of ES protein and mRNA in the Acupunct+medication group (not in the medication group) were notably lower (P<0.05, P<0.01), while the expression levels of VEGF and bFGF proteins and mRNAs in the Acupunct +medication group (not in the medication group) were considerably higher (P<0.01, P<0.05). No significant differences were found between medication and model groups in the CI percentage, VEGF, bFGF and ES proteins and mRNAs (P>0.05). The therapeutic effect of Acupunct +medication group was significantly superior to that of medication in lowering neurological deficit score, percentage of CI volume and expression of ES protein and mRNA and in up-regulating the expression of VEGF and bFGF proteins and mRNAs (P<0.05, P<0.01). Acupuncture and timely intervention can prolong the time window of thrombolysis in CI rats, which may be related to its function in up-regulating the expression of VEGF and bFGF mRNAs and proteins and in down-regulating the expression of ES mRNA and protein in ischemic cerebral cortex.

Bioactive strontium ions/ginsenoside Rg1-incorporated biodegradable silk fibroin-gelatin scaffold promoted challenging osteoporotic bone regeneration.

Autogenous healing of osteoporotic fractures is challenging, as the regenerative capacity of bone tissues is impaired by estrogen reduction and existed pro-inflammatory cytokines. In this study, a biofunctional ginsenoside Rg1 and strontium-containing mineral (SrHPO4, SrP)-incorporated biodegradable silk fibroin-gelatin (SG) scaffold (Rg1/SrP/SG) was developed to stimulate the osteoporotic bone repair. The incorporation of 15 wt% SrP significantly enhanced the mechanical strength, stimulated the osteogenic differentiation of mouse bone marrow mesenchymal stem cells, and suppressed the osteoclastogenesis of RAW264.7 in a concentration-related manner. The loading of Rg1 in SG and 15SrP/SG scaffolds obviously promoted the angiogenesis of human umbilical vein endothelial cells via activating the expression of vascular endothelial growth factor and basic fibroblast growth factor genes and proteins. The bioactive strontium ions (Sr2+) and Rg1 released from the scaffolds together mediated lipopolysaccharide-treated macrophages polarizing into M2 type. They downregulated the expression of inflammatory-related genes (interleukin (IL)-1β, tumor necrosis factor α, and IL-6) and stimulated the expression of genes related to anti-inflammation (Arginase and IL-10) as well as bone repair (BMP-2 and PDGF-BB) in the macrophages. The in vivo results also displayed that SrP and Rg1 significantly promoted the bone repair effect of SG scaffolds in osteoporotic critical-sized calvarial defects. Besides, the degradation rate of the scaffolds was close to the bone regeneration rate. Therefore, the simultaneous addition of SrP and Rg1 is a promising way for facilitating the osteoporotic bone repair activity of SG scaffolds via promoting the osteogenesis and angiogenesis, as well as inhibiting the osteoclastogenesis and inflammation.

Comparative Evaluation of Viability and Function of Albino Rabbit Periodontal Ligament Fibroblasts in Various Media (In Vitro Study)

Background: Tooth avulsion is one of traumatic injuries that cause damage to periodontal ligament (PDL) tissues. Avulsed tooth should be stored in a suitable storage medium to preserve the vitality of the PDL fibroblasts (PDLFs).Aim: To assess and compare the effect of various media on albino rabbit’s PDLFs viability and basic fibroblast growth factor (b-FGF) protein and gene expressions at different time intervals.Material and methods: periodontal ligament stem cells (PDLSCs) were isolated from molars of albino rabbit then differentiated into PDLFs. The PDLFs were cultured in different media (contact lens solution, skimmed milk, Dentosafe and alcohol free mouthwash) for 1h, 12h and 24h. Phosphate buffered saline (PBS) was used as control medium. Cell viability was assessed by MTT assay. b-FGF protein and gene expression levels were detected by immunofluorescence (IF) staining and Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) respectively. Data from MTT assay and H-score of IF staining were statistically analyzed using ANOVA test and multiple two groups comparisons pos hoc test.Results: examination of isolated PDLSCs at different passages showed various shapes of cells from rounded to spindle shaped. The differentiated PDLFs showed positive IF reaction. Statistical analysis of tested media at different time intervals represented that; PBS had the highest mean of fibroblastic cell viability% and H-score, followed by Dentosafe, skimmed milk, contact lens solution and mouthwash respectively. Gene expression of b-FGF was up-regulated in Dentosafe and skimmed milk while it was down-regulated in mouthwash.Conclusions: Dentosafe is more effective in maintenance of PDLFs viability and function than the other tested media. Mouthwash is not recommended as a storage media for PDLFs.

Growth factors enhanced angiogenesis and osteogenesis on polydopamine coated titanium surface for bone regeneration

Angiogenesis and osteogenesis have been considered as inseparable and critical processes for bone formation and fracture healing. One promising strategy to improve the capability of angiogenesis and osteogenesis on a titanium (Ti) surface involves the fabrication of stable multifunctional release interfaces. This study developed a combined sequential bio-interfacial release system with basic fibroblast growth factor (bFGF) and bone morphogenetic protein 2 (BMP-2) using a polydopamine (PDA) coating on the Ti surface to enhance angiogenesis and osteogenesis. Their surfaces were characterized and their biological properties were evaluated to provide sufficient supporting evidence for successfully fabricating the combined release system. The novel bio-interface demonstrated a significant improvement in surface hydrophilicity; highly effective growth factor adsorption; and moderate, sustained release on the surface of modified Ti substrates. Furthermore, this system surpassingly promoted the initial proliferation, migration and late angiogenic and osteogenic differentiation of HUVECs and MC3T3-E1 in vitro while also augmenting the osseointegration between the implant surface and the surrounding host tissues in vivo. All these results suggest that the fabrication of this novel bio-interface is a practical modification strategy for Ti implants to stimulate angiogenesis and osteogenesis and further optimize its clinical performance.

The Mechanism Underlying the Protective Effects of Tannic Acid Against Isoproterenol-Induced Myocardial Fibrosis in Mice.

Tannic acid (TA) belongs to a class of complex water-soluble polyphenolic derivatives that show anticarcinogenic, antiinflammatory, antioxidant, and scavenging activities. Here, we investigate the protective effects of TA against isoproterenol (ISO)-induced myocardial fibrosis (MF) in mice. Mice received TA and ISO dosing and were sacrificed 48 h later. The activities of creatine kinase (CK), creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), and mitochondria enzymes were measured. Cardiac histopathology was done using H&E, Sirius red, and Masson’s Trichrome staining. Immunohistochemical staining was applied to indicate changes in B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and basic fibroblast growth factor (bFGF) protein expressions in cardiac tissue. RT-PCR was used to measure the expression of atrial and brain natriuretic peptides (ANP and BNP, respectively), c-fos, and c-jun. Western blotting was used to measure the expression of nuclear factor-κB (NF-κB) p65, phosphorylated NF-κB p65), toll-like receptor 4 (TLR4), p38, phosphorylated p38, Bax, Bcl-2, and caspase-3. Compared to the ISO group, the TA group had reduced levels of TLR4, p38, p-p38, NF-κB (p65), p-NF-κB (p-p65), caspase-3, Bax, and Bcl-2, as well as CK, CK-MB, and LDH. These results indicate that TA protects against ISO-induced MF, possibly through its ability to suppress the TLR4-mediated NF-κB signaling pathway.

Recombinant protein production associated growth inhibition results mainly from transcription and not from translation

BackgroundRecombinant protein production can be stressful to the host organism. The extent of stress is determined by the specific properties of the recombinant transcript and protein, by the rates of transcription and translation, and by the environmental conditions encountered during the production process.ResultsThe impact of the transcription of the T7-promoter controlled genes encoding human basic fibroblast growth factor (hFGF-2) and green fluorescent protein (GFP) as well as the translation into the recombinant protein on the growth properties of the production host E. coli BL21(DE3) were investigated. This was done by using expression vectors where the promoter region or the ribosome binding site(s) or both were removed. It is shown that already transcription without protein translation imposes a metabolic burden on the host cell. Translation of the transcript into large amounts of a properly folded protein does not show any effect on cell growth in the best case, e.g. high-level production of GFP in Luria–Bertani medium. However, translation appears to contribute to the metabolic burden if it is connected to protein folding associated problems, e.g. inclusion body formation.ConclusionThe so-called metabolic burden of recombinant protein production is mainly attributed to transcription but can be enhanced through translation and those processes following translation (e.g. protein folding and degradation, heat-shock responses).

Modified bacterial outer membrane vesicles induce autoantibodies for tumor therapy

Using monoclonal antibodies to block tumor angiogenesis has yielded effective antitumor effects. However, this treatment method has long cycles and is very expensive; therefore, its long-term and extensive application is limited. In this study, we developed a nanovaccine using bacterial biomembranes as carriers for antitumor therapy. The whole basic fibroblast growth factor (BFGF) molecule (154 amino acids (aa)) was loaded onto bacterial outer membrane vesicles (OMVs) using gene recombination technology. The strong adjuvant effect of OMVs was used to induce the host to produce anti-BFGF autoantibodies. We proved that persistent anti-BFGF autoantibodies can be induced in mice after only 3 immunizations to antagonize BFGF functions. The effects included multiple tumor suppression functions, including inhibition of tumor angiogenesis, induction of tumor cell apoptosis, reversal of tumor immune barriers, and promotion of tumor-specific cytotoxic T lymphocytes (CTLs), eventually causing tumor regression. We confirmed that bacterial biomembranes can be used as a vaccine delivery system to induce the production of antibodies against autoantigens, which may be used for tumor therapy. This study expands the application fields of bacterial biomembrane systems and provides insight for tumor immunotherapy other than monoclonal antibody technology. Statement of SignificanceIn this study, we proved that bacteria-released outer membrane vesicles (OMVs) modified via genetic engineering can be used as a vaccine carrier to break autoimmune tolerance and induce the body to produce autoantibodies to antagonize pathological molecules and block pathological signaling pathways for tumor therapy. OMVs naturally released by bacteria were used to successfully load the full-length BFGF protein (154 aa). We proved that persistent anti-BFGF autoantibodies can be induced in tumor-bearing mice after only 3 immunizations to effectively inhibit tumors. Furthermore, the production of these antibodies successfully inhibited tumor angiogenesis, promoted tumor cell apoptosis, reversed the tumor immunosuppressive microenvironment, increased the cytotoxic T lymphocyte (CTL) reaction, and eventually inhibited tumor growth.

Initiation of Conceptus Elongation Coincides with an Endometrium Basic Fibroblast Growth Factor (FGF2) Protein Increase in Heifers.

Fibroblast growth factors (FGF) play an important role during embryo development. To date, the role of FGF and the respective receptors (FGFR) during the preimplantation phase in cattle are not fully characterized. We examined FGF1, FGF2, FGFR1, FGFR2, and FGFR3 in cyclic and early pregnant heifers at Days 12, 15, and 18 after insemination (Day 0). Endometrial FGF1 mRNA transcript abundance in heifers varied significantly with respect to the day after insemination, the pregnancy status, and their interaction. The expression was higher in nonpregnant than in pregnant heifers at Day 18. The conceptus transcripts abundance of FGFR2 and FGFR3 were significantly lower at Day 15 than 18. In the endometrium, FGF1 protein abundance significantly decreased from Day 12 onwards and FGF2 protein abundance showed a minor, but a significant increase at Day 15 in comparison to Days 12 and 18. We concluded that the decrease in FGF1 mRNA expression in pregnant heifers at Day 18 points towards a potential contribution of FGF1 in the preimplantation process. Additionally, successful embryo elongation might require a spatiotemporal FGF2 protein increase in the endometrium.

Scale up of a chromatographic capture step for a clarified bacterial homogenate – Influence of mass transport limitation and competitive adsorption of impurities

A model-based approach for scaling up chromatographic capture step was developed. The purification of human basic fibroblast growth factor protein 2 (FGF2) from an E. coli homogenate on a cation exchange resin was selected as a case study. Non-ideal effects accompanying the capture operation were examined, including: reduction in the protein diffusivity in the presence of the homogenate, competitive adsorption between FGF2 and undefined impurities, and flow behavior in external column volumes. The viscosity of the homogenate was measured as a function of dilution degree and shear stress, and its contribution to the diffusivity reduction was quantified. A dynamic model was formulated which accounted for underlying kinetic and thermodynamic dependencies. The model parameters were determined for a lab scale system using a small 2-mL column. The model was successfully used to scale up the capture operation from the lab scale column to a preparative bench scale column of about 1 L volume.

Electroacupuncture combined with rehabilitation training improves regional cerebral blood flow and reduces infarct volume by promoting expression of angiogenesis-related factors in acute cerebral ischemia rats

To observe the effect of electroacupuncture (EA) combined with rehabilitation training on regional cerebral blood flow (rCBF) and angiogenesis in rats with acute cerebral ischemia (ACI), so as to explore its mechanisms underlying improvement of ACI. A total of 135 male SD rats were divided into 5 groups: sham-operation (sham), model, EA, rehabilitation training and EA+rehabilitation training (combined treatment) groups (n=27 rats in each group). The ACI model was established by occlusion of the middle cerebral artery with thread embolus. EA (2 Hz/20 Hz, 3-5 V) was applied to "Baihui" (GV20), "Shuigou" (GV26) and bilateral "Neiguan" (PC6) for 20 min, once daily for 14 days. The rehabilitation training including hair-brushing in an enriched environment (10 min), round wooden-stick turning (10 min), grid-board climbing (10 min), and treadmill running (30 min/d) was condacted once daily for 14 days. The rCBF was measured by Doppler ultrasound. The cerebral infarct volume (CIV) was measured after 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. The expression of CD34+ in the ischemic penumbra region of brain tissue was detected by immunohistochemistry, and the expressions of angiogenesis-related factors as vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2) and basic fibroblast growth factor (bFGF) proteins in the ischemic brain tissue were detected by Western blot. Following modeling, the rCBF levels at the 5 min, 3rd, 7th and 14th day were significantly decreased in the model group relevant to the sham group (P<0.01). After the intervention, the rCBF levels were significantly increased on day 3, 7 and 14 in the combined treatment group and on day 7 and 14 in both the EA and rehabilitation training groups in comparison with the model group (P<0.01). The CIV was obvious in the model group in comparison with the sham group (P<0.01), but was markedly smaller in the EA, rehabilitation training and combined treatment groups on day 3,7 and 14 than in the model group (P<0.01). The number of CD34+ positive cells, and the expression levels of VEGF, VEGFR2, and bFGF proteins in ischemic brain tissues were significantly higher on day 3, 7 and 14 in the model group than in the sham group (P<0.01, P<0.05), and were further up-regulated considerably at the 3 time-points in the 3 treatment groups (P<0.01, P<0.05). The therapeutic effect of EA+rehabilitation training was significantly superior to that of simple EA and simple rehabilitation training in up-regulating rCBF, CD34+ positive cell number, and expression levels of VEGF, VEGFR2 and bFGF, and in down-regulating the CIV on day 3,7 and 14 (P<0.05, P<0.01). No significant differences were found between the EA and rehabilitation groups in the above-mentioned 6 indexes (P>0.05). EA combined with rehabilitation training can reduce the infarct volume and increase rCBF in ACI rats, which is probably associated with its effects in promoting the expression of angiogenesis-related factors of ischemic brain tissues. The effect of EA combined with rehabilitation training is markedly better than that of EA and rehabilitation training alone.

Effects of mature rehmannia root extract on PDGF and bcl-2 in rat myocardial tissue after myocardial infarction

Objective To observe the effect of mature rehmannia extract on platelet derived growth factor (PDGF) and b-cell lymphoma-2 (bcl-2) 2 in myocardial tissue of rats with myocardial infarction. Methods A total of 30 healthy SD rats were randomly divided into blank group, model group and rehmannia glutinosa group with 10 rats in each group. Myocardial infarction models were established in rats of model group and prepared Rehmannia glutinosa group. The rats in the prepared rehmannia glutinosa group were subcutaneously injected with the extract of the prepared Rehmannia glutinosa 4 g/kg. The rats in the blank group and the model group were subcutaneously injected with the same volume of saline. Once a day for 15 days. The cardiac function was measured by echocardiography in rats. The HE staining was used to observe the histopathological changes of myocardium. The levels of PDGF and Bcl-2 in myocardium were detected by ELISA. The levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (BFGF) and CD34 protein were detected by immunoturbidimetry. Results Compared with the model group, the levels of LVDd, LVDs, LVEDs and LVESV in the Rehmannia glutinosa group were significantly decreased (P<0.05) and LVEF was significantly increased (P<0.05), while the levels of PDGF (0.53 ± 0.02 g/ml vs. 0.35 ± 0.01 g/ml), Bcl-2 (1.04 ± 0.20 g/ml vs. 0.84 ± 0.12 g/ml), VEGF (85.24 ± 12.45 pg/ml vs. 65.26 ± 10.06 pg/ml), BFGF (86.27 ± 6.56 pg/ml vs. 62.26 ± 4.37 pg/ml) and CD34 (102.36 ± 10.52 pg/ml vs. 26.37 ± 3.94 pg/ml) in in the Rehmannia glutinosa group were significantly increased (P<0.05). Conclusions The Rehmannia glutinosa extract can improve the cardiac function of rats with myocardial infarction, Promote the formation of new blood vessels and improve myocardial damage. Key words: Rehmanniae radix praeparata; Extracts; Myocardial infarction; Platelet derived growth factor; Apoptosis; Rats

Paracrine Effects of Adipose-Derived Stem Cells Promote Lymphangiogenesis in Irradiated Lymphatic Endothelial Cells.

There is currently no reliable treatment for secondary lymphedema caused by lymph node dissection or radiotherapy; however, stem cell-based regenerative medicine is emerging as a promising remedy for such complications. The purpose of this study was to examine the effects of adipose-derived stem cells on lymphangiogenesis involving human dermal lymphatic endothelial cells exposed to ionizing radiation. Proliferation, migration, and tube formation were analyzed in human dermal lymphatic endothelial cells that were co-cultured with adipose-derived stem cells or cultured in adipose-derived stem cell-conditioned medium. The levels of lymphangiogenic factors secreted from adipose-derived stem cells were analyzed by enzyme-linked immunosorbent assays and Western blotting. Co-culturing with adipose-derived stem cells and the use of adipose-derived stem cell-conditioned medium both significantly promoted proliferation, migration, and tube formation in nonirradiated human dermal lymphatic endothelial cells. The authors also found that irradiated adipose-derived stem cells had similar alleviative effects on irradiated human dermal lymphatic endothelial cells. Enzyme-linked immunosorbent assays and Western blotting analysis revealed that irradiating adipose-derived stem cells increased their secretion of basic fibroblast growth factor in a dose-dependent manner, whereas it caused no detectable change in their secretion of vascular endothelial growth factor A or C, or hepatocyte growth factor. These results demonstrated that factors secreted by adipose-derived stem cells contribute to the promotion of lymphangiogenesis in irradiated human dermal lymphatic endothelial cells. The authors' findings also suggest that radiation potentiates the paracrine effects of adipose-derived stem cells by stimulating basic fibroblast growth factor protein expression.

Local supplementation with plant-derived recombinant human FGF2 protein enhances bone formation in critical-sized calvarial defects.

Numerous studies have demonstrated the advantages of plant cell suspension culture systems in producing bioactive recombinant human growth factors. This study investigated the biological activity of recombinant basic human fibroblast growth factor (rhFGF2) protein produced by a plant culture system to enhance new bone formation in a bone defect mouse model. The human FGF2 cDNA gene was cloned into a plant expression vector driven by the rice α-amylase 3D promoter. The vector was introduced into rice calli (Oryza sativa L. cv. Dongjin), and the clone with the highest expression of rhFGF2 was selected. Maximum accumulation of rhFGF2 protein (approximately 28mg/l) was reached at 13day post-incubation. Male C57BL/6 mice underwent calvarial defect surgery and the defects were loaded with absorbable collagen sponge (ACS) only (ACS group) or ACS impregnated with 5μg of plant-derived rhFGF2 (p-FGF2) protein or E. coli-derived rhFGF2 (e-FGF2) protein. Similar to the effects of e-FGF2, local delivery with p-FGF2 enhanced bone healing in the damaged region to higher levels than the ACS group. Exogenous addition of p-FGF2 or e-FGF2 exhibited similar effects on proliferation, mineralization, and osteogenic marker expression in MC3T3-E1 cells. Together, the current findings support the usefulness of this plant-based expression system for the production of biologically active rhFGF2.

Ghrelin Ameliorates Traumatic Brain Injury by Down-Regulating bFGF and FGF-BP.

Traumatic brain injury (TBI) is a primary cause of disability and mortality. Ghrelin, a gastrointestinal hormone, has been found to have protective effects for the brain, but the molecular mechanism of these neuroprotective effects of ghrelin remains unclear. In this study, an electronic cortical contusion impactor was used to establish a rat TBI model and we investigated the effect of ghrelin on brain repair by neurological severity score and histological examination. An antibody array was employed to uncover the molecular mechanism of ghrelin’s neuroprotective effects by determining the alterations of multiple proteins in the brain cortex. As a result, ghrelin attenuated brain injury and promoted brain functional recovery. After TBI, 13 proteins were up-regulated in the brain cortex, while basic fibroblast growth factor (bFGF) and fibroblast growth factor-binding protein (FGF-BP) were down-regulated after ghrelin treatment. It is known that bFGF can induce angiogenesis in the brain and accelerate wound healing, which can be further enhanced by FGF-BP. Based on the previous studies, it is hypothesized that the exogenous ghrelin curing TBI might cause the closure of bFGF and FGF-BP functions on wound healing, or ghrelin might exert the neuroprotective effects by competitively inhibiting bFGF/FGF-BP-induced neovascularization. Whether the combinational administration of ghrelin and bFGF/FGF-BP can enhance or weaken the therapeutic effect on TBI requires further research.

B-cell leukemia/lymphoma 10 promotes angiogenesis in an experimental corneal neovascularization model

PurposeCorneal neovascularization (CrNV) arises from many causes including corneal inflammatory, infectious, or traumatic insult, and frequently leads to impaired vision. This study seeks to determine the role of B-cell leukemia/lymphoma 10 (BCL-10) in the development of experimental CrNV.MethodsCorneas from BCL-10 knockout (KO) mice and wild-type (WT) mice were burned by sodium hydroxide (NaOH) to create the CrNV model and neovascular formation in the corneas was assessed 2 weeks later. Intracorneal macrophage accumulation and the expression of angiogenic factors were quantified by flow cytometric analysis (FCM) and real-time PCR, respectively.ResultsThe amount of CrNV was determined 2 weeks after alkali burn. Compared to WT mice, the amount of CrNV in BCL-10 KO mice was significantly decreased. FCM revealed that F4/80-positive macrophages were markedly decreased in BCL-10 KO mice compared with WT mice. Reverse transcription PCR showed that the mRNA expression levels of intracorneal vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (bFGF) and monocyte chemotactic protein 1 were reduced in BCL-10 KO mice compared with WT mice.ConclusionBCL-10 KO mice exhibited reduced alkali-induced CrNV by suppressing intracorneal macrophage infiltration, which subsequently led to decreased VEGF-A and bFGF expression, suggesting that BCL-10 may become a potential clinical intervening target of CrNV.

Effects of Noggin-Transfected Neural Stem Cells on Neural Functional Recovery and Underlying Mechanism in Rats with Cerebral Ischemia Reperfusion Injury

To investigate neuroprotection of noggin-transfected neural stem cells (NSCs) against focal cerebral ischemia reperfusion injury (IRI) in rats. Eighty Wistar rats were randomly divided into the sham, IRI, NSCs, and noggin + NSCs groups. Noggin containing adenoviral vectors was transfected into rat NSCs. Rats were subjected to 2.0 hours middle cerebral artery occlusion and reperfusion 1.0 hour, followed by infusion into the lateral ventricles of NSCs alone, noggin-transfected NSCs, and saline at 3 days in the NSCs, noggin + NSCs, and sham groups, respectively. All rats were sacrificed on 1, 3, 7, and 28 days after transplantation; the colorimetric method was used to detect the levels of superoxide dismutase (SOD) and the malondialdehyde (MDA) content after the behavior capability determined. Western blot was performed for detecting the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) proteins. The TUNEL-positive and BrdU/nestin double-positive cells were observed under a light microscope and quantitative analysis was performed by morphometric technique. Noggin-transfected NSCs significantly decreased the infarct volume and improved the neurological scores. Noggin-transfected NSCs also reduced the percentage of apoptotic neurons and relieved neuronal morphological damage. Noggin-transfected NSC transplantation markedly decreased the MDA levels and increased the SOD activity, and simultaneously downregulated the BMP4 (bone morphogenesis protein), VEGF, and bFGF proteins. The present study demonstrates that grafting NSCs modified by noggin gene provides better neuroprotection for cerebrovascular disease.