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Combined effect of bradykinin B₂ and neurokinin-1 receptor activation on endothelial cell proliferation in acute synovitis ¹

Academic Rheumatology, University of Nottingham, City Hospital, Nottingham, UK

²Correspondence: E-mail: helene.seegers{at}univ-angers.fr

SPECIFIC AIM

Angiogenesis and neurogenic mechanisms facilitate inflammation in a variety of disorders, including arthritis. In the present study we have investigated the in vivo modulation of early endothelial cell proliferation by bradykinin and substance P using specific receptor antagonists in rat models of knee synovitis.

PRINCIPAL FINDINGS

First the endothelial proliferative potential of substance P (SP), bradykinin B₂ and B₁ receptor agonists (BK and [des-Arg⁹] BK respectively) were tested in rat knees. Then we investigated kinin receptor involvement in synovitis by using antagonists specific for NK₁ (SR140333), B₁ (SR240612A) or B₂ (FR172357) receptors and by assessing receptor mRNA expression.

1. Substance P increases synovial endothelial cell proliferation via both neurokinin NK₁ and bradykinin B₂ receptors
Intra-articular injection of substance P (SP, 10 nmol) was followed 24 h later by an increase in the percentage of endothelial cells that were proliferating (EC proliferation index) and an increase in macrophage infiltration (macrophage percentage area) in the synovium, as detected by immunohistochemistry for proliferating cell nuclear antigen and for clone ED1 respectively (Fig. 1 A, B). Co-administration of SP with either NK₁ or B₂ receptor antagonist (1 µmol/knee and 30 mg/kg respectively) abolished proliferative and inflammatory effects of SP injection (Fig. 1A, B ).

View larger version (49K): [in this window] [in a new window]   Figure 1. : Synovial endothelial cell proliferation and macrophage percentage area after intra-articular injection of substance P (SP, 10 nmol), or 3% carrageenan and 3% kaolin (CK). Endothelial cell (EC) proliferation index, detected using antibodies to proliferating cell nuclear antigen (PCNA) (A, C) and macrophage percentage area, detected using antibody clone ED1 (B, D) in synovia 24 h after intra-articular injection of normal saline (control), or SP or CK, either alone, or together with one or more selective receptor antagonists. Co-administered receptor antagonists were: intra-articular injection of SR140333 1 µmol (+NK1), oral administration of SR240612A 30 mg/kg (+B1) and/or oral administration of FR172357 50mg/kg (+B2). Data represent arithmetic means (+SEM) of 6 rats per group (except "+NK1&B2," n=12). Data for saline controls from the groups were combined, as antagonists had no effect on saline injected knees (SP group control n=18; CK group control n=42). Analysis was by Student’s t test with Duncan’s correction for multiple comparisons. * P < 0.05 compared with SP or CK-injected knees. # P < 0.05 compared with FR172357-treated knees (+B2).

2. Mild synovitis is required for bradykinin to increase endothelial cell proliferation via B₂ receptors
BK intra-articular injection alone (1000 nmol) did not produce any significant change in EC proliferation index (mean difference: – 8% [95% CI, –44% to +51%], n = 6) when compared with the appropriate saline injected knees. Bilateral injection of 0.03% carrageenan in the knee joint cavities increased macrophage infiltration fivefold over saline after 48 h (mean difference: +400% [95% CI, +50% to +1600%] n=6; P=0.005) without enhancing endothelial cell proliferation (mean difference: –12% [95% CI, –50% to +53%], n=6). B₁ or B₂ selective agonists ([des-Arg⁹] BK or BK respectively), or saline were injected into the right mildly inflamed knees 48h after bilateral carrageenan injection. EC proliferation index was dose-dependently increased 24h after injection of the B₂ agonist (BK 100 nmol, mean difference: +250% [95% CI, +50% to +800%], n=6; P<0.05), but not after the B₁ agonist ([des-Arg⁹] BK, 100 nmol, mean difference: –18% [95% CI, –41% to +12%], n=6) when compared with the mildly inflamed, saline injected knees.

3. In moderate synovitis endothelial cell proliferation is inhibited by the combined blockade of both NK₁ and B₂ receptors
Injection of a mixture of 3% carrageenan and 3% kaolin (CK) into the joint cavity increased EC proliferation index and macrophage infiltration after 24 h (Fig. 1C, D ). Attenuation of 27% of the CK-enhanced EC proliferation index was observed when CK was co-administrated with B₂ receptor antagonist alone (FR172357, Fig. 1C ). Co-administration of NK₁ and B₂ receptor antagonists (SR140333 and FR172357 respectively) resulted in a greater inhibition (64%) of CK-enhanced EC proliferation (Fig. 1C ). When CK was co-administrated with either NK₁ or B₁ receptor antagonist alone, no significant modification of endothelial cell proliferation was observed (Fig. 1C ). No significant modification of macrophage infiltration was observed 24 h after injection of receptor antagonists either alone or together (Fig. 1D ).

4. B₁ and B₂ receptor expression
B₂ receptor PCR product was detected at a moderate intensity level in synovia from all carrageenan-, CK- and saline-injected knees (Fig. 2 A). B₂ receptor expression did not significantly differ between groups. The B₁ receptor PCR product was undetectable in saline-injected control knees and weak but detectable expression was found in 5 of 8 carrageenan-inflamed knees. In contrast, a strong induction of B₁ receptor PCR product was obtained in every CK-inflamed synovium, as shown by the 213% increase in intensity observed when compared with the carrageenan injected group (Fig. 2B ).

View larger version (33K): [in this window] [in a new window]   Figure 2. : Bradykinin B2 and B1 receptor mRNA expression in mild and moderate synovitis. Band intensities and products of semiquantitative PCR for bradykinin B2 (A) and B1 (B) receptor mRNAs in rat synovia 24 h after intra-articular injection of 3% carrageenan and 3% kaolin (CK, moderate inflammation), normal saline or 0.03% carrageenan (Carr., mild inflammation). Normalized GAPDH PCR products for each sample are shown. All PCR products were separated on a 2% (w/v) agarose gel. Each analysis was performed twice and the resulting arithmetic means (+SEM) of n = 4 synovia for the CK and the saline injected knees and n = 8 synovia for the carrageenan injected knees were plotted. * P < 0.01 compared with carrageenan-injected knees. The comparison of PCR product band intensities between groups was assessed using a Mann-Whitney U-test.

CONCLUSIONS AND SIGNIFICANCE

Our findings demonstrate that administration of exogenous SP (10 nmol) or BK (100 nmol) each can enhance in vivo EC proliferation in synovium. SP was able to induce both EC proliferation and inflammation, whereas the proliferative effect of BK was dependent on the prior induction of inflammation by additional agents such as carrageenan. This requirement for mild inflammation may indicate the contribution of other endogenous inflammatory mediators, in addition to BK-mediated B₂ receptor activation. The proliferative effects of SP or BK each were inhibited by co-administration with B₂ receptor antagonist, suggesting a requirement for BK generation in SP-enhanced angiogenesis. A similar finding was observed in the CK synovitis model, where our data indicate that B₂ receptor activation contributes to EC proliferation. The B₁ receptor antagonist did not significantly inhibit EC proliferation in these studies, despite a strong induction of B₁ receptor mRNA expression in CK-induced synovitis. Other studies have indicated angiogenic and proinflammatory roles of B₁ receptors in established inflammation, although our data suggest that B₂ receptors mediate angiogenesis in the initial stages of synovitis.

Our data are consistent with generation or release of endogenous BK and SP during the moderate inflammation that is induced by CK, since anti-proliferative effects were observed both with B₂ receptor antagonist, and with NK₁ receptor antagonist. However, neurogenic mechanisms are probably not the predominant source of BK generation in CK-induced synovitis, as the NK₁ receptor antagonist inhibited EC proliferation only when administered together with B₂ receptor antagonist.

Neural enhancement of angiogenesis in CK-induced synovitis seems to be masked by the angiogenic effects of endogenously generated BK acting on B₂ receptors (Fig. 3 ). Where B₂ receptors are pharmacologically inhibited, high indices of CK induced-EC proliferation were maintained by the action of endogenous SP on NK₁ receptors. As a result, there is synergy between the anti-angiogenic effects of B₂ and NK₁ receptor antagonists in acute synovitis. This may be explained by the existence of common second messengers that mediate the enhancement of EC proliferation both by B₂ receptors and by NK₁ receptors (Fig. 3 ).

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic diagram of how endogenous substance P and bradykinin released during inflammation can have a concerted action to stimulate endothelial cell proliferation. Exogenous substance P (SP) can directly induce inflammation and endothelial cell (EC) proliferation via its vascular NK1 receptor that can be blocked by its antagonist (SR 140333). This neurogenic inflammation will result in the generation of bradykinin (BK), which stimulates EC proliferation in the inflamed synovium via its B2 receptor. In CK-induced synovitis, where endogenous peptides are released alongside other angiogenic factors, endothelial cell proliferation is again mediated by activation of the BK/B2 pathway. Although neurogenic mechanisms are not the predominant source of BK generation in CK-induced synovitis, SP is released into the synovium from endogenous sources. SP/NK1 receptor activation takes on a pro-angiogenic role when the B2 receptor is blocked by its antagonist (FR172357), suggesting a common second messenger. Thicker arrows indicate a greater contribution to EC proliferation in CK-induced synovitis.

In conclusion, combined blockade of B₂ and NK₁ receptors can substantially inhibit in vivo EC proliferation during the early stage of synovitis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0727fje; doi: 10.1096/fj.03-0727fje

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