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Rabbits with naturally high levels of cholesterol ester transfer protein (CETP), unlike rodents, have become an interesting animal model for the study of lipid metabolism and atherosclerosis, as they have similarities to humans in lipid metabolism, cardiovascular physiology and susceptibility to develop atherosclerosis. Rodents, such as mice, are not prone to atherosclerosis as they lack the mass and activity of CETP, as a key player in lipoprotein metabolism. Recently, APOE-knockout in rabbits has been shown to promote atherosclerosis and associated premature IVD degeneration that mimic the symptoms of atherosclerosis and structural changes of IVDs in humans. Here we examined whether APOE-knockout promoted IVD degeneration in rabbits is associated with imbalanced inflammatory catabolic activities, as the underlying problem of biological deterioration that mimic the symptoms of advanced IVD degeneration in humans. We analysed in lumbar nucleus pulposus (NP) of APOE-knockout rabbits the cell viabilities and the intracellular levels of inflammatory, catabolic, anti-catabolic and anabolic proteins derogating IVD matrix. Grades of IVD degeneration were evaluated by magnetic resonance imaging. NP cells were isolated from homozygous APOE-knockout and wild-type New Zealand White rabbits of similar age. Three-dimensional cell culture with low-glucose was completed in alginate hydrogel. Cell proliferation and intracellular levels of target proteins were examined by MTT and ELISA assays. Alike human NP cells of different disc degeneration grades, NP cells of APOE-knockout and wild-type rabbits showed significantly different in vivo cell population densities (p<0.0001) and similar in vitro proliferation rates. Furthermore, they showed differences in overexpression of selective inflammatory and catabolic proteins (p<0.0001) similar to those found in human NP cells of different disc degeneration grades, such as IL-1β, TNF-α, ADAMTS-4, ADAMTS-5 and MMP-3. This study showed that premature IVD degeneration in APOE-knockout rabbits was promoted by the accumulation of selective inflammatory catabolic factors that enhanced imbalances between catabolic and anabolic factors mimicking the symptoms of advanced IVD degeneration in humans. Thus, APOE-knockout rabbits could be used as a promising model for therapeutic approaches of degenerative disc disorders. [ABSTRACT FROM AUTHOR]

Intervertebral disc (IVD) degeneration that accelerates the loss of disc structural and functional integrities is recognized as one of the major factors of chronic back pain. Cardiovascular risk factors, such as deficits of apolipoproteins that elevate the levels of cholesterol and triglycerides, are considered critical for the progress of atherosclerosis; notably in the abdominal aorta and its lumbar branching arteries that supply lumbar vertebrae and IVDs. Obstruction of the lumbar arteries by atherosclerosis is presumed to promote lumbar disc degeneration and low back pain. APOE-knockout rabbits have recently been shown to generate hyperlipidemia with increased levels of cholesterol and triglycerides that mimic the symptoms of atherosclerosis in humans. Here, we analysed IVD degeneration in the lumbar spines of ten homozygous APOE-knockout and four wild-type New Zealand White rabbits of matching age to prove accelerated IVD degeneration in APOE-knockout rabbits, since APOE-knockout rabbits could be a beneficial model for therapeutic approaches of degenerative IVD disorders. Experiments were performed using T1/T2-weighted magnetic resonance imaging, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, glucose-oxidase assay, enzyme-linked immunosorbent assay, quantitative reverse transcription PCR and western blot. APOE-knockout lumbar spines showed more advanced IVD degeneration, obstructed lumbar arteries and lower enhancement of contrast agent in IVDs. Moreover, lower concentration of glucose, lower number of viable cells and lower concentrations of aggrecan, collagen II and higher concentration of collagen I were detected in APOE-knockout IVDs (p < 0.0001). APOE-knockout in rabbits could induce structurally deteriorating premature IVD degeneration that mimics the symptoms of accelerated IVD degeneration in humans. APOE-knockout rabbits could be used as beneficial model, as they can bypass the standard surgical interventions that are commonly applied in research animals for the induction of enhanced IVD degeneration. Their parallel use in therapeutic approaches of IVD disorders and atherosclerosis could reduce the number of research animals to be used and contribute to the principles of 3Rs (Replacement, Reduction and Refinement). [ABSTRACT FROM AUTHOR]

Inhibition of intervertebral disc (IVD) degeneration, which is often accompanied by painful inflammatory and immunopathological processes, is challenging. Current IVD gene therapeutic approaches are based on adenoviral gene delivery systems, which are limited by immune reactions to their viral proteins. Their applications in IVDs near to sensitive neural structure could provoke toxicity and immunological side-effects with neurological deficits. Self-complementary adeno-associated virus (scAAV) vectors, which do not express any viral gene and are not linked with any known disease in humans, are attractive therapeutic gene delivery vectors in degenerative IVDs. However, scAAV-based silencing of catabolic or inflammatory factor has not yet been investigated in human IVD cells. Therefore, we used scAAV6, the most suitable serotype for transduction of human nucleus pulposus (NP) cells, to knockdown the major catabolic gene (ADAMTS4) of IVD degeneration. IVD degeneration grades were determined by preoperative magnetic resonance imaging. Lumbar NP tissues of degeneration grade III were removed from 12 patients by nucleotomy. NP cells were isolated and cultured with low-glucose. Titre of recombinant scAAV6 vectors targeting ADAMTS4, transduction efficiencies, transduction units, cell viabilities and expression levels of target genes were analysed using quantitative PCR, fluorescence microscopy, fluorescence-activated cell sorting, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assays, quantitative reverse transcription PCR, western blot and enzyme-linked immunosorbent assays during 48 days of post-transduction. Transduction efficiencies between 98.2% and 37.4% and transduction units between 611 and 245 TU/cell were verified during 48 days of post-transduction (p<0.001). scAAV6-mediated knockdown of ADAMTS4 with maximum 87.7% and minimum 40.1% was confirmed on day 8 and 48 with enhanced the level of aggrecan 48.5% and 30.2% respectively (p<0.001). scAAV6-mediated knockdown of ADAMTS4 showed no impact on cell viability and expression levels of other inflammatory catabolic proteins. Thus, our results are promising and may help to design long-term and less immunogenic gene therapeutic approaches in IVD disorders, which usually need prolonged therapeutic period between weeks and months.