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. Author manuscript; available in PMC: 2013 Aug 1.
Published in final edited form as: Osteoarthritis Cartilage. 2012 Apr 21;20(8):896–905. doi: 10.1016/j.joca.2012.04.010

Spine degeneration in a murine model of chronic human tobacco smokers

Dong Wang 1,2, Luigi A Nasto 2,8, Peter Roughley 3, Adriana S Leme 4, McGarry Houghton 4, Arvydas Usas 5, Gwendolyn Sowa 2,6, Joon Lee 2, Laura Niedernhofer 7, Steven Shapiro 4, James Kang 2, Nam Vo 2
PMCID: PMC3389285  NIHMSID: NIHMS379026  PMID: 22531458

Abstract

Objective

To investigate the mechanisms by which chronic tobacco smoking promotes intervertebral disc degeneration (IDD) and vertebral degeneration in mice.

Methods

Three months old C57BL/6 mice were exposed to tobacco smoke by direct inhalation (5 cigarettes/day, 5 days/week for 6 months) to model long-term smoking in humans. Total disc proteoglycan content (DMMB assay), aggrecan proteolysis (immunobloting analysis), and cellular senescence (p16INK4a immunohistochemistry) were analyzed. Proteoglycan and collagen syntheses (35S-sulfate and 3H-proline incorporation, respectively) were measured using disc organotypic culture. Vertebral osteoporosity was measured by micro-computed tomography.

Results

Disc proteoglycan content of smoke-exposed mice was 63% of unexposed control, while new proteoglycan and collagen syntheses were 59% and 41% of those of untreated mice, respectively. Exposure to tobacco smoke dramatically increased metalloproteinase-mediated proteolysis of disc aggrecan within its interglobular domain (IGD). Cellular senescence was elevated two folds in discs of smoke-exposed mice. Smoke exposure increased vertebral endplate porosity, which closely correlates with IDD in humans.

Conclusions

These findings further support tobacco smoke as a contributor to spinal degeneration. Furthermore, the data provide a novel mechanistic insight, indicating that smoking-induced IDD is a result of both reduced PG synthesis and increased degradation of a key disc extracellular matrix protein, aggrecan. Cleavage of aggrecan IGD is extremely detrimental as this result in the loss of the entire glycosaminoglycan-attachment region of aggrecan, which is vital for attracting water necessary to counteract compressive forces. Our results suggest identification and inhibition of specific metalloproteinases responsible for smoke-induced aggrecanolysis as a potential therapeutic strategy to treat IDD.

Keywords: Tobacco smoking, intervertebral disc degeneration, matrix proteoglycans, aggrecan, matrix metalloproteinases

INTRODUCTION

Intervertebral disc degeneration (IDD) is responsible for many spine related disorders, including back pain, that cause both temporary and permanent disability in a significant fraction of workers, aged 18 to 641, 2. Back pain is the second most common reason for a doctor's visit among Americans today3. Tobacco smoking is associated with spine-related pain. A prospective study of the Icelandic population documented higher frequency of back pain and disc degeneration in smokers than nonsmokers4. Likewise a longitudinal study of 5180 Finnish forest industry workers revealed smoking as a key contributing factor to radiating neck pain and sciatic pain5. Heavy smokers who are overweight or repeatedly perform hard physical labor were also reported to have significantly higher frequency of lower back pain6. Tobacco smoking has enormous negative health and economic impact, including a strong correlation with back pain and degenerative spinal disorders. Nevertheless, over 1.1 billion people worldwide continue to smoke7.

Several epidemiological studies implicate tobacco smoking as a causal factor in intervertebral disc degeneration (IDD). An 11-year follow-up study of nearly 60,000 adolescents reported tobacco smoking as a major risk factor for lumbar discectomy8. Smoking was also a positive predictor of hospitalization for back disorders in a cohort study of 902 metal industry employees9. Other studies indicate that smoking exacerbates pre-existing IDD and significantly delays recovery from spinal surgery10. Battie and coworkers reported about 20% greater disc degeneration via MRI in smokers compared to nonsmoking identical twins in whom genetic and other environmental differences were accounted for11.

Despite this strong correlative evidence, the mechanism by which tobacco smoke promotes IDD is not known. IVDs of rats exposed to passive tobacco smoke for 8 weeks exhibited decreased collagen expression, increased interleukin-1β expression and annular disorganization12. Early work by Holm and Nachemson on porcine models suggests that tobacco smoking induces vasoconstriction and thereby reduces the nutrient uptake within the disc, leading to IDD13. This idea was supported by subsequent studies on rabbits treated with nicotine, a major tobacco smoke constituent responsible for addiction and vasoconstriction14. This study showed decreased density of vascular buds and narrowing of the vascular lumen in the vicinity of the vertebral endplate in response to smoke exposure. These observations led to the current theory which posits that tobacco smoke causes disc degeneration indirectly through nicotine-induced vasoconstriction, resulting in decreased nutrient and waste exchange between discs and the surrounding vascular system.

Herein, we investigated the mechanism of smoking-related spinal degeneration by exposing mice to tobacco smoke inhalation for six months to mimic chronic heavy smoking in humans. Exposed mice in this direct inhalation model exhibited severe loss of vertebral bone and loss of disc matrix proteoglycan, a well-established hallmark of IDD. This chronic tobacco smoking exposure induced matrix metalloproteinase (MMP)- and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)- mediated proteolysis of disc aggrecan interglobular domain, which is particularly pathological due to the resulting loss of the entire GAG attachment region from the proteoglycan aggregate, revealing a novel molecular mechanism for IDD.

METHOD

Exposure to tobacco smoke

Three month-old C57BL/6 mice (n = 25) were exposed to tobacco smoke by direct inhalation (5 unfiltered cigarettes per day × 5 days/week for 6 months) using a smoking apparatus as previously described15. University of Kentucky 3R4F research referenced cigarettes were used in this study. The animals tolerated the treatment without evidence of toxicity (carboxyhemoglobin levels <10%). Unexposed age- and sex- matched littermates (n = 20) kept in the same facility environment were used as controls.

Isolation of nucleus pulposus (NP), annulus fibrosus, and whole mouse intervertebral discs

The experiments involving mice were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Isolation of mouse spines and intervertebral disc tissue were done as described previously16.

Histological staining

Isolated spines were decalcified and embedded in paraffin (Tissue Tek processor and Leica embedder). 7 μm sections were stained with either hematoxylin and eosin (H&E) or safranin O and fast green dyes (Fisher Scientific) by standard procedures and photographed under 40–200× magnification (Nikon Eclipse Ts100).

1,9-dimethylmethylene blue (DMMB) colorometric assay for sulfated glycosaminoglycans (GAG)

NP and AF tissue separately isolated from six lumbar IVDs of each mouse was pooled together and digested using papain at 60 °C for two hours. GAG content was measured in duplicate by the DMMB procedure17 using chondroitin-6-sulfate (Sigma C-8529) as a standard. The DNA concentration of each sample was measured using the PicoGreen assay (Molecular Probes) and used to normalize the GAG values.

Quantitation of matrix synthesis

Disc organ cultures of isolated functional spine units (FSU), each consisting of vertebra, disc, vertebra, were established as previously described18. Proteoglycan synthesis was measured by 35-S-sulfate incorporation and collagen synthesis was determined from collagenase-sensitive 3-H-proline incorporation, both as described previously19. The rate of proteoglycan and collagen synthesis was calculated as the fmoles of sulfate or 3-H-proline incorporated per μg DNA.

Western Analysis

Entire lumbar discs were removed en bloc by creating an incision along the endplates/vertebral bone interface. Five to ten discs from each mouse were used to extract proteins at 4°C under continuous agitation for 48 hrs, using 30 volumes (volume to weight of disc tissue) of 4 M guanidinium chloride, 50 mmol/L sodium acetate, pH 5.8, 10 mmol/L EDTA, and COMPLEAT proteinase inhibitor cocktail (Roche). Aliquots of disc extracts (100 μL) were precipitated with 9 volumes of ethanol and recovered by centrifugation. Pellets were washed twice in 75% ethanol, lyophilized and redissolved in 100 μL, 50 mM sodium acetate (pH 6.0), then digested overnight with 1 mU keratinase II. The solution was then adjusted to 100 mM Tris, 100 mM sodium acetate (pH 7.3), and digested for 6 hrs with 10 mU chondroitinase ABC. Digested protein extract were resolved on a SDS/polyacrylamide gel electrophoresis (PAGE) (4–12% gradient gel, Invitrogen). To control for loading, the same amount of tissue wet weight (1 mg) was loaded per well, i.e., 33 μL of the 100 μL contained the protein extracted from 3 mg tissue was used for SDS/PAGE. Samples were analyzed by immunoblot as previously described20 using antibodies (1:1000 dilution 1° Ab, 1:5000 dilution 2° Ab) raised against the ADAMTS-generated neoeptitope NITEGE (Ab1320)21 and MMP-generated neoepitope VDIPEN (Ab1319)22. The anti-NITEGE neoepitope antibody cross-reacts with the NVTEGE neoepitope generated from mouse aggrecan.

Immunohistochemistry

Paraffin embedded sections were used to probe for ADAMTS-generated neoeptitope NVTEGE and MMP-generated neoepitope VDIPEN using the same antibodies as in Western analysis (1:750 dilution of Ab1319 and 1:250 dilution of Ab1320). The immunohistochemical procedure was performed as described23. Forty-two day-old rat tibial growth plate was used as a positive control for anti-VDIPEN and anti-NITEGE detection. A negative control without addition of the primary antibodies was also performed.

Measuring apoptosis and cell senescence

Frozen sections of discs were treated with the TUNEL reagents (Roche) to detect apoptotic cells as previously described by the manufacturer and counterstained with the Hoechst nuclear stain to detect total cells in the specimens. Immunohistochemistry was used to localize the senescence marker P16INK4a in disc tissue using procedure previously described24. At least three random fields in three sections from each tissue sample were imaged at 200× to quantify the percent P16INK4a- and TUNEL-positive cells. Average values from 9 fields (3 field/NP × 3 mice) were calculated with 95% confidence intervals.

MicroCT

Micro-computed tomography scans of the spines isolated from mice were acquired using a VivaCT 40 (Scanco Medical) with 15 μm isotropic voxel size resolution, 70 kVp of energy and 114 μA of current. After the acquisition of transverse two-dimensional image slices, three-dimensional reconstruction of the lumbar vertebrae was performed using a constant threshold value which was selected manually for the bone voxels by visually matching the threshold areas to the gray-scale images. Trabecular bone was evaluated in the region approximately 150 slices below the cranial and above the caudal growth plates, as described25. Briefly, the anatomic region of interest was selected by manually drawing contours on each slice and merging them together. Trabecular bone parameters were assessed using Scanco evaluation software provided by the manufacturer which calculates bone volume fraction BV/TV (where BV is bone volume and TV is total volume). Porosity was calculated using formula 1-BV/TV. The porosity of proximal and distal endplates of L3–L5 vertebrae was calculated similarly by drawing contours on axial slices.

Statistical analysis

Values represent the average of 6–9 trials from different mice with 95% confidence intervals calculated to determine statistical significance at p=0.05. The confidence intervals were calculated based on the t-distribution because of the small sample size.

RESULTS

Exposure to tobaccos smoke

The goal of this study was to investigate the effects of long-term tobacco smoking on extracellular matrix proteoglycan homeostasis in intervertebral discs. For this we exposed three month-old mice to tobacco smoke by direct inhalation (5 cigarettes/day × 5 days/week for 6 months) to model chronic first-hand smoking in humans. This regiment was chosen for several reasons. At three months of age mice begin to reach skeletal maturity, and initiation of smoke exposure at this age mimics young human adults when they most likely begin to smoke. Exposure to five unfiltered cigarettes per day in mice correspond to a smoking regimen of more than one pack per day in humans26, 27, and six months of smoke exposure induces the development of pulmonary emphysema which models chronic smoking in humans (equivalent of 15–20 years)26. Under these conditions, carbon monoxide does not reach toxic levels (peak CO-Hb ~ 10%), and the mice appear grossly normal (without weight loss). We have examined the effects of smoking on discs of mice after two months of exposure, which also exhibited IDD features, albeit to a much lesser extent than those observed in mice exposed to smoke for six months. We chose to report the results obtained from the six month exposure regimen in this study.

Mice exposed to tobacco smoke displayed loss of matrix proteoglycan in their intervertebral discs

Mice chronically exposed to tobacco smoke showed loss of cellularity in the endplate and dramatic loss of matrix proteoglycan in the intervertebral discs (Fig 1). Safranin O staining of sulfated PGs revealed a substantial reduction of PGs in the discs of smokers compared to those from nonsmoking controls (Fig. 1A). This was confirmed by the quantitative DMMB assay measuring sulfated glycosaminoglycans (GAG) (Fig. 1B). The GAG content of in exposed mice (329 ± 100 μg GAG/ng DNA in NP and 60 ± 13 μg GAG/ng DNA in AF) was much reduced compared to unexposed mice (516 ± 79 μg GAG/ng DNA in NP and 88 ± 34 μg GAG/ng DNA in AF). Compared to AF tissue, NP proteoglycan was reduced to a much greater extent by tobacco smoking. This is perhaps due to differential response of different tissue to smoking-induced stress.

Figure 1. Chronic tobacco smoke exposure decreased disc matrix proteoglycan.

Figure 1

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A, Representative images of H&E (left panel, bar = 250 μm) and Safranin O/fast green stained discs (right panel, bar = 50 μm). Decreased cellularity in AF (yellow arrows) and endplate (black arrows) in H&E stained discs are indicated. Decreased safranin O staining of PG (red stain) in nucleus pulposus (NP) was observed in smoke-exposed mice compared to unexposed mice (black arrow). B, DMMB assay for total GAG content from AF and NP tissue of smoke-exposed mice and untreated controls. NS=nonsmokers, S=smokers. Average values from seven exposed mice and seven unexposed controls are shown with 95% confidence interval.

Chronic tobacco smoke exposure reduced disc matrix protein synthesis

Net loss of disc PGs could be caused by decreased PG synthesis and/or increased PG degradation. We determined PG synthesis by measuring the level of 35S-sulfate incorporated into the discs of mice ex vivo after chronically exposing the mice to cigarette smoke. Exposed mice had 11 ± 2 fmoles sulfate/ng DNA, a 35% reduction compared to that of unexposed control (17 ± 4 fmoles sulfate/ng DNA) (Fig. 2A). In addition, the discs of exposed mice incorporated 2.5 fold less collagenase-sensitive 3-H-proline than unexposed mice (0.17 ± 0.03 fmoles proline/ng DNA in smokers vs. 0.41 ± 0.12 fmoles proline/ng DNA in nonsmokers), suggesting that new collagen synthesis was also compromised in discs of mice exposed to tobacco smoking (Fig. 2B).

Figure 2. Chronic exposure to tobacco smoke decreased new matrix protein synthesis in intervertebral disc.

Figure 2

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Proteoglycan synthesis as measured by 35S-sulfate incorporation (A) and collagen synthesis by collagenase-sensitive incorporation of 3-H-proline (B) in intervetebral discs of mice exposed to tobacco smoking (S) and non-exposed control (NS). Average values from seven exposed mice and seven unexposed controls are shown with 95% confidence interval.

Chronic exposure to cigarette smoke increased proteolytic cleavage of aggrecan interglobular domain in discs

To determine if PG breakdown also contributed to PG loss in smoke-exposed mice, we performed immunoblot analysis using antibodies against the VDIPEN and NITEGE neo-epitopes of aggrecan22,21 to detect proteolytic cleavage of the interglobular domain of aggrecan, the primary PG constituent responsible for the osmotic turgidity of the disc (Fig. 3A). The ADAMTS-generated aggrecan G1 fragments terminating in NVTEGE−392 were detected in the discs of smoke-exposed mice, but not unexposed control mice (Fig. 3B, lanes 2–3). Similarly, the MMP-generated aggrecan G1 fragment terminating in VDIPEN−360 was detected in higher levels (~2 fold by densitometry) in smoke-exposed mice compared to unexposed (Fig. 3B, lanes 4–5). Similar results were observed when anti-G1 antibodies were used to probe these aggrecan proteolytic fragments (Fig. 3B, lanes 6–7). The immunoblot data were further confirmed by immunohistochemistry, where aggrecan fragments containing the NVTEGE−392 and VDIPEN−360 neo-epitopes were detected at higher levels in discs of smoke-exposed mice compared to unexposed controls (Fig. 3C).

Figure 3. Chronic cigarette smoke exposure increased disc aggrecan proteolysis.

Figure 3

Figure 3

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A, schematic representation of mouse proteoglycan aggregate consisting of the core aggrecan protein bound to hyaluronan via a link protein. The MMP-mediated cleavage site (yielding VDIPEN neo-epitope) and ADAMTS-mediated cleavage site (yielding NVTEGE neo-epitope) within the interglobular domain residing between the G1 and G2 domain of aggrecan are indicated. B, Immunoblot analysis of G1 fragments bearing the NVTEGE and VDIPEN neo-epitopes. Protein size marker (M), nonsmokers (N), smokers (S). To control for loading, proteins extracted from 1mg of disc tissue wet weight were loaded per well. The anti-NITEGE neoepitope antibody cross-reacts with the NVTEGE neoepitope generated from mouse aggrecan. C, Immunohistochemical detection of aggrecan fragments using antibodies raised against the NITEGE and VDIPEN neo-epitopes. 42 day-old rat tibial growth plate was used as a positive control for anti-VDIPEN (C.1) and anti-NITEGE (C.2) detection. A negative control without addition of the primary antibodies to the disc section is shown in C.3. Arrow indicates sites of detection of positive signal. The bars represent 100 μm.

It should be noted that the same amount of tissue wet weight (1 mg) was loaded per well in our immunoblotting analysis. Tissue wet weight is typically used for loading normalization for analysis of aggrecan fragments in intervertebral disc tissue because no known matrix protein in the extracellular matrix of disc tissue that exists in abundant and constant quantity that could be used as loading control, unlike a loading control such as actin for analysis of cellular protein. We did not use total GAG or total protein extract from disc tissue as loading control as either of these constituents can be variable in discs of treated and untreated mice.

Mice chronically exposed to tobacco smoke show higher level of cell senescence in their intervertebral discs

The most obvious cause of reduced PG synthesis is if functional cells are lost. Hence we measured apoptotic cells by TUNEL assay and senescent cells by immunodetection of a cellular senescence marker, P16INK4a. More p16-positive cells (27 ± 7 %) were detected in discs of mice exposed to smoke compared to unexposed control (14 ± 7 %), indicating increased cellular senescence in discs of smokers (Fig. 4). There was no detectable difference in the percent of apoptotic cells (0.6–0.7%) between the two groups of mice (Fig. 4).

Figure 4. Effects of exposure to tobacco smoking on cell senesecence and cell death in mouse intervetebral discs.

Figure 4

Figure 4

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A, left panel, immunohistochemical detection of P16INK4a, a senescence marker, to distinguish senescent (brown, arrow) from non-senescent (blue) cells in nucleus puloposus tissue. Right panel, TUNEL assay to identify apoptotic cells (green) in disc annulus fibrosus tissue. Insets, nuclear DAPI stain to reveal tissue cellularity. The bars represent 20 μm. B, quantitative assessment of the percent immunopositive cells for P16INK4a and TUNEL. Average values from seven random fields are shown with 95% confidence interval.

Smoking induced vertebral bone loss

Degenerative changes in the disc highly correlate with the degenerative changes in its surrounding tissue, including the vertebral endplate and the vertebral bodies28. Recent mCT analysis of human lumbar region revealed that the vertebral endplate becomes progressively porous and that the trabecular thickness decreases with disc aging and advancing disc degeneration28. To determine if these changes also occur in our tobacco smoke exposed mice, we utilized mCT to analyze the bone microstructure of the vertebral endplates and vertebral bodies. Mice exposed to tobacco smoke also showed degenerative changes in their vertebrae as assessed by mCT. These include increased bone porosity and significantly reduced trabecular bone thickness (Fig. 5). Vertebral bone porosity of exposed mice was 0.730 ± 0.012 compared to 0.698 ± 0.036 for unexposed controls. Trabecular bone thickness decreased from 0.078 ± 0.003 mm in nonsmokers to 0.072 ± 0.001 mm in smokers. Total trabecular bone volume also decreased (3.2 ± 0.05 mm3 in smokers vs. 3.4 ± 0.07 mm3 in nonsmokers). Porosity also increased significantly within the endplate region, with an average value of 0.36 ± 0.03 observed for smokers compared to 0.18 ± 0.03 for nonsmokers.

Figure 5. Smoking exposure effects on vertebral bone.

Figure 5

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A, Representative 3D reconstruction of the micro-computed tomographical images of the spine (top) and vertebral trabecular bone (bottom). All panels are at the same magnification and the bar represents 500 μm. B, smoking-induced changes in trabecular thickness, vertebral bone porosity, and vertebral endplate porosity. Quantitative bone parameters (TV= total volume, BV= bone volume, Tb.T=trabecular thickness). Average values from seven exposed mice and seven unexposed controls are shown with 95% confidence interval.

DISCUSSION

Previous studies link tobacco smoking to disc degeneration, spinal disorders and lower back pain, but most of them are correlative and do not necessarily establish a direct cause-effect relationship between smoking and IDD. The objective of this study was to determine if chronic first-hand exposure to cigarette smoke causes spine degenerative changes, particularly the intervertebral discs. We demonstrated unambiguously that long-term exposure of healthy adult mice to tobacco smoke significantly depletes disc matrix proteoglycan content, a well-established hallmark of IDD. PG synthesis was decreased and PG degradation increased, providing novel insight into the mechanism of PG loss as a consequence of smoking. Discs of exposed mice exhibited increased MMP- and ADAMTS- mediated proteolysis of the aggrecan interglobular domain (IGD). Cleavage within the IGD is considered most pathological because it leads to loss of the entire GAG-containing region that is vital for disc function29. We also demonstrated that chronic exposure to smoke leads to vertebral bone loss, which is consistent with results recently reported in smoke-exposed rats30. These results provide definitive experimental evidence that tobacco smoke negatively impacts the overall spine health.

Disc degenerative features in smoke-exposed mice in this study generally matched those observed in human IDD. In this study, smoke-exposed mice exhibited greater percentage of cells expressing the cellular senescent marker, P16INK4a, in their discs, consistent with the reported finding of accelerated cellular senescence as a correlative factor of disc degeneration in humans31. Decreased matrix proteoglycans and collagens have been reported in human disc degeneration32, and these features were also observed discs of mice exposed to tobacco smoke in our study. Furthermore, we demonstrated that long-term exposure to tobacco smoking in mice resulted in increased disc aggrecan proteolysis, a well-established hallmark of human disc aging and disc degeneration33, 34. Degenerative changes in the other structural components of the spines were also observed in smoke exposed mice. These included increased porosity in the vertebral endplate and vertebral body and decreased vertebral trabecular thickness, which highly correlate with disc aging and advancing disc degeneration in humans28. Careful quantitative analysis of human spine specimens by Rodriguez and coworkers28 recently showed that vertebral endplate porosity increased with advancing disc aging and degeneration, and this inversely correlates with disc proteoglycan content. Our study also showed that the endplate became more porous with tobacco smoking and this correlates with the degenerative changes in the intervetebral disc. It is still not known how increased endplate porosity affects the number and/or quality of vertebral capillaries and how this in turn influences nutrient/waste exchange between the disc and endplate tissue. These questions await further studies.

Smoking-induced aggrecan proteolysis (Fig. 3) is a novel and important finding. Until now smoke-induced cleavage within the aggrecan IGD has not previously been reported in any other animal models or tissue types after cigarette smoke exposure, including articular cartilage and lung, which contain abundant matrix proteoglycans. The proteolytic aggrecan fragments containing the neoepitope NVTEGE in discs were detected after six months of smoke exposure, but not after two months of exposure (data not shown). This suggests that long-term exposure is required for ADAMTS-mediated cleavage of the aggrecan IGD. The presence of the G1 fragment containing the VDIPEN neoepitope in nonsmokers (Fig. 3B, lane 4) suggest occurrence of MMP-mediated proteolysis of aggrecan IGD with advancing age. However, additional environmental stress caused by tobacco smoking further increases MMP-mediated proteolysis of aggrecan (Fig. 3B, lane 5), contributing to the decreased disc proteoglycan content. It should be noted that metalloproteinase expression is not specifically induced by smoking, but is up-regulated by a number of other stimuli. However, metalloproteinase induction does appear to be important in smoking induced aggrecanolysis.

The mechanism by which smoking promotes proteolysis of disc aggrecan is not known. Expression of MMPs and ADAMTS are greatly enhanced whereas TIMP expression is suppressed when disc cells are exposed directly to tobacco smoke extract (TSE) in vitro35. Direct exposure of disc cells to toxic, water-soluble by-products of tobacco combustion in vivo is conceivable as infiltration of these smoke constituents could occur in peripheral AF, which is vascularized, or into the NP by diffusion through the endplate. Aged and degenerated discs, especially those found in older smokers, could have increased neovascularization36–38 as a consequence of tissue fissures, which could in turn increase exposure of disc cells to the toxic soluble smoke chemicals in the circulatory system. The presence of tobacco smoke-induced DNA damage in a variety of tissues (e.g, bone marrow, bladder, and urethra) far from the primary respiratory sites of initial contact, support infiltration of harmful tobacco smoke components into a variety of tissues39–41. Thus, direct interaction of disc cells with tobacco smoke chemicals in vivo could cause up-regulation of MMP and ADAMTS and down-regulation of TIMP expression, leading to aggrecan degradation observed in smoke-exposed mice.

Inflammatory stress is another key factor causing perturbation of disc PG homeostasis via increasing matrix catabolism and decreasing matrix anabolism42–44. Given the existence of numerous pro-inflammatory substances in tobacco smoke45, it is also possible that smoke-induced cleavage of aggrecan is mediated through the inflammatory pathways. Rats exposed to passive cigarette smoking for eight weeks showed a three-fold increase in the pro-inflammatory cytokine interleukin-1β (IL-1β) in their intervertebral disc tissue12. PCR analysis also revealed a two-fold increase in IL-1β mRNA in discs of our smoke-exposed mice (data not shown). Similarly, disc cells exposed to tobacco smoke extract produce high amounts of the inflammatory mediator prostaglandin F2α, which stimulates MMP expression35. Thus smoking-induced inflammation could up-regulate expression and activities of the metalloproteinases which act on disc aggrecan to produce the observed proteolysis. On the other hand, decreased PG synthesis in discs of smoke-exposed mice could also be due to cellular senescence (Fig. 4). Senescent chondrocytes have reduced capacity for PG synthesis46. Cellular senescence can be driven directly or indirectly by DNA damage resulting from the many mutagenic compounds in tobacco smoke. Furthermore senescent cells secrete pro-inflammatory cytokines47 which could upregulate catabolism in neighboring cells and extracellular matrix.

Tobacco smoking promotes nicotine-induced vasoconstriction, increases CO-hemoglobin content48, and accelerates aortic atherosclerosis and stenosis of the arteries feeding the spine49, 50, all of which lead to the decrease of nutrient and oxygen levels in the disc. The avascular nature of disc tissue and its relatively poor nutrition supply make it exceptionally susceptible to the decrease in nutrient level in smokers, where disc cells would have to reduce matrix synthesis to meet their nutrient input. Because the PG synthesis capacity of disc cells also is extremely sensitive to changes in pH, Urban and coworkers suggested that the fall in oxygen level in the disc as a result of smoking raises the lactate level, causing disc pH to drop, which would then suppress PG synthesis51. Thus, although our study clearly established a direct cause-effect relationship between smoking and IDD in mice, it remains to be investigated whether or not smoking causes IDD directly by the interaction of its toxic chemicals with disc tissue or indirectly through the systemic changes, i.e., vasoconstriction, atherosclerosis, DNA damage, bone marrow alteration…etc., which in turn adversely affect disc matrix homeostasis.

Unlike all previous studies of IDD which exposed rats or porcine to passive tobacco smoking, our approach of using the mouse experimental model of direct smoke inhalation is innovative as it is a more pertinent model system to study the effects of tobacco smoking on IDD because it mimic first-hand smoking in humans. Moreover, adapting our experimental smoking model to mice offers a significant advance over the former model systems in that one can use gene-targeted mice to dissect the mechanism of smoke-induced IDD in future studies. For instance, many different MMP and ADAMT genetic knockout mouse strains exist, and exposing these strains to tobacco smoke allows for identification and delineation of the relative contribution of specific MMPs and ADAMTSs in smoking-induced disc aggrecanolysis.

In summary, mice exposed to direct smoke inhalation to mimic chronic first-hand human smokers exhibit considerable loss of vertebral bone and disc matrix proteoglycan, thus providing clear and compelling evidence that prolonged exposure to tobacco smoke can lead to spinal degeneration. Our mouse model of direct smoke-induced IDD is useful for dissecting and understanding the molecular and cellular mechanisms underlying the development of smoke-related IDD. The discovery of smoke-induced proteolysis within the disc aggrecan IDG at MMP- and ADAMTS-specific cleavage sites also provides novel enzyme targets for inhibitors aimed at delaying the onset or ameliorating the severity of IDD in chronic smokers.

Figure 6. A summary of effects of tobacco smoking on spine degeneration.

Figure 6

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Smoking negatively impacts the major structures of the spine, including vertebral bone, endplate, and intervertebral disc. Within the intervertebral disc, smoking affects disc cellular function as well as induces matrix aggrecan breakdown. PG, proteoglycan. Col, collagen.

ACKNOWLEDGEMENTS

The authors would like to thank Lisa Lamplugh for assistance with the immunohistochemical analysis, Yeqing Geng for Western analysis of aggrecan proteolysis, Qing Dong for histological studies, Kevin Ngo for mCT analysis, and Dr. Rebecca Studer for proof reading this manuscript. This work was supported in part by the Albert B. Ferguson, Jr. M.D. Orthopaedic Fund of the Pittsburgh Foundation, 2010 ORS Collaborative Exchange Award to Nam Vo, and NIH grants AG033046 to Nam Vo., ES016114 to Laura Niedernhofer.

Footnotes

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AUTHOR CONTRIBUTIONS The contributions from the following individuals are gratefully acknowledged. Sowa G, Lee J, Niedernhofer L, Kang J (experimental design, data interpretation, intellectual inputs, and manuscript preparation), Wang D, Nasto L (matrix synthesis, histology and immunohistochemistry). Usas A (radiography), Roughley P (aggrecan proteolysis), Houghton M, Lemme A (breeding, smoking of mice). All authors were involved in drafting and critically editing this article. Vo N, Shapiro S, Kang J, and Roughley P were responsible for obtaining funding for this work.

CONFLICT OF INTEREST None

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