| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 8, 2003 as doi:10.1096/fj.02-0514fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,
* A. I. Virtanen Institute,
Department of Surgery,
Department of Pathology and Forensic Medicine,
|| Department of Biochemistry and
¶ Gene Therapy Unit, University of Kuopio, Kuopio, and
Department of Pathology, and Tampere University Hospital, Tampere, Finland
2Correspondence: Department of Molecular Medicine, A. I. Virtanen Institute, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio Finland E-mail: Seppo.Ylaherttuala{at}uku.fi
SPECIFIC AIMS
Local gene transfer offers a promising alternative for treatment of various acquired diseases. In orthopedics, potential targets for gene therapy are osteoporosis, arthritis, tissue repair, and tumors. In this paper we focus on osteoblast recruitment and activation by vascular endothelial growth factor (VEGF-A) gene transfer to bone marrow cells in order to enhance bone repair and regeneration.
PRINCIPAL FINDINGS
1. Gene transfer efficiency in vitro
Viral and nonviral vectors were compared for gene transfer efficiency on osteoblast and osteosarcoma cell lines. Both vectors carried lacZ as a marker gene and detection was made using X-Gal staining. It was found that adenovirus was able to transfect 92.7% ± 1.0 of osteoblasts (HNO cell line) with MOI 500 and 97.7 ± 1.1 with MOI 1000. In osteosarcoma cell line (MG-63), the corresponding percentages were 46.7% ± 2.0 and 77.1% ± 1.7. Using plasmid (5 µg/50,000 cells)/Fugene complexes, the transfection efficiency in osteoblast cell line (HNO) was 5.7% ± 1.0 and in osteosarcoma cell line (MG-63) 6.2% ± 0.3.
2. Transfection efficiency in the bone marrow
For the gene transfer technique, see Fig. 1
. Twenty New Zealand White rabbits were used. Gene transfer was performed and the animals were killed 1 or 3 wk later. Number of rabbits in both study groups (lacZ and VEGF-A) was 5 at both times. The localization of the needle in bone marrow was confirmed under fluoroscopical control. Virus titer of 1.4 x 1010 pfu was used in the final volume of 2 mL in 0.9% saline and the gene transfer was performed for 2 min (1.0 mL/min). VEGF-A (murine VEGF164) and nuclear-targeted lacZ adenoviruses were constructed as described. Replication-deficient E1/partial E3 deleted, clinical GMP-grade adenoviruses were produced in 293T cells. Adenoviruses were determined to be free from helper viruses, lipopolysaccharide, and bacteriological contaminants. One and 3 wk after the injections the femurs were collected for analysis. Transfection efficiency 1 wk after the gene transfer was 22% ± 4 according to the X-Gal staining method for ß-galactosidase activity. ß-Galactosidase activity was detected at the 3 wk point at a level of 17% ± 3. mRNA expression of the transfected genes was verified using RT-PCR.
|
3. Expression of transfected lacZ gene in ectopic organs
Liver, spleen, heart, lung, cerebra, kidney, and ovaries were analyzed for nuclear ß-galactosidase expression using X-Gal staining. High level of ectopic marker gene expression was found in liver and spleen. In the liver, positive cells were localized around the central veins. In the spleen, positive cells were seen mostly in the red pulp, but also to a lesser extent in the white pulp. No X-Gal-positive cells were seen in other tissues.
4. Effect of VEGF-A on bone histomorphometry
The distal femurs and proximal tibiae were embedded in methylmethacrylate. After polymerization, the blocks were cut longitudinally with a hard tissue microtome. Histomorphometry of trabecular bone was performed on the upper part of tibia and 3 µm thick slices were cut and stained by the Masson-Goldner technique. A semiautomatic quantitative method was used for static histomorphometrical evaluation. Trabecular bone hard tissue histomorphometry of the distal femurs was performed to study the effect of gene transfer on trabecular bone turnover. Results are shown in Fig. 2
. Compared with the contralateral saline-injected side at wk 1, VEGF-A-injected trabecular bone had 4% higher bone volume, 12% higher osteoblast number, 17% higher osteoblast surface, 26% higher osteoid volume, and 24% less resorption surface. When compared with unilateral lacZ-transfected trabecular bone, VEGF-A bone had 8% less bone volume, 90% higher osteoblast number, 100% higher osteoblast surface, 125% higher osteoid volume, and 70% less resorption surface.
|
Compared with contralateral saline-injected side at wk 3, VEGF-A-injected trabecular bone had 60% higher bone volume, 15% higher osteoblast number, 11% higher osteoblast surface, 35% higher osteoid volume, and 15% less resorption surface. When compared with unilateral lacZ-transfected trabecular bone, VEGF-A bone had 70% higher bone volume, 7% higher osteoblast number, 30% higher osteoblast surface, 22% higher osteoid volume, and 49% less resorption surface.
CONCLUSIONS AND SIGNIFICANCE
Current methods to treat osteoporosis include anti-resorptive and osteogenic drugs. Anti-resorptive agents are an elegant way to prevent further bone loss and even slightly increase bone density. Osteogenic drugs on the other hand, replenish lost bone tissue. Some problems relate, however, to the use of osteogenic drugs. Tolerance and production of bone of poor quality should be corrected before wider use of these therapies. Therefore, there is a clear need for new therapeutic strategies that replenish the lost bone to a level compatible with normal bone mass and quality without development of tolerance. We have demonstrated that local gene transfer of VEGF cDNA enhances bone formation parameters such as osteoblast number and osteoid volume and increases bone volume.
Bone formation is connected with angiogenesis, and vascular invasion is a prerequisite for endochondral bone formation and fracture healing. VEGF is known as an angiogenetic factor produced by many types of cells, including endothelial cells and osteoblasts. VEGF has been shown to stimulate primary human osteoblast chemotaxis and differentiation. It was shown that these cells express VEGFR-1 and VEGFR-2. Furthermore, osteoblast apoptosis can be inhibited by VEGF. A recent study suggests that VEGF stimulates bone repair in vivo and is an essential mediator in bone healing.
In this study, gene transfer instead of recombinant protein administration was used in order to produce a therapeutic effect for several days. For many conditions, such as fracture healing, only a temporary expression of the transgene is probably needed to obtain a therapeutic effect. Although the gene transfer efficiency in bone marrow is limited, the therapeutic effect can be achieved by using genes encoding secreted gene products such as VEGF. Increased bone formation was found after VEGF-A gene transfer. This is caused by recruitment of osteoblasts and enhancement of the osteoblast differentiation via direct activation of osteoblasts. The effect of VEGF gene transfer on angiogenesis was not investigated in our study, but it is probable that angiogenesis also contributes an important role in recruitment and survival of osteoblasts. Surprisingly, it was shown that resorption surface was significantly smaller in VEGF group. Whether VEGF has inactivated osteoclasts remains unknown. This would not be consistent with previous findings where VEGF has been shown to stimulate chemotaxis of osteoclasts.
Problems related to the use of adenoviral vectors include immunological and inflammatory reactions and spread of virus to other organs. Immunological reactions may be due to induction of NF-
B expression and activation of a CTL response. Some problems related to the use of adenoviruses may be due to impurities or replication-competent viruses in the virus lots. No major immunological reactions were seen in this study, perhaps because we used only clinical grade viruses and short exposure time in the bone marrow. However, immunostimulatory properties of adenoviruses may limit the use of very high titer viruses or repeated gene transfers.
It is concluded that adenovirus-mediated gene transfer to bone marrow is feasible and that VEGF-A gene transfer is highly osteogenic. Thus, VEGF-A is a potential candidate for gene therapy of osteoporosis and other diseases that demand efficient osteogenic therapy.
|
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0514fje; to cite this article, use FASEB J. (April 8, 2003) 10.1096/fj.02-0514fje 
This article has been cited by other articles:
|
|
 |
|
  J. Dai and A.B.M. Rabie VEGF: an Essential Mediator of Both Angiogenesis and Endochondral Ossification J. Dent. Res., October 1, 2007; 86(10): 937 - 950. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.-L. Shiau, S.-Y. Chen, M.-Y. Chang, C.-H. Su, S.-Y. Chung, Y.-T. Yo, C.-R. Wang, and C.-L. Wu Prothymosin {alpha} Lacking the Nuclear Localization Signal as an Effective Gene Therapeutic Strategy in Collagen-Induced Arthritis J. Immunol., April 1, 2007; 178(7): 4688 - 4694. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Kitagawa, J. Dai, J. Zhang, J. M. Keller, J. Nor, Z. Yao, and E. T. Keller Vascular Endothelial Growth Factor Contributes to Prostate Cancer-Mediated Osteoblastic Activity Cancer Res., December 1, 2005; 65(23): 10921 - 10929. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Yen, J. L. Su, C. L. Chien, K. W. Tseng, C. Y. Yang, W. F. Chen, C. C. Chang, and M. L. Kuo Diosgenin Induces Hypoxia-Inducible Factor-1 Activation and Angiogenesis through Estrogen Receptor-Related Phosphatidylinositol 3-kinase/Akt and p38 Mitogen-Activated Protein Kinase Pathways in Osteoblasts Mol. Pharmacol., October 1, 2005; 68(4): 1061 - 1073. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Sivakumar, L. E. Harry, and E. M. Paleolog Modulating Angiogenesis: More vs Less JAMA, August 25, 2004; 292(8): 972 - 977. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
