Abstract
IntroductionThe etiology of degenerative disc disease is unknown. Several investigators have reported the presence of proteolytic enzymes, such as the matrix metalloproteinase (MMP) and ADAMTS (a disintegrin and metalloprotease with thrombospondin-like repeats) families, in degenerated human discs. Glasson and colleagues recently reported that a significant reduction occurs in the severity of cartilage destruction in ADAMTS5 knockout mice compared with wild-type mice. The purpose of this study was to evaluate the suppressive effects of injections of ADAMTS5 small interference RNA (siRNA) oligonucleotide on intervertebral disc degeneration in the rabbit anular needle-puncture model.MethodsRabbit nucleus pulposus (NP) cells were transfected with siRNA oligonucleotides specific for ADAMTS5 or the control. The suppression of the ADAMTS5 gene by siRNA transfection was assessed by using real-time polymerase chain reaction (PCR), both in monolayer and alginate bead cultures with or without interleukin-1β (IL-1β) stimulation. The effect of siRNA was determined in vivo by using the rabbit anular needle-puncture model (control group: n = 8; ADAMTS5 group: n = 8). One week after the initial anular puncture, the animals received an injection of the control or anti-ADAMTS5 oligonucleotide (100 μg each at the L2/3 and L4/5 level; 16 discs/group). Disc height, magnetic resonance imaging (MRI) (Thompson classification and signal intensity), and safranin-O staining (histologic grade) were assessed.ResultsIL-1β treatment significantly increased the ADAMTS5 mRNA level in NP cells (P < 0.01). ADAMTS5 gene suppression was 70% compared with the control oligonucleotide in both monolayer and alginate bead culture with or without stimulation with IL-1β. The injection of anti-ADAMTS5 oligonucleotide in vivo resulted in improved MRI scores with increased signal intensity and improved histologic grade scores with statistical significance (P < 0.05). No significant change in disc height was observed.ConclusionsA single injection of ADAMTS5 siRNA induced the suppression of degradation in NP tissues, as shown by significantly improved MRI and histologic grades. The mechanism of response to siRNA may be worthy of exploration for possible therapeutic purposes.
Highlights
The etiology of degenerative disc disease is unknown
A single injection of ADAMTS5 small interfering RNA (siRNA) induced the suppression of degradation in nucleus pulposus (NP) tissues, as shown by significantly improved magnetic resonance imaging (MRI) and histologic grades
In vitro study Rabbit NP cells were chosen because their response to IL-1β was more consistent than that of anulus fibrosus (AF) cells and because the injection site in the NP has a higher concentration of proteoglycans
Summary
Several investigators have reported the presence of proteolytic enzymes, such as the matrix metalloproteinase (MMP) and ADAMTS (a disintegrin and metalloprotease with thrombospondin-like repeats) families, in degenerated human discs. Glasson and colleagues recently reported that a significant reduction occurs in the severity of cartilage destruction in ADAMTS5 knockout mice compared with wildtype mice. Aggrecan is cleaved at a specific "aggrecanase" site [1,2]; this cleavage results from the action of several members of the ADAMTS (adisintegrin and metalloprotease with thrombospondin-like repeats) family [3,4,5,6]. In a murine model of osteoarthritis, ADAMTS5-knockout mice have a significantly reduced level of cartilage destruction, compared with wildtype mice [7]. Glasson and colleagues [8] reported that no effect in ADAMTS4 knockout mice was noted on the progression or severity of osteoarthritis after surgical induction of joint instability. The dual deletion of ADAMTS4 and ADAMTS5 provided significant protection against proteoglycan degradation ex vivo and decreased the severity of murine osteoarthritis in vivo [9]
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