Spine degeneration in a murine model of chronic human tobacco smokers

Abstract

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

Methods: Three month old C57BL/6 mice were exposed to tobacco smoke by direct inhalation (4 cigarettes/day, 5 days/week for 6 months) to model long-term smoking in humans. Total disc proteoglycan (PG) content [1,9-dimethylmethylene blue (DMMB) assay], aggrecan proteolysis (immunobloting analysis), and cellular senescence (p16INK4a immunohistochemistry) were analyzed. PG and collagen syntheses ((35)S-sulfate and (3)H-proline incorporation, respectively) were measured using disc organotypic culture. Vertebral osteoporosity was measured by micro-computed tomography.

Results: Disc PG content of smoke-exposed mice was 63% of unexposed control, while new PG 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-fold 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 results 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.

Figure 1. Chronic tobacco smoke exposure decreased disc matrix proteoglycan

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.

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

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.

Figure 3. Chronic cigarette smoke exposure increased disc aggrecan proteolysis

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.

Figure 3. Chronic cigarette smoke exposure increased disc aggrecan proteolysis

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.

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

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.

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

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.

Figure 5. Smoking exposure effects on vertebral bone

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.

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

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.