Abstract
Early angiogenesis is a key step in the transition from acute to persistent inflammation. The nervous system has long been known to play a role in inflammation, in part through the release of substance P from peripheral nerve terminals (neurogenic inflammation). Application of substance P can stimulate vessel growth in a variety of angiogenesis assays, although it was previously not known whether endogenous substance P released from sensory nerves could modulate angiogenesis. We hypothesized that endogenous substance P can initiate angiogenesis during acute neurogenic inflammation. Here we show that 10 nmol of substance P can stimulate angiogenesis within the rat knee synovium, as shown by increased endothelial cell proliferation index [PCNA index, 19% (95% confidence interval (CI), 17 to 20%)] compared with saline injected knees [6% (95% CI, 4% to 8%), p < 0.05]. Moreover, this was prevented by coadministration of an antagonist of the neurokinin-1 (NK1) subtype of neurokinin receptor SR140333 (nolpitantium), 1 μmol [8% (95% CI, 5% to 11%)]. Capsaicin 0.5%, which stimulates release of endogenous substance P from sensory nerves, was also found to enhance synovial angiogenesis, [PCNA index 17% (95% CI, 14% to 19%)] compared with saline injected control knees [2% (95% CI, 1% to 3%), p < 0.05], and this also was inhibited by 1 μmol of SR140333 [11% (95% CI, 8 to 16%)]. Inhibition of capsaicin-enhanced angiogenesis was incomplete, and this may indicate a contribution of other neuropeptides, in addition to substance P-NK1 receptor interactions, in capsaicin-enhanced angiogenesis. NK1 receptor antagonists could have therapeutic potential in conditions where neurogenic angiogenesis contributes to disease.
Angiogenesis, the growth of new vessels from a pre-existing vasculature, is a common feature of chronic inflammation (for review, see Weber and De Bandt, 2000; Walsh and Haywood, 2001) and synovitis (Walsh et al., 1998a). Using the rat model of carrageenan synovitis, we have previously shown that angiogenesis during the acute phase can predict persistent inflammation (Walsh et al., 1998a). Understanding and targeting early angiogenesis may then be of potential benefit for the treatment of chronic inflammation diseases.
Sensory nerves and nerve-derived neuropeptides have been implicated in peripheral inflammation (for review, see Holzer, 1988) and involvement of substance P in joint inflammation has been extensively studied (for review, see Garrett et al., 1992). Substance P is released from the peripheral terminals of fine unmyelinated sensory nerves in vivo, where it mediates acute neurogenic inflammation (for review, see Richardson and Vasko, 2002). Its release can also be stimulated by application of capsaicin, the pungent component of chili pepper (Szallasi and Blumberg, 1999). Through its primary action on tachykinin NK1 receptors on the surface of vascular endothelial cells, substance P induces vascular permeability, plasma extravasation, and edema, i.e., neurogenic inflammation (Eglezos et al., 1991; Xu et al., 1992). Substance P released from endogenous sources has not previously been reported to enhance angiogenesis, however.
Substance P-enhanced endothelial cell proliferation and in vivo angiogenesis are mimicked by selective NK1 receptor agonists and inhibited by antagonists of neurokinin receptors (Ziche et al., 1990; Fan et al., 1993; Ziche et al., 1994). Substance P may also stimulate neurokinin-2 receptors (NK2) in some peripheral tissues, although it binds NK2 receptors with lower affinity than NK1 receptors. Specific, nonpeptide antagonists of neurokinin receptors have been developed, of which SR140333 and SR144190 display high affinity, stereoselectivity, and receptor subtype specificity at rat NK1 and NK2 receptors, respectively (Emonds-Alt et al., 1993; Oury-Donat et al., 1994; Emonds-Alt et al., 1997). The effects of these have not been tested on neurogenic angiogenesis, however.
We have investigated the effects of exogenous and endogenous substance P on angiogenesis in the rat knee, the receptor subtypes that mediate these effects, and the ability of antagonists of these receptors to inhibit angiogenesis during a model of synovitis. We hypothesized that endogenous substance P initiates angiogenesis during the early phase of synovitis through an action on NK1 receptors.
Materials and Methods
Experiments, licensed under the UK Home Office regulations, were performed on male Wistar rats (180 g).
Animal Models. On day 0, rats were injected in the right knee with substance P in 0.9% normal saline or 0.5% capsaicin in 10% ethanol, 10% Tween 80, and 80% normal saline (v/v/v) (Mapp et al., 1996), each either alone, or mixed and injected together with pharmacological agents. Neurokinin receptor antagonists and inactive enantiomers were dissolved in ethanol to a stock concentration of 100 mM and then diluted in normal saline to give a final ethanol concentration ≤10% (v/v). Preliminary controls with the capsaicin vehicle demonstrated no increase in angiogenesis (data not shown). Left knees were injected with normal saline alone, which does not increase indices of angiogenesis, macrophage infiltration, or knee diameter at 24 h compared with naive animal knees (Walsh et al., 1998a). Volumes for all intra-articular injections were 100 μl, performed under neuroleptanalgesia [0.15 ml kg–1 Hypnorm (0.315 mg ml–1 fentanyl citrate, 10 mg ml–1 fluanisone); Janssen-Cilag Ltd., High Wycombe, UK]. Knee diameters (millimeters) were measured with a digital electronic caliper (Mitutoyo UK Ltd., Andover, UK).
Tissue Collection and Preparation. Twenty four hours after intra-articular injection, rats were killed by asphyxiation in carbon dioxide, and the right and left knee synovia with patellae were immediately harvested. Synovia were embedded in Tissue Tek (Miles, Inc., Elkhart, IN), frozen in melting isopentane onto cork block mounts, and stored at –80°C until use.
Staining Procedures. Multiple sequential staining was used to identify endothelial proliferation. Sections (4 μm) were first immunostained for proliferating cell nuclear antigen (PCNA) using monoclonal antibody clone PC10 and then for endothelium using a monoclonal antibody directed against CD31 (Waseem and Lane, 1990; Male et al., 1995). Nuclei were counterstained by immersion of sections in the fluorescent DNA ligand, 4′-6′-diamidino-2-phenylindole hydrochloride (DAPI) (Sanna et al., 1992). Macrophage infiltration was identified by immunoreactivity for the monoclonal antibody clone ED1 (Dijkstra et al., 1985). Staining procedures were similar to those previously described (Walsh et al., 1998a). Proliferation markers and ED1 were developed with diaminobenzidine using the glucose oxidase/nickel-enhanced method. Endothelium markers were developed using Sigma-Aldrich Fast Red (Poole, UK). H&E stains were performed on sections consecutive to those used for immunohistochemistry to assist in identification of tissue structures.
Quantification. Quantification was performed by an observer blinded to experimental details using a Zeiss microscope (Carl Zeiss GmbH, Jena, Germany) with a 16× objective lens. Transmitted light and fluorescence images of the same field were each captured using a 3-CCD camera and analyzed using a KS300 image analysis system (Imaging Associates Ltd., Thame, UK).
Synovium was delineated according to morphology, and synovial area was measured. Within this synovial region, a mask of the endothelial area was created that included CD31-positive blood vessels. This endothelial mask was applied over corresponding images of PCNA-positive and DAPI-positive nuclei. Nuclei falling within endothelium were counted as PCNA-positive endothelial nuclei and total endothelial nuclei, respectively.
Endothelial cell PCNA index was defined as the percentage of endothelial nuclei positive for PCNA. Vascular density was defined as the percentage of synovial area immunoreactive for endothelium and the macrophage percentage area as the percentage of synovial area that was ED1-positive.
Data Analysis. The optimum number of fields per section and sections per case were determined in previous experiments (Walsh et al., 1998a). Data are presented on six knees per experimental group, except for data on vascular densities and effect of NK1 receptor antagonist on macrophage infiltration (n = 12 knees/group).
For statistical analysis, endothelial cell PCNA indices and endothelial and macrophage percentage areas were logarithmically transformed. All data were analyzed using one-way analysis of variance. Univariate comparisons were made using Student's t test with Duncan's correction for multiple comparisons. Numerical data are quoted as means [95% confidence interval] in the text and arithmetic means ± S.E.M. in the figures.
Materials. Monoclonal antibody to PCNA (clone PC10) was obtained from DAKO Ltd. (High Wycombe, UK). Biotinylated ratadsorbed horse anti-mouse antibody and avidin-biotin complexes were obtained from Vector Laboratories Ltd. (Peterborough, UK). Monoclonal antibodies to rat CD31 (clone TLD-3A12) and to macrophages (clone ED1) were from Serotec Ltd. (Oxford, UK). Substance P, capsaicin, DAPI, bovine serum albumin, and other chemicals were obtained from Sigma-Aldrich.
Results
PCNA Index. Synovia from saline-injected knees displayed low levels of endothelial cell proliferation (Fig. 1). There was a dose-dependent increase in endothelial cell PCNA index in synovia 24 h after injection of substance P (Fig. 1 and 2). For all the experiments with administration of 10 nmol of substance P, the mean endothelial cell PCNA index was 19% [95% CI, 17 to 20%] and was higher than in the saline injected knee [6% (95% CI, 4 to 8%), p < 0.05]. Intra-articular injection of 0.5% capsaicin was also followed by an increase in endothelial cell PCNA index [17% (95% CI, 14 to 19%)] compared with saline injected control knees [2% (95% CI, 1 to 3%), p < 0.05].
Coinjection of the NK1 receptor antagonist SR140333 (0.01 to 1 μmol) together with 10 nmol of substance P or 0.5% capsaicin dose dependently attenuated the increases in endothelial cell PCNA indices (Figs. 3 and 4). At the highest dose of SR140333 (1 μmol), endothelial cell PCNA indices were lower than in synovia from knees that had been injected with either substance P or capsaicin alone. The inactive enantiomer of SR140333, SR140603 (1 μmol), did not significantly attenuate the increased endothelial cell PCNA index observed 24 h after injection of substance P or capsaicin (Figs. 3 and 4).
Coinjection of the neurokinin-2 receptor antagonist SR140190 (1 μmol) did not significantly attenuate the increased endothelial cell PCNA index observed 24 h after injection of substance P [18% (95% CI, 13 to 23%)]. In the absence of substance P or capsaicin, intra-articular injection of either NK1 or NK2 receptor antagonist did not affect endothelial cell PCNA indices when compared with saline-injected control knees [7% (95% CI, 4 to 10%)] and [10% (95% CI, 6 to 14%)] respectively.
Vascular Densities (Endothelial Percentage Areas). Synovia from saline-injected knees were highly vascularized. The mean vascular densities in saline injected knees for each experiment was in the range 3.8 to 5.8%. Vascular densities were higher 24 h after intra-articular injection of substance P (10 nmol) [6.2% (95% CI, 5.0 to 7.6%)] or 0.5% capsaicin [6.5% (95% CI, 5.9 to 7.2%)] than after saline injection [4.5% (95% CI, 3.1 to 5.7%), p < 0.05] or [5.2% (95% CI, 4.5 to 6.0%), p = 0.01], respectively.
Inflammation (Macrophage Infiltration and Knee Diameter). Intra-articular injections of substance P (10 nmol) or 0.5% capsaicin induced significant increases in knee joint diameter and macrophage infiltration (ED1 percentage area) at 24 h compared with saline-injected knees (Table 1).
Coinjection of either NK1 or NK2 antagonists (SR140333 or SR140190, respectively) or the inactive enantiomer of SR140333, SR140603 (1 μmol each), did not significantly attenuate the increased diameter or macrophage infiltration observed 24 h after injection of substance P or capsaicin (Table 1).
Discussion
In this study, we have shown that exogenous application of substance P (10 nmol) and capsaicin (0.5%) each enhanced endothelial cell proliferation and increased vascular density, which could be inhibited by the coadministration of specific NK1 receptor antagonist. Collectively our results strongly suggest that the effect of endogenous substance P on NK1 receptors is an important component of the neurogenic enhancement of angiogenesis in rat knee synovitis.
Normal synovium has evolved to be one of the most highly vascular tissues in the body to meet the metabolic requirements of the avascular articular cartilage. In inflamed synovia, edema, cellular hyperplasia, and vascular regression blunt the increase in vascular density that would otherwise accompany synovial angiogenesis (Stevens et al., 1991; Walsh et al., 1998a,b). We have found that endothelial cell PCNA index is a more sensitive measure of synovial angiogenesis than is vascular density, and its use increases statistical power for demonstrating effects of interventions (Walsh et al., 1998a,b).
Enhancement of angiogenesis by substance P was dose dependently and, at the highest dose, completely inhibited by the nonpeptide NK1 receptor antagonist SR140333 but not by its inactive enantiomer SR140603 or by the NK2 receptor antagonist SR144190. These findings are consistent with a proangiogenic action of substance P via the NK1 receptor. We have previously localized NK1 receptors by quantitative in vitro receptor autoradiography to microvascular endothelium in normal rat and human synovium (Walsh et al., 1992, 1993). Substance P and selective NK1 receptor agonists enhance endothelial cell proliferation in vitro, suggesting that this may be a direct effect (Ziche et al., 1990, 1994). Mediation of substance P-enhanced angiogenesis by NK1 receptors was also suggested by the use of the NK1 receptor antagonist RP67580 (Fan et al., 1993). Some nonpeptide NK1 receptor antagonists, such as RP67580, have been found to also inhibit calcium channels (Schmidt et al., 1992; Rupniak et al., 1993). This has limited the ability of some early studies to predict NK1 receptor-mediated effects (Rupniak et al., 1993). NK1 receptor antagonism is stereospecific, whereas calcium channels are inhibited by both stereoisomers with equal affinity. We found that SR140609, the enantiomer of SR140333, did not inhibit substance P- and capsaicin-enhanced angiogenesis. Antiangiogenic effects of NK1 receptor antagonists therefore are unlikely to be mediated by calcium channel blockade.
Intra-articular injection of capsaicin was followed by an increase in synovial angiogenesis, as indicated both by increased endothelial cell proliferation and increased vascular density. Capsaicin induces release of neuropeptides, including substance P, from the peripheral terminals of fine, unmyelinated sensory nerves (Theriault et al., 1979). Capsaicin-enhanced endothelial cell PCNA index was dose dependently inhibited by the NK1 receptor antagonist SR140333 but not by its inactive enantiomer. This suggests that the increase in angiogenesis in this model is mediated, in part, by endogenous substance P acting through NK1 receptors.
Inhibition of capsaicin-enhanced angiogenesis was incomplete, even at the highest dose of NK1 receptor antagonist that was found to completely inhibit substance P-enhanced angiogenesis. This may indicate a contribution of other mechanisms in addition to substance P-NK1 receptor interactions in capsaicin-enhanced angiogenesis. Calcitonin gene-related peptide may also have angiogenic activity and is coreleased with substance P from sensory nerves following exposure to capsaicin (Haegerstrand et al., 1990; Villablanca et al., 1994; Grant et al., 2002). Moreover, sustained release of substance P beyond the duration of action of coadministered antagonists and non-neurogenic effects may also contribute to capsaicin-enhancement of synovial angiogenesis.
Intra-articular injection of substance P or capsaicin was followed by joint swelling and synovial macrophage infiltration, indicative of acute and chronic inflammation. This suggests another potential role for neurogenic mechanisms in chronic inflammation. Our data indicate that macrophage infiltration in these models is mediated, at least in part, by pathways that do not require the NK1 receptor.
In conclusion, our findings indicate that substance P, either exogenously applied or released from endogenous sources, can stimulate synovial angiogenesis through an action on NK1 receptors. Selective NK1 receptor antagonists can inhibit angiogenesis under these circumstances. Further work is required to determine the contributions of substance P and NK1 receptors to angiogenesis in specific pathologies.
Footnotes
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This work was supported by Arthritis Research Campaign Project Grant W584 (Sanofi-Synthelabo, France) for the neurokinin-1 and -2 antagonists. H.S. is supported by the Marie Curie Fellowship.
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DOI: 10.1124/jpet.103.050013.
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ABBREVIATIONS: NK1, neurokinin-1; PCNA, proliferating cell nuclear antigen; DAPI, 4′-6′-diamidino-2-phenylindole hydrochloride; CI, confidence interval; SR140333, nolpitantium; SR140333,1-[2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidi-n-3-yl]ethyl]-4-phenyl-1-azoniabicyclo[2.2.2]octane, chloride; SR144190, (R)-3-(1-[2-(4-benzoyl-2-(3,4-difluorophenyl)-morpholin-2-yl)-ethyl]-4-phenylpiperidin-4-yl)-1-dimethylurea; SR160603, R enantiomer of SR140333; RP-67580 {2-[1-imino-2-(2-methoxyphenyl)ethyl-7,7-diphenyl-4-perhydroisoindolone(3aR,7aR).
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↵1 Current address: AstraZeneca R&D, Charnwood, Loughborough, LE11 5RH, UK.
- Received February 4, 2003.
- Accepted March 14, 2003.
- The American Society for Pharmacology and Experimental Therapeutics