The expression of NG2 proteoglycan in the human intervertebral disc
ABSTRACT Immunohistochemical and biochemical analyses of NG2 proteoglycan in the human intervertebral disc.
To determine if the human intervertebral disc expresses NG2 proteoglycan.
In the nervous system, NG2 has been reported to play an important role as an interactive extracellular matrix component and membrane receptor for growth factors. NG2 is also found in non-neuronal tissues, such as cartilage and bone; however, the expression of NG2 within the human intervertebral disc is unknown.
NG2 expression in the intervertebral disc was examined through Western blotting, reverse transcriptase polymerase chain reaction, and immunohistochemistry. Confocal microscopy was used to assess the spatial association of NG2 with type VI collagen. To reveal changes in the content of NG2 with disc degeneration, Western blot analysis was used to assess the relative content of NG2 in human intervertebral disc tissues with varying degrees of degeneration.
NG2 was clearly identified in cells from both the anulus fibrosus and nucleus pulposus, and colocalized with both type VI collagen and beta-integrin, located in the inner area of the cell-associated matrix. Throughout the anterior and posterior regions of the disc tissues, most cells were confirmed to be NG2 positive. Cells expressed NG2 messenger ribonucleic acid, and Western blot confirmed the presence of the core protein of the NG2 protein, 250 kDa. A study comparing the different grades of disc degeneration showed that the content of NG2 was elevated in disc tissues in an advanced stage of degeneration compared to tissues in an early stage of degeneration.
Although the biologic role of NG2 remains to be elucidated, the colocalization of NG2 with type VI collagen in the pericellular area suggests that NG2 may play an important role in cell-matrix interactions. The high level of NG2 expression in advanced degeneration also suggests an important role of NG2 in the loss of disc integrity.
SourceAvailable from: Anshu Rastogi[Show abstract] [Hide abstract]
ABSTRACT: INTRODUCTION: Degeneration of the intervertebral disc (IVD) is characterized by marked degradation and re-structuring of the annulus fibrosus (AF). While several matrix metalloproteinases (MMPs) have been found to be more prevalent in degenerate discs, their coordination and function within the context of the disease process are still not well understood. In this study, we sought to determine whether MMP-2 is associated with degenerative changes in the AF, and to identify the manner by which AF cells utilize MMP-2. METHODS: Two established animal models of disc degeneration, static compression and trans-annular needle puncture of rodent caudal discs, were examined for MMP-2 immunopositivity. Using lentiviral transduction of an shRNA expression cassette we screened and identified an effective shRNA sequence for generating stable RNA interference to silence MMP-2 expression in primary rat AF cells. Gelatin films were used to compare gelatinase activity and spatial patterns of degradation between transduced cells, and both non-infected and nonsense shRNA controls. The functional significance of MMP-2 was determined by assessing the ability for cells to remodel collagen gels. RESULTS: Both static compression and 18g annular puncture of rodent caudal discs stimulated an increase in MMP-2 activity with concurrent lamellar disorganization in the AF, while 22g and 26g needle injuries did not. To investigate the functional role of MMP-2, we established lentivirus-mediated RNAi to induce stable knock down of transcript levels by as much as 88%, and protein levels by as much as 95% over 10 days. Culturing transduced cells on gelatin films confirmed that MMP-2 is the primary functional gelatinase in AF cells, and that MMP-2 is utilized locally in regions immediately around AF cells. In collagen gels, transduced cells demonstrated an inability to remodel collagen matrices. CONCLUSIONS: Our study indicates that increases in MMP-2 observed in human degenerate discs are mirrored in experimentally-induced degenerative changes in rodent animal models. AF cells appear to utilize MMP-2 in a very directed fashion for local matrix degradation and collagen remodeling. This suggests that MMP-2 may have a functionally significant role in the etiology of degenerative disc disease, and could be a potential therapeutic target.Arthritis research & therapy 04/2013; 15(2):R57. DOI:10.1186/ar4224 · 4.12 Impact Factor
Manuelle Medizin 04/2008; 46(2):77-81.
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ABSTRACT: Die Bandscheiben altern schneller als fast jedes andere Gewebe, da ihre Ernährung in einem avaskulären Gewebe behindert ist [25, 96, 83]. Die maßgeblichste strukturelle Veränderung, die im Degenerationsprozess abläuft, ist die Abnahme von Wassergehalt und osmotischem Druck, vor allem im Nucleus pulposus und im inneren Teil des Anulus fibrosus [95, 25, 57], sowie die Abnutzung der Matrix . Der abnehmende osmotische Druck in alternden Bandscheiben verstärkt die Öffnung existierender Risse trotz der Abnahme der Scherkräfte im Anulus . Fibröse Veränderungen des Nukleus, Desorganisation des Anulus sowie Veränderungen des Wirbelkörpers und der Endplatten geschehen vor allem im 5.–7. Lebensjahrzehnt. Diese Veränderungen sind in den unteren Bandscheiben stärker ausgeprägt als in den oberen und gehen der Formierung von Rissen und Spalten voraus. Der zeitliche Ablauf weist auf eine strenge Korrelation von Spalt- und Rissbildungen hin, die in der ersten Dekade im Nukleus beginnen, während Randläsionen unabhängig davon entstehen und deutlich später eintreten . Intervertebral discs age more rapidly than most other tissues because the nutritional supply is hindered by avascular tissue [25, 96, 83]. The most decisive structural alterations in the degenerative process are a decrease in water content and osmotic pressure, especially in the nucleus polposus and in the inner part of the annulus fibrosus [95, 25, 57], as well as wear of the matrix . The decrease in osmotic pressure in aging intervertebral discs strengthens the opening of existing tears despite a decrease in shearing forces in the annulus . Fibrous changes of the nucleus, disorganization of the annulus and changes to the vertebra and endplates occur in particular in the 5th–7th decades of life. These alterations are more intensive in the lower regions than in the upper and precede the formation of tears and clefts. The time scale indicates a strong correlation to tear and cleft formation, which begin in the nucleus in the first decade, whereas marginal lesions are formed independently and occur much later .Manuelle Medizin 01/2008; 46(2):77-81. DOI:10.1007/s00337-007-0569-y