Basic ScienceMitochondrial gene expression in the human annulus: in vivo data from annulus cells and selectively harvested senescent annulus cells
Introduction
Although it is well recognized that apoptosis, senescence, and increased production of inflammatory cytokines and catabolic products are important factors in degeneration of the human intervertebral disc, there is poor understanding of the underlying cause at the cellular level. Mitochondria are essential for normal energy metabolism because they contain the oxidative phosphorylation system, which is the major source of adenosine triphosphate (ATP) for the cell. The low bioenergetic level of human disc cells recognized in vivo is reflected in the small number of mitochondria within disc cells [1], [2], [3]; this characteristic is even present in vitro, where it can take up to a month to expand disc cell numbers for experimental use.
Negative mitochondrial contributions to cell pathophysiology occur when mitochondria are a source of reactive oxygen species that damage critical molecules, promote oxidative stress, and lead to pathology associated with mitochondrial dysfunction. Well recognized is the critically important role of apoptosis (programmed cell death) as a mechanism of cell loss in the aging/degenerating disc; apoptosis in this disc has a known mitochondrial involvement [4], [5], [6], [7], [8], [9].
Cell senescence (also termed replicative senescence) is another important event that occurs during disc aging and degeneration. A number of studies have now shown the presence of cell senescence in the human disc [10], [11], [12], [13], [14], including studies of the nucleus pulposus [15]. Recent data have shown that the proportion of senescent cells rises significantly with increasing stages of disc degeneration (p<.0001) [13]. Senescent cells are viable but cannot divide and exhibit alterations in phenotype and altered gene expression patterns [16], [17], [18], [19]. Senescent cells may have altered responsiveness to external stimuli, and they may secrete factors that can influence neighboring cells or their nearby extracellular matrix. There is currently a great deal of interest in the manner in which cell senescence may contribute to age-associated loss of function or age-related pathology in vivo, and molecular studies are directed toward elucidating mechanisms and pathways that activate the senescence program in cells [20]. Mitochondrial dysfunction and oxidative stress are now known to be key underlying problems in cell senescence and aging [21], [22], [23], [24], [25], [26], [27].
Several recent studies have provided evidence for the presence of mitochondrial dysfunction in human osteoarthritis, and an overview has been presented by Blanco et al. [28]. Terkeltaub et al. [29] have suggested that mitochondrial dysfunction is involved in a number of pathogenic features of osteoarthritis, including oxidative stress, increased proinflammatory cytokines and matrix degradation, increased apoptosis, pathologic matrix calcifications, and impaired chondrocytes mitochondrial ATP synthesis. Strong parallels exist in disc degeneration for these osteoarthritic features. Mitochondrial analysis that pointed to dysregulation in human osteoarthritic chondrocytes included decreased mitochondrial superoxide dismutase (reflecting a redox imbalance) in the work of Ruiz-Romero et al. [30]. Cillero-Pastor et al. [31] have shown involvement of nuclear factor κB acting as an intermediary between reactive oxygen species generated by mitochondria and the transactivation of cyclooxygenase 2. However, as in the human disc, the role of mitochondrial dysfunction in osteoarthritis pathology is a relatively new research area, which is just now beginning to be revealed [32].
In the present study, our objective was to use two approaches to analyze gene expression patterns related to mitochondria in the human annulus. First, we assessed human annulus cells in a genome-wide microarray analysis approach to evaluate mitochondrial gene expression in annulus tissue from degenerated compared with healthier discs. Second, we used laser capture microdissection (LCM) to selectively isolate senescent versus nonsenescent annulus cells and then evaluate their mitochondrial gene expression patterns.
Section snippets
Clinical study population
Experimental study of human disc specimens was approved prospectively by the authors’ Human Subjects Institutional Review Board at Carolinas Medical Center. The need for informed consent was waived because disc tissue was removed as part of routine surgical practice. Scoring of disc degeneration used a modification of the Thompson scoring system [33] incorporating author’s (ENH) radiologic, magnetic resonance imaging, and surgical findings. The Thompson system scores disc degeneration over the
Findings in mitochondrial genes in annulus cells from degenerated discs versus healthier discs
Demographic data on the 20 lumbar annulus specimens used in this portion of our study are presented in Table 1. In this set of subjects, a significant positive correlation was present between subject age and Thompson grade (p=.006, r2=0.34).
The study design in our gene expression analyses compared gene expression patterns in more degenerated annulus cells (from Thompson Grades IV and V discs) with healthier annulus cells (derived from Grade II–III discs).
Analyses of gene expression patterns
Discussion
The aging and degenerating intervertebral disc is avascular, with a markedly reduced cell population, which experiences apoptosis and cell senescence. This unique physiological setting challenges disc cell function with respect to matrix production and remodeling and makes cellular energy production by mitochondria even more crucial than in normal disc tissue.
In the present analysis, we chose to take advantage of the strengths of genome-wide microarray analysis to assess changes in genes
Acknowledgments
The authors wish to thank the Brooks-Carolina Back Pain Research Endowment for general laboratory support. We also wish to thank Nury Steuerwald, PhD, and Judy Vachris for expert assistance with our molecular studies.
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FDA device/drug status: Not applicable.
Author disclosures: HEG: Nothing to disclose. JAW: Nothing to disclose. GLH: Nothing to disclose. SFB: Nothing to disclose. JAI: Nothing to disclose. NSZ: Nothing to disclose. ENH: Stock Ownership: Medtronic (800 shares, 1%); Board of Directors: Journal of Bone & Joint Surgery (C); Other Office: American Orthopaedic Association (Nonfinancial); Research Support (Staff/Materials): AO Foundation (D, Paid directly to institution/employer); Fellowship Support: Synthes (E).
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