Abstract
Introduction
Proteoglycans, described as ground substance of the intervertebral disk tissue, are proteins rich in glycosaminoglycans (GAGs) providing stiffness and elasticity to the IVD. During IVD degeneration, a decrease of GAG is observed. This decrease results in loss of water content which alters its mechanical properties1. Glucuronyltransferase I (GT-I) and Xylosyltransferase I (XT-I) are two key enzymes involved in the synthesis of GAGs. Both enzymes have been shown to be downregulated in degenerated cartilage2,3. While GT-I has been reported to have a crucial role in GAG synthesis in the IVD, the implication of XT-I has not yet been demonstrated. Nonviral-based gene therapy is an attractive therapeutic approach because of its relative safety compared to viral therapies. The hypothesis of this study is that nonviral mediated delivery of GT-I and XT-I plasmids will increase the GAG production in IVD cells. The specific objectives of this study were1 to investigate the expression of GT-I and XT-I in relation to aging within the IVD tissue and2 to upregulate GT-I and XT-I expression using a polymeric transfecting agent for gene delivery of p GT-I and p XT-I.
Materials and Methods
(1)Expression of GAGs, GT-I and XT-I in relation to aging IVD tissues were harvested from 6-month-old, 1-, 2-, and 8-year-old bovine tails. One half of the IVD tissue was fixed by 4% paraformaldehyde. After sucrose treatment and sectioning, safranin O/Fast green staining and immunostaining of XT-I and GT-I were carried out. The other half was digested by 0.5 mg/mL of proteinase K for biochemical (DMMB assay) and HPLC analyses.
(2)Polymeric transfection-mediated gene delivery of pGT-I and pXT-I Nucleus pulposus and annulus fibrosus cells (passage 1) and adipose derived stem cells (ADSCs) (passage 3), extracted from 6-month-old bovine caudal disks and rabbit adipose tissue, respectively, were seeded at a density of 50,000 cells/cm2. After 24 hours, cells were treated with XT-I and/or GT-I TOPO TA plasmids DNA complexed with commercial partial degraded polyamidoamine dendrimer (SuperFect, Qiagen) for 30 minutes before addition to cells (1 µg of plasmid DNA final concentration). p Luc was used as the nontargeting plasmid and control for transfection (luciferase activity) (empty vector (EV)). Medium was then changed daily until the completion of the study. Cellular metabolic activity (AlamarBlue) and proliferation (Picogreen), GAG quantification (DMMB), gene expression (XT-I, GT-I and aggrecan), and enzyme activity analyses were conducted on day 2 and 4 posttransfection.
Results
(1)Expression of GAGs, XT-I, and GT-I with aging A decrease of the intensity of GAG staining associated with a disorganization of the tissue was observed with aging. GAG downregulation was confirmed by a significant decrease of sulfated GAGs (6092 ± 393 µg GAG/µg DNA for 6-month-old IVD to 1033 ± 530 µg GAG/µg DNA for 8-year-old IVD) within the tissue (Fig. 1). Associated with this phenomenon, a decrease of XT-I and GT-I expression was noted by immunohistochemistry. Indeed, the high expression of XT-I and GT-I observed within 6-month-old bovine IVD decreased with aging to become nondetectable within 8-year-old bovine IVD.
(2)Nonviral mediated pXT-I and pGT-I delivery After 2 and 4 days posttreatment, using polymer and pDNA complexes (polyplexes), over 80% cell viability was seen regardless of the plasmid and cell types. ADSCs, NP, and AF cells showed luciferase activity 2 and 4 days posttransfection. The expression of both plasmids GT-I and XT-I was observed in the three cell types.
I-sGAG content within NP and AF tissues with aging. p < 0.05, Anova one-way test. II-GAG distribution in 6-month-old, 1-, 2-, and 8-year-old bovine IVD tissues (Safranin O (GAG, orange), Fast Green (collagen fibers, green) staining) (A-D) NP tissue (E-H) AF tissue.
Conclusion
A downregulation of GAGs was observed within bovine IVDs with aging. This decrease is associated with a downregulation of XT-I and GT-I expression known to be implicated in the GAG production. The use of a nonviral system shows promising results for gene transfer by their ability to transfect IVD cells.
No
None declared
Halloran D, et al. Tissue Engineering 2007;13:1927–1954
Venkatesan N, et al. Proc Natl Acad Sci USA 2004;101(52):18087–18092
Venkatesan N, et al. FASEB J 2009;23(3):813–822