Beta-nerve growth factor promotes neurogenesis and angiogenesis during the repair of bone defects

We previously showed that the repair of bone defects is regulated by neural and vascular signals. In the present study, we examined the effect of topically applied β-nerve growth factor（β-NGF） on neurogenesis and angiogenesis in critical-sized bone defects filled with collagen bone substitute. We created two symmetrical defects, 2.5 mm in diameter, on either side of the parietal bone of the skull, and filled them with bone substitute. Subcutaneously implanted osmotic pumps were used to infuse 10 μgβ-NGF in PBS（β-NGF ＋ PBS） into the right-hand side defect, and PBS into the left（control） defect, over the 7 days following surgery. Immunohistochemical staining and hematoxylin-eosin staining were carried out at 3, 7, 14, 21 and 28 days postoperatively. On day 7, expression of β III-tubulin was lower on the β-NGF ＋ PBS side than on the control side, and that of neurofilament 160 was greater. On day 14, β III-tubulin and protein gene product 9.5 were greater on the β-NGF ＋ PBS side than on the control side. Vascular endothelial growth factor expression was greater on the experimental side than the control side at 7 days, and vascular endothelial growth factor receptor 2 expression was elevated on days 14 and 21, but lower than control levels on day 28. However, no difference in the number of blood vessels was observed between sides. Our results indicate that topical application of β-NGF promoted neurogenesis, and may modulate angiogenesis by promoting nerve regeneration in collagen bone substitute-filled defects.

CO-TRANSFECTION OF RAT BONE MARROW MESENCHYMAL STEM CELLS WITH HUMAN BMP2 AND VEGF165 GENES

Objective To explore the feasibility and efficacy of lentivirus-mediated co-transfection of rat bone marrow mesenchymal stem cells （MSCs） with human vascular endothelial growth factor 165 （hVEGFI65） gene and human bone morphogenetic protein 2 （hBMP2） gene. Methods The hVEGF165 and hBMP2 cDNAs were obtained from human osteosarcoma cell line MG63 and cloned into lentiviral expression vectors designed to co-express the copepod green fluorescent protein （copGFP）. The expression lentivector and packaging Plasmid Mix were co-transferred to 293TN cells, which produced the lentivirus carrying hVEGF165 （Lv-VEGF） or hBMP2 （ Lv-BMP） , respectively. MSCs of Wistar rats were co-transfected with Lv-BMP and Lv-VEGF （BMP ＋ VEGF group）, or each alone （BMP group and VEGF group）, or with no virus （ Control group）. The mRNA and protein expressions of hVEGF165 and hBMP2 genes in each group were detected by real-time PCR and enzyme linked immunosorbent assay （ELISA）. Results Lentiviral expression vectors carrying hVEGF165 or hBMP2 were correctly constructed and confirmed by restriction endonucleses analysis and DNA sequencing analysis. A transfer efficiency up to 90% was archieved in all the transfected groups detected by the fraction of fluorescent cells using fluorescent microscopy. From the results generated by real-time PCR and ELISA, VEGF165 and BMP2 genes were co-expressed in BMP ＋ VEGF group. No significant difference of BMP2 expression was detected between BMP ＋ VEGF and BMP groups （ P 〉 0. 05）. Similarly, there was no significant difference of VEGF165 expression between BMP ＋ VEGF and VEGF groups （ P 〉 0. 05）. Conclusion VEGF165 and BMP2 genes were successfully co-expressed in MSCs by lentivirus-mediated co-transfection, which provided a further foundation for the combined gene therapy of bone regeneration.

Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells to Myogenic Cells and Expression of VEGF After Gene Transfection

Objectives To induce the differentiation of rabbit bone marrow mesenchymal stem cells （MSCs） to myogenic cells in vitro, and to investigate the expression of vascular endothelial growth factor （VEGF） gene in MSCs transfected by AdTrackCMV-VEGF165. Methods MSCs were isolated and purified from rabbit bone marrow by percoll （11）73 g/ml） and then cultured in low glucose DMEM with 10% FBS. AdTrackCMV-VEGF165 eukaryotic expression vector was constructed and transfected into the MSCs. After being incubated with 5-azacytidine （5- Aza）, the expression of troponin I in MSCs was assayed by immunohistochemistry and the expression of VEGF gene was identified by northern blot and western blot. The concentration of VEGF in the supernatant was measured by ELASA. Results MSCs were isolated and cultured successfully from rabbit bone marrow. The positive cTnI stain of some MSCs after the induction of 5-aza indicated that the cells were differentiated to myogenic cells. Northern blot and western blot showed that the expression of VEGF 165 mRNA was much higher in the hVEGF165 gene transfected cells than that of the control. The concentration of VEGF in the supernatant got to the peak 3-5 days after hVEGF165 gene transfection （1011- 1027 pg/ml） and decreased gradually thereafter, but still higher than that of control group or pAdTrackCMV group （349 pg/mLvs 116 pg/mL or 125pg/ml respectively, MSCs could be induced P〈 0.01）. Conclusions to differentiate to myogenic cells by 5-aza in vitro and could express VEGF by VEGF gene transfection.

ADENOVIRUS MEDIATED GENE TRANSFER OF VASCULAR SMOOTH MUSCLE CELLS AND ENDOTHELIAL CELLS IN VITRO

Introducing foreign gene(s) into vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) is the pre-require-ment of gene therapy for cardiovascular diseases. We have explored the use of adenoviral vectors (Adv-CMV / LacZ) to transfer LacZ gene into cultured VSMCs and ECs. Our results demonstrated that adenoviral vectors transfered foreign gene into VSMCs and ECs high-efficiently with dose-dependent response pattern. The frequencies of transfection reached 100% at the viral titer of 109 pfu / ml. Comparing the sensitivities of VSMCs and ECs to adenoviral vectors, we found that ECs were more sensitive than VSMCs, of which the frequencies of transfection in ECs reached 80% while in VSMCs only 40% for 8 hrs after transfection. In addition, the transfection of ECs and VSMCs with adenoviral vectors was partly blocked by monoclonal antibodies to Fiber and Core protein of the adenoviral capsid, but not by monoclonal antibody to Hcxon protein. It is suggested transfection of ECs and VSMCs with adenovirus vectors is mediated by Fiber or Core protein of adenoviral capsid proteins.

Identification of a Novel VEGFR-3 Missense Mutation in a Chinese Family with Hereditary Lymphedema Type I

A novel mutation of vascular endothelial growth factor receptor gene （VEGFR-3）, was identified in a four-generation Chinese family with hereditary lymphedema type I （HL-I）. Genetic linkage analysis was performed on the known genetic locus for HL-I with a panel of polymorphic markers, and then mutations were screened out by direct sequencing. By genotyping, the family showed the linkage to HL-I locus on 5q35.3. Mutation screening analysis of the exons encoding the intracellular kinase domains of VEGFR-3, revealed a novel missense mutation D1055V. This mutation cosegregated with the disease phenotype in the family and was not found in 100 normal controls. This finding has expanded the spectrum of the VEGFR-3 gene mutations causing HL-I, and will be useful for further genetic consultation and genetic diagnosis.