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From the Division of Rheumatology,*
the Department of
Medicine,
and the University Medical
Policlinic,
Centre Hospitalier Universitaire
Vaudois (CHUV) University Hospital, Lausanne, Switzerland; and
the Northwest Lipid Research Laboratory,
Seattle, Washington
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The exact mechanism whereby apo(a) is atherogenic remains to be elucidated. Apo(a) has a high affinity for lysine-binding sites on fibrin(ogen)9,10 and may therefore compete with plasminogen at sites of fibrin deposition and thus interfere with the fibrinolytic system.11 In the same line apo(a), or fragments of apo(a) that have been found in human plasma and urine,12 may antagonize the effect of angiostatin, a biologically active fragment of plasminogen that inhibits angiogenesis.13,14 Finally, Lp(a) may contribute to lipid delivery to atherosclerotic plaques.
In arthritis, pathological mechanisms that may be modulated by Lp(a) in the joint include the equilibrium between extravascular coagulation and fibrinolysis, plasmin-mediated cartilage and bone matrix degradation, and angiogenesis.15-22 Despite numerous reports on elevated concentrations of Lp(a) in plasma from patients with rheumatoid arthritis (RA)19,23-25 or other rheumatological diseases,23,26,27 little is known on the biology and the role of Lp(a) within the joint.
In the present study, we examined human arthritic synovial fluid for the presence of apo(a). We demonstrate that apo(a) is present in human synovial fluid as part of Lp(a) and we provide evidence that Lp(a) in synovial fluid originates from the liver. Next, because of difficulties in examining the function of apo(a) in humans, we tested whether apo(a) is beneficial or deleterious to the joints by using transgenic mice expressing human apo(a)28,29 in an experimental model of immunological arthritis, antigen-induced arthritis (AIA), which recapitulates some of the features of RA.30
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Patients were diagnosed with osteoarthritis (OA) based on clinical and radiological criteria (n = 8), with RA if they fulfilled at least four of the seven American Rheumatism Association revised criteria for RA (n = 12), or with other types of inflammatory arthritis (n = 12) (two patients had Still’s disease, two had gout, three had spondylarthritis, one had Reiter’s disease, one had chondrocalcinosis, and three had inflammatory monoarthritis of unknown origin). For all patients, synovial fluid, as well as venous blood, were collected into citrated tubes, subjected to centrifugation for 10 minutes at 1500 x g and supernatants were stored at -70°C until use. In addition, specimens of synovial tissue from seven OA and seven RA patients undergoing joint surgery of the knee or the hip for advanced disease were obtained from the local Department of Orthopedics. All tissues were cut into small pieces, embedded in Tissue-Tek OCT, and immediately frozen in precooled hexane to be stored at -70°C until use.
Immunological Analysis of Apo(a) in Human Plasma and in Synovial Fluid and Tissue
Apo(a) was quantitated in plasma, synovial fluid, and tissue using a sandwich enzyme-linked immunosorbent assay using antibodies IgG-a6 as capture antibody and horseradish peroxidase-conjugated IgG-1-1 as detecting antibody, two mouse monoclonal antibodies of well-defined specificity against apo(a),31 as described.32 Immunoblot analysis of apo(a) was performed after size-fractionation of synovial fluid and plasma proteins using a 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis33 and immunoblotting using IgG-a5, a mouse anti-human apo(a) monoclonal antibody directed against K4-type 2.31 This analysis was performed on untreated samples. In addition, we incubated plasma and synovial fluid samples with heparin-coated beads, and analyzed the heparin-bound fraction [corresponding to apo(a) bound to apoB-100 of LDL to form Lp(a)] and the heparin-unbound fraction [corresponding to apo(a) or fragments of apo(a) circulating free of Lp(a)], exactly as described.33 Concentrations of total cholesterol, high-density lipoprotein (HDL)-cholesterol, triglycerides, and albumin were measured in cell-free synovial fluid and plasma samples using the Unimate5-chol (Roche, Basel, Switzerland), the HDL-kit (Behring, Deerfield, IL), the Unimate7-trig kits (Roche), and by nephelometry (Behring), respectively, as described.32 LDL-cholesterol was calculated using the Friedewald formula. D-dimer concentration in mouse plasma was measured by a commercially available enzyme-linked immunosorbent assay kit designed for human D-dimer (Asserachrom D-Di, Diagnostica Stago, Asnières, France), which cross-reacts with murine D-dimer. The content of murine D-dimer was calculated according to the human D-dimer standard curve.
Immunohistochemical Analysis of Apo(a) and Fibrin
Immunohistochemistry on human tissues was performed on air-dried 5-µm cryostat synovial tissue sections, fixed for 10 minutes in acetone at 4°C before use. Each slide was incubated for 30 minutes with 10% normal human serum, 10% normal goat serum, and 1% bovine serum albumin (BSA). For fibrin immunohistochemistry, slides were then overlaid for 30 minutes at room temperature with a murine monoclonal antibody against fibrin (Y22 monoclonal antibody, recognizing fibrin but not fibrinogen, TNO), used at 5 µg/ml final concentration. For apo(a) immunohistochemistry, slides were overlaid for 2 hours at room temperature with IgG-a5, used at 5 µg/ml final concentration. Immunohistochemical analyses on murine tissues were performed on paraffin-embedded sections of knee joints that were deparaffinized and rehydrated, then incubated for 30 minutes at room temperature with 5% BSA and 20% normal goat serum. Endogenous peroxidase activity was blocked with 3% H2O2 for 10 minutes. Slides were then overlaid with rabbit anti-mouse fibrin(ogen) antiserum (diluted 1/1000) for 30 minutes at room temperature or with rabbit anti-human apo(a) polyclonal antibody (Europa, Cambridge, UK), at a concentration of 5 µg/ml, for 2 hours and 30 minutes at room temperature. Bound antibodies were visualized using an adequate biotinylated secondary antibody and the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA). The color was developed by 3,3'-diaminobenzidine (Sigma, St. Louis, MO) containing 0.01% hydrogen peroxide. After extensive washing in water, slides were counterstained with Papanicolaou and mounted in Merckoglass. Staining specificity for apo(a) and fibrin immunohistochemistry was confirmed using, as primary antibodies, preadsorbed antibodies onto clotted fibrin or Lp(a)-containing serum, respectively. Incubation in which the first antibody was omitted served as a negative control.
Apo(a) mRNA Expression in Synovial Tissues
Cryostat sections from synovial or liver tissues
were homogenized in Trizol reagent (Gibco BRL, Basel, Switzerland) and
total RNA was extracted according to the manufacturer’s instructions.
A total of 
5 µg of total RNA was hybridized overnight at 52°C
with 3 x 105
cpm of the
32P-UTP labeled apo(a) probe, obtained by
in vitro transcription of a linearized pGEM-T plasmid
(Promega, Madison, WI) containing an insert corresponding to sequence
10,467 through 10,807 of the apo(a) cDNA.8
A labeled GAPDH
riboprobe served to normalize the apo(a) gene expression. Samples were
then treated with an RNase cocktail (Ambion, Austin, TX), RNase was
removed by proteinase K treatment, and samples were purified by
phenol/chloroform extraction followed by ethanol precipitation using
glycogen as carrier. Protected fragments were resolved through a 5%
sequencing gel.
AIA in Genetically Modified Mice
LDL-receptor-deficient (LDL-R-/-) mice expressing a human apoB-100 transgene (hApoB +/+) and a human apo(a) transgene containing 17 K4 repeats under the control of the mouse transferrin promoter [hApo(a) +/-]29 were used in these experiments. [LDL-R-/-, hApoB +/+, hApo(a)-/-] littermates served as controls. Mice were of the haplotype H-2b, and were 10 to 12 weeks of age at the start of the experiment. AIA was elicited as previously described.30 Briefly, mice were immunized at day 0 and day 7 by intradermal injection of 100 µg of methylated BSA (mBSA, Sigma) emulsified in 100 µl of complete Freund’s adjuvant containing 200 µg of mycobacterial strain H37RA (GD Diagnostics, Sparks, MD), and by intraperitoneal injection of 2 x 109 heat-killed Bordetella Pertussis organisms (Berna, Bern, Switzerland). Arthritis of the right knee was induced at day 21 by intra-articular injection of 100 µg of mBSA in 10 µl of sterile phosphate-buffered saline (PBS), whereas the left knee was injected with sterile PBS alone. Grading of arthritis was performed after knees had been dissected and fixed in 10% buffered formalin for 4 days. Fixed tissues were decalcified for 5 days in a solution of 25% formic acid and 7.5% w/v sodium formate, dehydrated, and embedded in paraffin. Sagittal sections (6 µm) of the whole knee joint were stained with Safranin-O and counterstained with fast green/iron hematoxylin. Histological sections were graded by two observers unaware of the animal genotype. Infiltration of cells was scored on a scale from 0 to 3, depending on the amount of inflammatory cells in the synovial cavity and synovial tissues (0 = no inflammatory cells to 3 = massive exudate or infiltrate). Cartilage proteoglycan depletion, reflected by loss of Safranin-O-staining intensity, was scored on a scale from 0 (fully stained cartilage) to 3 (totally unstained cartilage) in proportion to severity. The intensity of fibrin(ogen) deposition in the joints was scored on a 0 to 6 scale where 0 corresponded to complete absence and 6 to massive deposition of fibrin(ogen).
Statistical Analyses
Nonparametric Mann-Whitney two-sample test was used to compare groups. Analysis of statistical correlation was performed with Spearman’s test of rank correlation. For animal studies, the Wilcoxon rank-sum test for unpaired variable (two-tailed) was used to compare differences in histological scoring between groups. P values < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apo(a) was quantitated in paired plasma and synovial fluid samples
collected from 32 arthritic patients (8 patients diagnosed with OA, 12
with RA, and 12 with miscellaneous inflammatory arthritides).
Concentrations of apo(a) in plasma varied widely between individuals
(from 0.7 to 145.1 nmol/L) and the distribution was markedly skewed
toward lower values (median, 11.5 nmol/L), as in other Caucasian
populations.7
In synovial fluid, the level of apo(a)
ranged from 0.5 to 43.1 nmol/L (median, 7.8 nmol/L) and rose in
proportion to plasma apo(a) levels (Figure 1
, r = 0.88). In addition
to apo(a), we detected the presence in synovial fluid of cholesterol
[from 0.5 to 4.4 mmol/L, 2.4 ± 0.2 mmol/L (mean ± SEM)],
triglycerides (0.1 to 1.6 mmol/L, 0.5 ± 0.1 mmol/L),
HDL-cholesterol (0.1 to 1.2 mmol/L, 0.7 ± 0.1 mmol/L), and
albumin (11 to 44 g/L, 23 ± 1 g/L).
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Immunoblot analysis of apo(a) was performed on paired plasma and
synovial fluid samples, either untreated or after incubation with
heparin-coated beads [that separate free apo(a) and apo(a) fragments
from Lp(a)].33
A representative blot performed on samples
from two different individuals with moderately elevated apo(a) levels
in plasma (28.4 and 18.1 nmol/L) and synovial fluid (10.6 and 8.2
nmol/L) is depicted in Figure 2
. In both
plasma samples, apo(a) was present as full-length apo(a) (lanes 1 and
7), and attached heparin as part of Lp(a) (lanes 3 and 9), whereas
apo(a) fragments, which correspond to a small fraction (2%) of
apo(a) immunoreactive material, were hardly visible on this exposure
(lanes 2 and 8). The size of apo(a) in untreated synovial fluid (lanes
4 and 10) was similar to the one of full-length apo(a) in plasma.
Moreover, as was the case in plasma, full-length apo(a) in synovial
fluid equally bound heparin (lanes 6 and 12), whereas apo(a) fragments
were hardly visible on this exposure (lanes 5 and 11). This data
demonstrated that apo(a) immunoreactive material in synovial fluid
corresponds to full-length apo(a) as part of Lp(a) particles.
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Concentrations of apo(a) and the other particles examined were
compared in arthritic synovial fluid and in plasma. The synovial
fluid-to-plasma (SF/P) ratio varied widely between individuals and
between the particles examined. However, patients who had a high ratio
for one particle tended to have equally high ratios for the other
particles, and a close correlation was observed between the SF/P ratios
for apo(a) and LDL-cholesterol (r = 0.63, Figure 3A
). A similar correlation was observed
between the SF/P ratio for apo(a) and triglycerides
(r = 0.61), total cholesterol
(r = 0.67), HDL-cholesterol
(r = 0.51), and albumin
(r = 0.62). In Figure 3A, a
lower SF/P ratio for
apo(a) was apparent for patients with OA (16 ± 4%), as compared
with patients suffering from RA or other inflammatory types of
arthritis (37 ± 4%, P < 0.001). Moreover, the
SF/P ratio for the particles examined rose in inverse proportion with
their size (Figure 3B)
, ranging from 27 ± 3% (range, 3 to 61%)
for triglycerides (mainly transported by large very low-density
lipoprotein particles) to 32 ± 4% (2 to 100%) for apo(a)
[transported as Lp(a) particles], 42 ± 5% (1 to 100%) for LDL
particles, 52 ± 3% (3 to 80%) for HDL particles, and 62 ±
3% (33 to 97%) for albumin. This data indicated that the SF/P ratio
was mainly dependent on the type of disease (inflammatory
versus noninflammatory) and the size of the particles
examined.
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To determine whether apo(a) is also present in human synovial
tissues, we quantitated apo(a) in homogenized synovial tissue collected
from seven patients with OA and seven patients with RA (Figure 4A)
. In this limited sample, the amount
of apo(a) in synovial tissues ranged from 52 to 1690 pmol/mg of
protein, and seemed to be higher in synovial tissues of patients with
RA compared to OA (897 ± 194 pmol/mg protein versus
182 ± 26 pmol/mg protein, P = 0.002).
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. As
positive controls, human and apo(a)-transgenic mouse liver mRNA was
used. In these two samples, apo(a) transcripts were found; in contrast,
apo(a) transcripts were undetectable in the human synovial tissues
examined, even after prolonged exposure of the gel. This data provided
strong evidence that the vast majority, if not all, apo(a) detected in
synovial fluid and tissue was derived from circulating Lp(a). Apo(a) Co-Localizes with Fibrin in Human Inflammatory Arthritic Joints and in a Murine Model of Experimental Arthritis
Immunohistochemical analysis was then performed to examine the
localization of apo(a) in arthritic synovial tissue. A representative
result of this analysis performed on human RA synovial tissues is
illustrated in Figure 5
. Abundant amounts
of apo(a) were identified in vascular and perivascular areas, in
association with the extracellular matrix and in scattered cells,
possibly macrophages and foam cells (Figure 5A)
. The specificity of the
apo(a) immunoreactivity was confirmed by a pronounced attenuation of
the signal when adjacent sections were incubated with the anti-apo(a)
antibody preadsorbed with an Lp(a)-containing serum (Figure 5B)
. Fibrin
staining performed on an adjacent section revealed a distribution of
immunoreactivity similar to that of apo(a) (compare Figure 5, C and D
),
indicating that apo(a) co-localized with fibrin.
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and in synovial fluid (Figure 6C)
, and the localization of apo(a) in synovial tissue closely
resembled the pattern observed in human specimen. Moreover, we observed
that apo(a) in mouse synovial tissue equally co-localized with
fibrin(ogen), as was observed in humans [compare Figure 6E
, apo(a),
and 6F, fibrin]. The reaction was specific to apo(a), as confirmed by
the absence of signal when examining the arthritic knee from a mouse
that did not express apo(a) (Figure 6, B and D)
.
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To evaluate the functional impact of apo(a) on the development of
experimental arthritis, we next compared the severity of AIA in mice
that expressed (n = 20) or did not express
(n = 19) human apo(a). A similar abundance of
inflammatory cells in synovial tissue (infiltrate) and fluid (exudate)
and a similar cartilage damage were observed in both groups (Figure 7)
. Moreover, equivalent fibrin(ogen)
content was found in the two groups. Finally, the concentration of
D-dimers in the circulation, which reflects ongoing fibrinolysis, as
measured 10 days after induction of AIA, was similar between mice
expressing or nonexpressing apo(a) (3.8 ± 1.0 ng/ml
versus 4.7 ± 0.9 ng/ml, P = 0.20).
Taken together, this data indicated that, in this particular mouse
model, apo(a) did not impact significantly on the development of
experimental arthritis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apo(a) was originally identified in human plasma, where its function remains elusive. Identification of fragments of apo(a) in urine33 raised the hypothesis that apo(a) function may reside outside the circulation, ie, in the kidney or in the urinary tract. More recently, we have reported a strong association between apo(a) and apoE in the development of Alzheimer’s disease.35 The present demonstration that apo(a) is present in arthritic synovial fluid and tissue, a compartment where plasminogen has an important role and where the contribution of lipids has recently been highlighted, adds further support for a tissular role for apo(a).
Apo(a) in human synovial fluid was detected as a full-length glycoprotein. This is a somewhat unexpected finding, given the elastolytic activity present in synovial fluid,36 and the fact that apo(a) is sensitive to digestion by elastase in vitro.37 This suggests that, at least in the samples examined, the elastolytic activity in synovial fluid was sufficiently inhibited by anti-proteases and/or that apo(a) is rather insensitive, in vivo, to digestion. As such, the present data are in line with our previous observation, in which we showed that no apo(a) fragments were generated in the circulation during cardiopulmonary bypass38 or sepsis,32 two conditions characterized by marked release of elastase by polymorphonuclear cells.
Apo(a) is not the first apolipoprotein to be detected in synovial fluid, as the presence of apoA1, apoE, and apoB in this fluid has already been reported.39 However, the mechanism responsible for the presence of these apolipoproteins in synovial fluid remained poorly understood. Here, we show that the concentration of lipoprotein particles in synovial fluid relative to plasma was smaller in OA than in inflammatory types of arthritis, and that the SF/P ratio inversely correlates with the size of the particles. This observation strongly suggests that lipoproteins diffuse from the circulation into synovial fluid and that the permeability of this barrier is increased in inflammatory types of arthritis. Whether the increased amount of apolipoproteins and their accompanying lipids contribute to the development of inflammatory types of arthritis, whether quantitation of these parameters in synovial fluid will allow, in clinical practice, to better evaluate the severity of the inflammatory reaction associated with arthritis, and whether diffusion of Lp(a) particles and binding to fibrin depends on the size of the apo(a) isoforms, remain to be established.
In the particular mouse model used here, the presence of apo(a) was not shown to impact on the severity of experimental arthritis or on the relative amount of fibrin(ogen) within the joint. This does not formally rule out, however, a role for apo(a) in the pathophysiology of arthritis in humans. First, mice do not express apo(a), and may not have the accessory machinery to allow the development of apo(a) function. Next, the concentration of apo(a) in the joint (which could not be assayed for technical reasons because of the very limited amount of material available) may not be sufficient in the mice examined here to inhibit locally the rate of plasmin generation. The similar plasma D-dimer levels in apo(a)-expressing or nonexpressing mice are in favor of this possibility. Moreover, it is conceivable that apo(a) in the joint may have contrasting effects, and that the net balance between the pro- and anti-arthritic effects may vary between species, or according to age, type of arthritis, or at different stages of the disease. Finally, mice expressing this human apo(a) transgene may not be an adequate model to study the function of apo(a). Indeed, despite ample demonstration that apo(a) is atherogenic in humans, no accentuation by apo(a) of the development of atherosclerosis has been observed in transgenic mice expressing apo(a).28,29
To conclude, the presence of Lp(a) in human synovial fluid and tissue, the larger abundance of Lp(a) in synovial fluid from inflammatory arthritides, and the co-localization of apo(a) with fibrin in inflammatory synovial tissues suggest that, in humans, apo(a) may modulate locally the fibrinolytic activity and may thus contribute to the persistence of intra-articular fibrin and bone matrix degradation in inflammatory arthritis.
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Acknowledgements |
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Footnotes |
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Supported by the Swiss National Foundation for Scientific Research (no. 44971.95 to V. M. and no. 56710.99 to N. B.), The Octave Botnar and Placide Nicod Foundation, and The Kamillo Eisner Foundation.
Accepted for publication July 10, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chains. J Biol Chem 2000, 275:38206-38212This article has been cited by other articles:
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