Mechanisms underlying clinical efficacy of Angiotensin II type 2 receptor (AT2R) antagonist EMA401 in neuropathic pain: clinical tissue and in vitro studies

Abstract

Background: The clinical efficacy of the Angiotensin II (AngII) receptor AT2R antagonist EMA401, a novel peripherally-restricted analgesic, was reported recently in post-herpetic neuralgia. While previous studies have shown that AT2R is expressed by nociceptors in human DRG (hDRG), and that EMA401 inhibits capsaicin responses in cultured hDRG neurons, the expression and levels of its endogenous ligands AngII and AngIII in clinical neuropathic pain tissues, and their signalling pathways, require investigation. We have immunostained AngII, AT2R and the capsaicin receptor TRPV1 in control post-mortem and avulsion injured hDRG, control and injured human nerves, and in cultured hDRG neurons. AngII, AngIII, and Ang-(1-7) levels were quantified by ELISA. The in vitro effects of AngII, AT2R agonist C21, and Nerve growth factor (NGF) were measured on neurite lengths; AngII, NGF and EMA401 effects on expression of p38 and p42/44 MAPK were measured using quantitative immunofluorescence, and on capsaicin responses using calcium imaging.

Results: AngII immunostaining was observed in approximately 75% of small/medium diameter neurons in control (n = 5) and avulsion injured (n = 8) hDRG, but not large neurons i.e. similar to TRPV1. AngII was co-localised with AT2R and TRPV1 in hDRG and in vitro. AngII staining by image analysis showed no significant difference between control (n = 12) and injured (n = 13) human nerves. AngII levels by ELISA were also similar in control human nerves (4.09 ± 0.36 pmol/g, n = 31), injured nerves (3.99 ± 0.79 pmol/g, n = 7), and painful neuromas (3.43 ± 0.73 pmol/g, n = 12); AngIII and Ang-(1-7) levels were undetectable (<0.03 and 0.05 pmol/g respectively). Neurite lengths were significantly increased in the presence of NGF, AngII and C21 in cultured DRG neurons. AngII and, as expected, NGF significantly increased signal intensity of p38 and p42/44 MAPK, which was reversed by EMA401. AngII mediated sensitization of capsaicin responses was not observed in the presence of MAP kinase inhibitor PD98059, and the kinase inhibitor staurosporine.

Conclusion: The major AT2R ligand in human peripheral nerves is AngII, and its levels are maintained in injured nerves. EMA401 may act on paracrine/autocrine mechanisms at peripheral nerve terminals, or intracrine mechanisms, to reduce neuropathic pain signalling in AngII/NGF/TRPV1-convergent pathways.

Figure 1

IHC in human DRG tissues. Serial sections of post-fixed human avulsion injured DRG immunostained with rabbit antibodies to AngII (a) and TRPV1 (c). Arrows indicate co-localising cells. Similar serial sections of post-fixed human avulsion injured DRG immunostained with antibodies to AngII (b) and AT₂R (d). Arrows indicate co-localising cells. Scale bar 50 microns.

Figure 2

IHC in human nerve tissue. Serial sections of post-fixed human peripheral nerve immunostained with antibodies to AngII (a, b) showing co-localisation with TRPV1 (c) and the structural nerve marker Neurofilament (NF) (d). Scale bar 100 microns. Graph showing image analysis (% area) of AngII immunoreactivity in control (n = 12) and injured (n = 13) nerves expressed as a ratio to the structural nerve marker neurofilaments (e). Graph showing AngII levels by ELISA were similar in control human nerves (n = 31), injured human nerve trunks (n = 7), and painful human neuromas (n = 12) (f).

Figure 3

Immunofluorescence in cultured hDRG neurons. IF images of cultured hDRG neurons positive for the neuronal marker PGP9.5 (a, red), co-localised with AngII expression in small diameter neuron (yellow in merged image, b), but not in large neuron, and AngII expression alone (c, green). Bar 50 μM. IF images showing co-localization of Gap43 (green, d), AT₂R (red, e), and AngII (blue, f) in small diameter cultured hDRG neurons; merged AT₂R and AngII (g). Bar  20 μm. Co-localization of AT2R (red, h), TRPV1 (blue, i), AngII (green, j) and the merged image (k). Bar 10 μm.

Figure 4

pp42/44 and pp38 expression in cultured rDRG neurons. IF images of pp42/44 expression (upper panel), and pp38 expression (lower panel) in vehicle treated rDRG neurons (control 0), or treated with NGF, AngII, or AngII + EMA401 (a). Graph showing quantitation of signal intensity for pp42/44 (b), and pp38 expression (c).

Figure 5

Effect on neurite length and calcium imaging. Image of an AngII treated DRG neuron showing β tubulin immunofluorescent cell soma and neurites (a), bar 200 μm. Graph showing comparative neurite lengths of neurons treated with BSF2 (medium alone), and significant increase with AngII + NTFs, Ang II or EMA1087 (b). AngII mediated sensitization of hDRG neurons was reversed in the presence of the MAPK inhibitor PD98059 (c). The commercial AT₂R agonist EMA1087 (Compound 21) also caused significant sensitization of capsaicin responses. AngII mediated sensitization was abolished in the presence of PD98059, staurosporine, and TrkA inhibitor GW441756 (d).

Figure 6

Diagram indicating the pathway involved in AngII and TRPV1 signalling, with activation of p38 (MAPK) and p44/42 (ERK) and their inhibition by EMA401 in DRG neurons.