P.I. Mapp

Angiogenesis and nerve growth factor at the osteochondral junction in rheumatoid arthritis and osteoarthritis

Objectives. The osteochondral junction can be a source of pain in both RA and OA. Growth of blood vessels and nerves from the subchondral bone into articular cartilage may mediate the association between joint pathology and symptoms. We have investigated associations between angiogenesis, inflammation and neurovascular growth factor expression at the osteochondral junction in human arthritis. Methods. Osteochondral junctions from medial tibial plateaux of patients undergoing arthroplasty for RA (n = 10) or OA (n = 11), or from non-arthritic post-mortem controls (n = 11) were characterized by immunohistochemistry for CD34 and smooth muscle α-actin (blood vessels), CD68 (macrophages), CD3 (lymphocytes), proliferating cell nuclear antigen, vascular endothelial, platelet-derived and nerve growth factor (NGF). Results. Osteochondral angiogenesis was demonstrated as increased endothelial cell proliferation and vascular density in non-calcified articular cartilage, both in RA and OA. Osteochondral angiogenesis was associated with subchondral bone marrow replacement by fibrovascular tissue expressing VEGF, and with increased NGF expression within vascular channels. RA was characterized by greater lymphocyte infiltration and PDGF expression than OA, whereas chondrocyte expression of VEGF was a particular feature of OA. NGF was observed in vascular channels that contained calcitonin gene-related peptide-immunoreactive sensory nerve fibres. Conclusions. Osteochondral angiogenesis in RA and OA is associated with growth factor expression by cells within subchondral spaces, vascular channels and by chondrocytes. NGF expression and sensory nerve growth may link osteochondral angiogenesis to pain in arthritis.

Mechanisms and targets of angiogenesis and nerve growth in osteoarthritis

During osteoarthritis (OA), angiogenesis is increased in the synovium, osteophytes and menisci and leads to ossification in osteophytes and the deep layers of articular cartilage. Angiogenic and antiangiogenic factors might both be upregulated in the osteoarthritic joint; however, vascular growth predominates, and the articular cartilage loses its resistance to vascularization. In addition, blood vessel growth is increased at—and disrupts—the osteochondral junction. Angiogenesis in this location is dependent on the creation of channels from subchondral bone spaces into noncalcified articular cartilage. Inflammation drives synovial angiogenesis through macrophage activation. Blood vessel and nerve growth are linked by common pathways that involve the release of proangiogenic factors, such as vascular endothelial growth factor, β-nerve growth factor and neuropeptides. Proangiogenic factors might also stimulate nerve growth, and molecules produced by vascular cells could both stimulate and guide nerve growth. As sensory nerves grow along new blood vessels in osteoarthritic joints, they eventually penetrate noncalcified articular cartilage, osteophytes and the inner regions of menisci. Angiogenesis could, therefore, contribute to structural damage and pain in OA and provide potential targets for new treatments.

Contributions of angiogenesis to inflammation, joint damage, and pain in a rat model of osteoarthritis

OBJECTIVE To determine the contributions of angiogenesis to inflammation, joint damage, and pain behavior in a rat meniscal transection model of osteoarthritis (OA). METHODS OA was induced in male Lewis rats (n=8 per group) by meniscal transection. Animals were orally dosed with dexamethasone (0.1 mg/kg/day), indomethacin (2 mg/kg/day), or the specific angiogenesis inhibitor PPI-2458 (5 mg/kg every other day). Controls consisted of naive and vehicle-treated rats. Synovial inflammation was measured as the macrophage fractional area (expressed as the percentage), thickness of the synovial lining, and joint swelling. Synovial angiogenesis was measured using the endothelial cell proliferation index and vascular density. Channels positive for vessels at the osteochondral junction were assessed (osteochondral angiogenesis). Medial tibial plateaus were assessed for chondropathy, osteophytosis, and channels crossing the osteochondral junction. Pain behavior was measured as weight-bearing asymmetry. RESULTS Dexamethasone and indomethacin each reduced pain behavior, synovial inflammation, and synovial angiogenesis 35 days after meniscal transection. Dexamethasone reduced, but indomethacin had no significant effect on, the total joint damage score. PPI-2458 treatment reduced synovial and osteochondral angiogenesis, synovial inflammation, joint damage, and pain behavior. CONCLUSION Our findings indicate that synovial inflammation and joint damage are closely associated with pain behavior in the meniscal transection model of OA. Inhibition of angiogenesis may reduce pain behavior both by reducing synovitis and by preventing structural change. Targeting angiogenesis could therefore prove useful in reducing pain and structural damage in OA.

Increased vascular penetration and nerve growth in the meniscus: a potential source of pain in osteoarthritis

Objectives Meniscal damage is a recognised feature of knee osteoarthritis (OA), although its clinical relevance remains uncertain. This study describes vascular penetration and nerve growth in human menisci, providing a potential mechanism for the genesis of pain in knee OA. Methods Menisci obtained post mortem were screened on the basis of high or low macroscopic tibiofemoral chondropathy as a measure of the presence and degree of OA. Forty cases (20 per group) were selected for the study of meniscal vascularity, and 16 (eight per group) for the study of meniscal innervation. Antibodies directed against α-actin and calcitonin gene-related peptide (CGRP) were used to localise blood vessels and nerves by histochemistry. Image analysis was used to compare vascular and nerve densities between groups. Data are presented as median (IQR). Results Menisci from knees with high chondropathy displayed degeneration of collagen bundles in their outer regions, which were more vascular than the inner regions, with an abrupt decrease in vascularity at the fibrocartilage junction. Vascular densities were increased in menisci from the high compared with low chondropathy group both in the synovium (3.8% (IQR 2.6–5.2), 2.0% (IQR 1.4–2.9), p=0.002) and at the fibrocartilage junction (2.3% (IQR 1.7–3.1), 1.1% (IQR 0.8–1.9), p=0.003), with a greater density of perivascular sensory nerve profiles in the outer region (high chondropathy group, 144 nerve profiles/mm2 (IQR 134–189); low chondropathy group, 119 nerve profiles/mm2 (IQR 104–144), p=0.049). Conclusion Tibiofemoral chondropathy is associated with altered matrix structure, increased vascular penetration, and increased sensory nerve densities in the medial meniscus. The authors suggest therefore that angiogenesis and associated sensory nerve growth in menisci may contribute to pain in knee OA.

Monoarthritis in the rat knee induces bilateral and time-dependent changes in substance P and calcitonin gene-related peptide immunoreactivity in the spinal cord.

Bilateral changes in the spinal cord and dorsal root ganglion content of the sensory peptides substance P and calcitonin gene-related peptide have been previously reported in animal models of arthritis which affect many joints within the body. The central nervous system has been implicated in the symmetry of joint involvement in human rheumatoid arthritis. We aimed to determine whether unilateral inflammation of the knee joint can also induce bilateral changes in the spinal cord. We have induced a monoarthritis in the knee joint of the rat and used quantitative immunocytochemistry to look at changes of these peptides in the dorsal horn of the spinal cord and the dorsal root ganglia. Furthermore we have examined the responses during the acute (three days) and the chronic (21 days) phases of the model. The data show that in the acute phase of the monoarthritis there is both an ipsilateral and contralateral response which increases the immunoreactive substance P and calcitonin gene-related peptide in the L4 level of the dorsal horn of the spinal cord. In the chronic phase of the monoarthritis, the contralateral side of the dorsal horn returned to control values whilst the ipsilateral side showed reduced amounts of immunoreactive substance P and calcitonin gene-related peptide compared to controls. We propose that the acute response, at three days, to unilateral inflammation is appropriate and has evolved to protect an organism against the original insult ipsilaterally, and the possibility of subsequent insult contralaterally.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of calcitonin gene-related peptide on formation of intra-articular oedema by inflammatory mediators.

1 The temporal and quantitative effects of inflammatory mediators on plasma extravasation in the rat knee were investigated by use of a perfusion technique. 2 Intra‐articular perfusion of substance P (SP), bradykinin or histamine over a 5 min test period produced rapid‐onset and prolonged plasma extravasation in a dose‐dependent fashion. The rank order of potency was bradykinin > SP > histamine. 3 Calcitonin gene‐related peptide (CGRP) did not induce plasma extravasation but enhanced substance P‐induced plasma extravasation in a dose‐dependent fashion. A 5 min co‐perfusion of the two agents produced short‐term enhancement lasting 10 min while continuous co‐perfusion produced enhancement for the duration of the perfusion. 4 A 5 min perfusion of CGRP enhanced plasma extravasation when co‐perfused with bradykinin but not histamine. However, when CGRP and histamine were continuously co‐perfused over a 20 min test period, an enhanced response was apparent. 5 The results indicate that intra‐articular perfusion of CGRP enhances synovial plasma extravasation induced by agents that increase vascular permeability, but suggest that the response is not uniform and is critically dependent on the duration of perfusion within the joint.

Effects of a metalloproteinase inhibitor on osteochondral angiogenesis, chondropathy and pain behavior in a rat model of osteoarthritis

Summary Objective To investigate the effects of a matrix metalloproteinase (MMP) inhibitor on joint pathology and pain behavior in the rat meniscal transection (MNX) model of osteoarthritis (OA) and evaluate which aspects of structural disease modification contribute to symptom improvement. Methods OA pathology was induced in male Lewis rats, by transecting the medial collateral ligament with (MNX) or without (SHAM) a full thickness cut through the meniscus. MNX animals were orally administered an equipotent MMP 2, 8, 9, 12, 13 inhibitor (0.25, 1 and 5 mg/kg/day) or vehicle from day 1. Chondropathy, osteophytosis, osteochondral vascularity were assessed from toluidine blue stained coronal sections of the total knee joint and weight-bearing asymmetry by incapacitance. Group differences were evaluated using 1-way analysis of variance (ANOVA) and associations as Spearmans correlation coefficients. Results Treatment with the MMP inhibitor reduced weight-bearing asymmetry from day 14 onwards, and attenuated chondropathy (both P < 0.05). Osteochondral vascularity was elevated in MNX compared with SHAM-operated animals (P < 0.001) and reduced, dose-dependently, by MMP inhibitor treatment (r = −0.89, P < 0.05). Reduced osteochondral vascularity and chondropathy were associated with the amelioration of weight-bearing asymmetry (both P < 0.05). Conclusion Here we show that treatment with a MMP inhibitor reduces joint damage, osteochondral angiogenesis and behavioral evidence of pain. The association between osteochondral angiogenesis and pain behavior may be explained by perivascular nerve growth or stimulation of subchondral nerves following loss of osteochondral integrity. Our data suggest that targeting angiogenesis may have utility in the treatment of pain associated with structural damage in OA.

Localisation and characterisation of substance P binding to human synovial tissue in rheumatoid arthritis.

The neuropeptide substance P is found in perivascular and free unmyelinated nerve fibres in human synovial tissue. Quantitative receptor autoradiography was used to show specific, high affinity (Kd = 0.75 (0.21), nmol/l (mean (standard error of the mean)), low capacity (Bmax = 27.8 (7.9) amol/mm2) binding sites for substance P Bolton Hunter-labelled with iodine-125 localised to vascular endothelial cells in human synovial tissue. The binding could be saturated, was reversible, and was dependent on the magnesium concentration. Unlabelled substance P and neurokinin A competitively inhibited specific binding with 50% inhibition at concentrations of 1.25 (0.21) and 175 (29) nmol/l respectively. Neurokinin B (mumol/l) and calcitonin gene related peptide (1 mumol/l) did not inhibit binding. These binding sites show characteristics of the neurokinin 1 tachykinin receptor subtype. This provides further evidence that substance P may play a part in the vascular control of human synovium and may influence inflammatory processes in joints.

Structural Associations of Symptomatic Knee Osteoarthritis

Structural changes of osteoarthritis (OA) may occur in the absence of pain. In this study, we aimed to identify histopathologic features that are associated with symptomatic knee OA.

Neurogenic influences on contralateral responses during experimental rat monoarthritis

Many inflammatory conditions show topographically precise symmetrical responses. In this study we assessed vascular and cellular responses of apparently normal knees following induction of monoarthritis on the opposite side. A strictly localised monoarthritis was induced in the right knee of experimental animals using intra-articular latex spheres. In both knee joints bradykinin-induced plasma extravasation was significantly enhanced increasing from 0.52 +/- 0.07 micrograms/ml Evans blue to 0.99 +/- 0.07 micrograms/ml and 0.88 +/- 0.1 micrograms/ml in the injected and uninjected, contralateral, knees respectively (P < 0.05). A bilateral increase in cellularity was also apparent with cell counts in the uninjected, and apparently normal, knee increasing from 512 +/- 42 cells/mm2 to a maximum of 812 +/- 125 cells/mm2 on day 10 (P < 0.05). Immunohistological analysis demonstrated that the infiltrating cells in both the ipsilateral and contralateral joints were predominantly macrophages. Cell counts were not increased in the other peripheral joints. Levels of the sensory neuropeptide substance P were significantly elevated in both the ipsilateral and contralateral dorsal root ganglia and prior inhibition of small unmyelinated nerve activity inhibited the cellular infiltrate on the contralateral side, suggesting that the effect was mediated, at least partially, by a specific neurogenic pathway. The data suggests the presence of a neurogenic mechanism able to induce a topographically precise response. This may serve to upregulate the cellular defences of at-risk tissues following a potentially damaging stimulus at another site.