Abstract
Background: It is well known that age-related fibrosis, or decreases
in the elastin-to-collagen ratio of the ligamentum flavum, along with
hypertrophy of the ligamentum flavum, are associated with lumbar spinal
stenosis. However, the molecular mechanism by which this fibrosis and
hypertrophy develop is unknown. Tissue inhibitors of matrix metalloproteinase
(TIMPs) are proteinase inhibitors that suppress extracellular matrix
degradation. Elevated TIMP-1 and TIMP-2 expression has been implicated in
various fibrotic diseases of the liver, kidney, lung, and heart. These TIMPs
can also induce cellular proliferation and inhibit apoptosis in a wide range
of cell types. These findings led us to postulate that TIMP-1 and TIMP-2 might
also be associated with hypertrophy and fibrosis of the ligamentum flavum in
lumbar spinal stenosis.
Methods: We quantified and localized TIMP expression in ligamentum
flavum tissues that had been obtained during surgery from thirty patients with
spinal stenosis and from thirty gender-matched control patients with disc
herniation. The thickness of the ligamentum flavum at the level of the facet
joint was measured on axial T1-weighted magnetic resonance images. In
addition, we examined ligamentum flavum tissues for the expression of markers
of cellular proliferation and apoptosis.
Results: The ligamentum flavum was significantly thicker in the
patients with spinal stenosis (mean, 5.68 mm) than in the patients with disc
herniation (mean, 2.70 mm) (p < 0.001). The concentration of TIMP-2 in the
ligamentum flavum was significantly higher in the patients with spinal
stenosis (mean, 12.62 ng/mL) than in those with disc herniation (mean, 8.85
ng/mL) (p = 0.028). TIMP-1 and TIMP-2 were detected in the cytoplasm of
ligamentum flavum fibroblasts. TIMP-1 and TIMP-2 concentrations were
associated with hypertrophy of the ligamentum flavum (p = 0.015 and p = 0.003,
respectively). None of the samples from the patients with stenosis had
evidence of proliferation of ligamentum flavum fibroblasts. The expression of
markers for apoptosis was significantly higher in the patients with spinal
stenosis (58.8%) than in those with disc herniation (26.6%) (p <
0.001).
Conclusions: Increased TIMP expression has been implicated in
fibrosis and hypertrophy of the extracellular matrix of several organs. Our
results suggest that increased expression of TIMP-2 in ligamentum flavum
fibroblasts is associated with fibrosis and hypertrophy of the ligamentum
flavum in patients with spinal stenosis.
Clinical Relevance: Gaining insights into the molecular pathogenesis
of hypertrophy and fibrosis of the ligamentum flavum may eventually result in
new therapeutic and potentially noninvasive alternatives to the treatment of
spinal stenosis.
The ligamentum flavum is composed of elastin and collagen fibers in a 2:1
ratio. The elastin fibers impart a yellow color to the structure and provide
the elasticity, while the collagen provides the stiffness and stability. With
age, the elastin-to-collagen ratio decreases, resulting in decreased
elasticity and increased stiffness or fibrosis. In addition, the ligamentum
flavum hypertrophies, and the combination sometimes results in lumbar spinal
stenosis1-5.
Although the hypertrophy and fibrosis have been postulated to be due to
degenerative changes secondary to the aging process and mechanical stress
secondary to instability, the pathogenesis remains undetermined.
Matrix metalloproteinases (MMPs) are a family of more than twenty enzymes
that digest proteins in the extracellular
matrix6-9.
There are also inhibitors of MMPs, called tissue inhibitors of matrix
metalloproteinase (TIMPs), and it is believed that, together, MMPS and TIMPs
regulate the integrity and homeostasis of the extracellular matrix. There are
four known TIMPs (TIMP-1 through 4) that suppress extracellular matrix
degradation by forming an inhibitory 1:1 complex with the
MMPs10-18.
Elevated expressions of TIMP-1 and TIMP-2 have been implicated in the
increased fibrosis found in a wide variety of human organs, including the
liver, kidney, lung, and heart. TIMPs are also known to increase cellular
proliferation19-21
and inhibit programmed cell death (apoptosis) in a wide range of cell
types22-26.
On the basis of what is known about TIMPs, we hypothesized that they might
also play a role in the hypertrophy of the ligamentum flavum seen in lumbar
spinal stenosis. We postulated that TIMPs might alter the ligamentum flavum in
three ways: by increasing the density of the extracellular matrix and
promoting fibrosis by inhibiting MMPs, by increasing fibroblast proliferation,
and by reducing the rate of fibroblast apoptosis. In the present study, our
goals were to determine (1) whether there is an increased concentration of
TIMPs in the ligamentum flavum of patients with spinal stenosis compared with
that in patients with disc herniation, and (2) whether the ligamentum flavum
in spinal stenosis has increased fibroblast proliferation and/or decreased
apoptosis, either of which would result in increased cellularity. To our
knowledge, no one has previously investigated the association between
hypertrophy of the ligamentum flavum and concentrations of TIMP-1 and
TIMP-2.
Thirty samples of ligamentum flavum tissue were obtained from thirty
patients who were undergoing decompression because of neurogenic claudication
due to lumbar spinal stenosis that had been unresponsive to conservative
measures for at least three months. None of these patients received epidural
or selective nerve-root blocks. Twenty-one patients were female, and nine were
male. The mean age of the patients at the time of surgery was 63.7 years
(range, fifty-two to seventy-eight years). Patients with degenerative
spondylolisthesis were excluded from the study. Seventeen patients underwent a
one-level operation; nine, a two-level operation; and four, a three-level
operation. We tried to obtain the entire layer of the central portion of the
ligamentum flavum, and we removed the epidural fat from the ligamentum flavum
tissues. Half of each ligamentum flavum specimen was fixed in 4% neutral
formalin, decalcified with 20% EDTA for six weeks, and embedded in paraffin;
the other half was kept in a freezer at —70°C for subsequent
enzyme-linked immunosorbent assay (ELISA).
We randomly selected thirty gender-matched control patients with lumbar
disc herniation from a group of 124 patients who were being operatively
managed for that disorder. The mean age of the control patients was 31.7 years
(range, nineteen to forty-three years), which was significantly younger than
the mean age of the patients with spinal stenosis (p < 0.001).
Measurement of the Thickness of the Ligamentum Flavum
An axial T1-weighted magnetic resonance image (repetition time, 600 msec;
echo time, 30 msec) was made, with a 1.5-T unit (Somatom Plus; Siemens,
Erlangen, Germany), at the facet joint level of the lesion for each patient.
The maximum thickness of the ligamentum flavum was traced with use of the
manual cursor technique and was computed automatically by the installed
software in the magnetic resonance imaging scanner. All of the radiographic
analyses were independently performed by two experienced spine surgeons who
were not involved in the care of the patients. Each observer independently
measured the thickness of the ligamentum flavum twice, and the average of the
four measurements was used as the final thickness.
Quantification of TIMP-1 and TIMP-2 Concentrations by ELISA
After thawing, 100 mg of the ligamentum flavum tissue was homogenized with
phosphate-buffered saline solution at 3000 rpm in a tissue homogenizer (model
985-370, Tissue-Tearor; Bio-Spec Products, Racine, Wisconsin). Centrifugation
to 15,000 rpm was then performed at 4°C for thirty minutes, and the
supernatant was obtained. Quantification of protein was performed according to
Bradford's method27
with use of a protein assay kit (catalogue number 500-0006; Bio-Rad
Laboratories, Hercules, California), and optical density was measured at 595
nm with a spectrophotometer (Ultrospec 3000; Pharmacia Biotech, Cambridge,
United Kingdom) and adjusted equally to levels of 0.5 mg/mL (total protein
concentration) for each sample.
TIMP-1 and TIMP-2 concentrations were measured twice in each sample with
use of an ELISA kit with antibodies that recognize human TIMP-1 and TIMP-2 (R
and D Systems, Minneapolis, Minnesota); the kit was used according to the
manufacturer's instructions. The average of the two measurements was
considered to be the final concentration. For calibration, we used human
recombinant TIMP-1 and TIMP-2 provided by the supplier to construct a standard
curve and to obtain absolute values.
Apoptosis: Analysis with In Situ Nick End-Labeling (TUNEL) Assay
In situ terminal deoxynucleotidyl transferase-mediated deoxyuridine
triphosphate nick end-labeling (TUNEL) reaction was performed with use of a
TACS 2 TdT DAB In Situ Apoptosis Detection Kit (Trevigen, Gaithersburg,
Maryland) according to the manufacturer's instructions. One section (4 µm
thick) was treated with proteinase K. Endogenous peroxidase was removed with
2% H2O2. The 3'-OH ends of the fragmented DNA were
marked with TdT dNTP and divalent cation by adding TdT. Two pathologists, who
were unaware of the clinical data, were responsible for counting total and
TUNEL-positive ligamentum flavum fibroblasts under ten randomly selected
high-power fields (×400). The apoptosis index was calculated as
TUNEL-positive cells/total cells × 100%. Human tonsil tissue was used as
a positive control.
Cellular Proliferation: Analysis with Immunohistochemistry for TIMPs
and Ki-67
Ki-67, a cellular proliferation marker, was assayed to determine if there
was evidence of proliferation of ligamentum flavum fibroblasts. Three
consecutive 4-µm-thick sections were cut on a microtome, deparaffinized in
xylene, and rehydrated. To determine the expression of TIMP-1, TIMP-2, and
Ki-67, the avidin-biotin-peroxidase complex method and a Histostain-plus SP
kit (Zymed Laboratories, South San Francisco, California) were used according
to the manufacturer's instructions. Purified mouse monoclonal antibodies to
TIMP-1 and TIMP-2 (NeoMarkers, Fremont, California) and Ki-67 (DakoCytomation,
Glostrup, Denmark) were used for this study at an optimum dilution of 1:20,
1:50, and 1:75, respectively. A human breast carcinoma and a human colon
carcinoma were used as positive controls for TIMP-1 and TIMP-2, and tonsil
tissue was used for Ki-67.
Statistical Analysis
The independent-sample t test was used to compare the patients with lumbar
spinal stenosis and those with disc herniation in terms of the patients' age,
TIMP-1 and TIMP-2 concentrations, ligamentum flavum thickness, and apoptosis
index. The relationship between the TIMP concentration and the thickness of
the ligamentum flavum was determined with the Pearson correlation test. A p
value of <0.05 was considered to be significant.
Thickness of the Ligamentum Flavum
The mean thickness of the ligamentum flavum (and standard deviation) was
5.68 ± 1.02 mm (range, 4.5 to 7.2 mm) in the patients with lumbar
spinal stenosis and 2.70 ± 0.35 mm (range, 2.2 to 3.3 mm) in those with
lumbar disc herniation. The difference was significant (p < 0.001)
(Table I).
TIMP-1 and 2 Concentrations
With the number of samples available, there was no significant difference
in the mean concentration of TIMP-1 between the patients with lumbar spinal
stenosis (6.33 ± 2.81 ng/mL) and those with lumbar disc herniation
(4.55 ± 2.06 ng/mL) (p = 0.08)
(Table I). In contrast, the
difference between the mean concentration of TIMP-2 in the patients with
lumbar spinal stenosis (12.62 ± 3.47 ng/mL; range, 6.9 to 19.1 ng/mL)
and that in the patients with disc herniation (8.85 ± 4.33 ng/mL;
range, 4.3 to 12.6 ng/mL) was significant (p = 0.028)
(Table I). Fibroblasts in the
ligamentum flavum of both groups of patients showed positive cytoplasmic
staining for TIMP-1 and TIMP-2 (Figs. 1-A
and 1-B).
Correlation Between TIMP Concentration and Ligamentum Thickness
The Pearson correlation test showed a positive correlation between the
TIMP-2 concentration and the thickness of the ligamentum flavum (correlation
coefficient = 0.631, p = 0.003) and between the TIMP-1 concentration and the
thickness of the ligamentum flavum (correlation coefficient = 0.534, p =
0.015).
Apoptosis of Ligamentum Flavum Cells
There was a mean of 35.7 ± 18.4 total ligamentum flavum fibroblasts
and 21.0 ± 10.7 TUNEL-positive ligamentum flavum fibroblasts per ten
high-power fields (×400) in the specimens from the patients with spinal
stenosis; the apoptosis index was 58.8% ± 18.3% (Figs.
2-A and
2-B). The specimens from the
patients with disc herniation showed a mean of 53.1 ± 17.7 total and
14.1 ± 5.63 TUNEL-positive ligamentum flavum fibroblasts per ten
high-power fields, and the apoptosis index was 26.6% ± 21.3%. The
apoptosis index was significantly higher for the group with lumbar spinal
stenosis than it was for the group with disc herniation (p < 0.001), which
disproved our theory that ligamentum flavum hypertrophy was due to a decrease
in cell death.
Fibroblast Proliferation in the Ligamentum Flavum
None of the fibroblasts within the ligamentum flavum samples from the
patients with spinal stenosis or from those with disc herniation stained
positively with Ki-67 (cellular proliferation marker), suggesting that
fibroblasts were not proliferating in any of the samples (Figs.
3-A and
3-B).
There are four types of tissue inhibitors of matrix metalloproteinase
(TIMP-1 through 4). They bind strongly but noncovalently to activated MMPs
(matrix metalloproteinases), enzymes that digest proteins in the extracellular
matrix. TIMPs are co-expressed with the MMPs and contribute to the regulation
of their local activity so that increases in TIMP levels reduce MMP activity.
Decreased MMP activity impairs matrix degradation, which has been demonstrated
to be associated with fibrotic diseases. As the activity of MMPs is regulated
by specific TIMPs, the imbalance between MMPs and TIMPs is thought to be an
important determinant of extracellular matrix deposition and breakdown. TIMPs
also have other important biological functions, including promotion of
cellular proliferation and inhibition of apoptosis (programmed cell death) in
a wide range of cell types.
On the basis of this information, we postulated that TIMPs may also play a
role in the ligamentum flavum hypertrophy associated with lumbar stenosis,
which is characterized by increased extracellular matrix and fibrosis in the
ligamentum. We also hypothesized that TIMPs might influence ligamentum
hypertrophy by increasing proliferation and decreasing apoptosis of the
ligamentum fibroblasts. To test our hypotheses, we measured the concentrations
of TIMP-1 and TIMP-2 in ligamentum flavum tissues from patients with lumbar
spinal stenosis and compared them with those from a gender-matched group of
patients who had lumbar disc herniation.
We found that the TIMP-2 concentration was significantly (1.43-fold) higher
in the patients with spinal stenosis than it was in those with disc herniation
(p = 0.028), and a significant association was observed between the TIMP-2
concentration and the thickness of the ligamentum flavum (p = 0.003). The
TIMP-1 concentration in the patients with spinal stenosis was also positively
associated with the thickness of the ligamentum flavum (p = 0.015). In
addition, TIMP-1 and 2 were positively stained in the cytoplasm of ligamentum
flavum fibroblasts. These results suggest that increased expression of TIMPs,
especially TIMP-2, in ligamentum flavum fibroblasts may be associated with
hypertrophy of the ligamentum flavum in spinal stenosis.
We had theorized that TIMPs might play a role in stenosis by decreasing the
rate at which fibroblasts undergo programmed cell death, resulting in
increased cellularity. We found that the expression of intracellular TIMP-2 in
the ligamentum flavum fibroblasts was higher in the patients with spinal
stenosis than in the patients with disc herniation. However, contrary to our
hypothesis, the programmed death rate of fibroblasts was significantly higher
and the number of total cells was lower in the patients with spinal stenosis
than they were in those with disc herniation. These findings suggest that the
ligamentum flavum hypertrophy in spinal stenosis is not caused by an increase
in cell number resulting from a delay in cell death. Furthermore, these
results suggest that TIMPs do not have the biologic function of inhibiting
apoptosis of ligamentum flavum fibroblasts.
We had also theorized that TIMPs might play a role in ligamentum flavum
hypertrophy not just by decreasing cell death, but also by inducing
fibroblastic cell proliferation. We utilized the Ki-67 antigen, a nuclear
protein, which is preferentially expressed during all active phases of the
cell cycles (G1, S, G2, and M-phase) as well as mitosis
but is absent in resting cells (G0-phase). The antigen is rapidly
degraded as the cell enters the nonproliferative state. Therefore, the Ki-67
antigen is accepted as an excellent marker for cellular proliferation. Again,
contrary to our hypothesis, the ligamentum flavum fibroblasts from the
patients with spinal stenosis and those with disc herniation tested negative
for the Ki-67 antigen, suggesting that they were not proliferating. These
results suggest that TIMPs do not have the biologic function of inducing
proliferation of ligamentum flavum fibroblasts.
Few authors have investigated the biochemical etiology of ligamentum flavum
hypertrophy. We previously demonstrated the expression of transforming growth
factor-beta 1 (TGF-ß1) in ligamentum flavum fibroblasts and found that
the concentration was significantly higher in patients with spinal stenosis
than it was in patients with disc
herniation28. In an
in vitro study, Nakatani et al. found that mechanical stretching force
promotes collagen synthesis by cultured cells from human ligamentum flavum
tissues through increased TGF-ß1
production29. These
studies support our finding that TGF-ß1 can influence the expression of
both MMPs and
TIMPs30,31.
Together, these studies suggest that biochemical as well as mechanical factors
should be considered as possible etiologies of ligamentum flavum hypertrophy
in lumbar spinal stenosis.
As with any investigation, our study had limitations. First, the patients
with spinal stenosis were significantly older than those with disc herniation.
Therefore, we cannot exclude the possibility that the natural aging process
had an impact on the hypertrophy of the ligamentum flavum or on the other
variables that we measured in this study. In other words, some (perhaps all)
of the changes that were noted in our study could have simply been related to
age. Second, it is not known whether the increased concentration of TIMP-2 in
the patients with spinal stenosis was a local or systemic phenomenon. To
address this issue, we are currently in the process of comparing serum TIMP-1
and TIMP-2 concentrations in patients with lumbar spinal stenosis with those
concentrations in normal healthy volunteers.
In conclusion, the increased expression of TIMPs, especially TIMP-2, in
ligamentum flavum fibroblasts might result in fibrosis and hypertrophy of the
ligamentum flavum in spinal stenosis by inhibiting MMP activity, but not by
causing proliferation of ligamentum flavum fibroblasts or inhibiting apoptosis
of ligamentum flavum fibroblasts. Gaining insights into the biochemical
pathophysiology of ligamentum flavum hypertrophy might elucidate novel
noninvasive therapeutic approaches to the treatment of spinal stenosis.
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