4:21 Gene delivery of the catabolic inhibitor tissue inhibitor of metalloproteinase–1 increases proteoglycan synthesis in human intervertebral disc cells

Abstract

Purpose of study: Intervertebral disc degeneration is characterized by a progressive loss of proteoglycan content and subsequent disc dehydration. Previous studies demonstrate increased matrix metalloproteinases in degenerated discs compared with controls and propose an intimate role of these catabolic proteins in the degenerative process. Gene therapy has been investigated as a method to induce endogenous synthesis of therapeutic growth factors to stimulate proteoglycan synthesis. Although many anabolic growth factors increase matrix synthesis, the use of catabolic inhibitors has yet to be explored. The objective of this study was to evaluate the effect of adenoviral-mediated delivery of the catabolic inhibitor tissue inhibitor of metalloproteinase–1 (TIMP–1), as well as the anabolic potential of bone morphogenic protein–2 (BMP–2), on proteoglycan synthesis.Methods used: Human nucleus pulposus cells were isolated by means of enzymatic digestion and cultured in monolayer. Patient samples were pooled and experimental groups normalized for cell number before transduction with either Ad-TIMP-1 or Ad-BMP-2 at 50, 75, 100 or 150 multiplicity of infection (MOI). After 48 hours of incubation, cells were incorporated into a three-dimensional “pellet” culture system. After an additional 48 hours of incubation, active proteoglycan synthesis was assessed with 35S radioactive sulfate incorporation using chromatography and scintillation count.of findings: Both Ad-TIMP-1 and Ad-BMP-2 increase proteoglycan synthesis compared with controls (p<.05). Cultures treated with Ad-BMP-2 demonstrated a progressive increase in synthesis with increasing viral MOI (190%, 212%, 310% and 466%). Cells treated with Ad-TIMP-1 exhibited a maximum response at a MOI of 100 (65%, 289%, 378% and 249%).Relationship between findings and existing knowledge: Although anabolic factors have previously been shown to increase proteoglycan synthesis in cultured disc cells, this is the first report demonstrating a similar upregulating capacity with the use of catabolic inhibitors.Overall significance of findings: This study demonstrates that both catabolic inhibitors (TIMP–1) and anabolic growth factors (BMP-2) are successful at increasing measured proteoglycan synthesis in cultured human intervertebral disc cells. The optimal regulation of matrix synthesis may be achievable with gene therapy using a combination of both anabolic factors and catabolic inhibitors. Further studies are warranted to assess the potential of combination therapy with these growth factors to upregulate proteoglycan synthesis, as well as to determine if such combinations can minimize the total vector load required to elicit a beneficial, and potentially therapeutic, response (Table 1)Table 1Stimulatory effect on proteoglycan synthesis50 MOI75 MOI100 MOI150 MOITIMP–165%289%378%249%BMP–2190%212%310%466%.Disclosures: No disclosures.Conflict of interest: No conflict.