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

Anand U, Yiangou Y, Sinisi M, Fox M, MacQuillan A, Quick T, Korchev YE, Bountra C, McCarthy T, Anand P - Mol Pain (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Peripheral Neuropathy Unit, Centre for Clinical Translation, Hammersmith Hospital, Imperial College London, Area A, Ground Floor, Du Cane Rd, London, W12 ONN, UK. u.anand@imperial.ac.uk.

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.

No MeSH data available.


Related in: MedlinePlus

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).
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Fig2: 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).

Mentions: Serial sections of human peripheral nerve immunostained with antibodies to AngII (Figure 2a), and TRPV1 (Figure 2c) showed positive nerve fibres. AngII was present in injured human nerve fibres (Figure 2b), also shown with the structural nerve marker neurofilaments (Figure 2d). Image analysis (% area) of nerves immunostained with AngII antibodies showed no significant difference between control (n = 12) and injured (n = 13) human nerves (Figure 2e). AngII levels by ELISA were also similar in control human nerves (4.09 ± 0.36 pmol/g, n = 31), injured nerve trunks (3.99 ± 0.79 pmol/g, n = 7), and painful neuromas (3.43 ± 0.73 pmol/g, n = 12) (Figure 2f). AngIII-specific and Ang-(1-7) ELISA levels were undetectable (below <0.03 and <0.05 pmol/g respectively), indicating that the major angiotensin analogue in these human nerve tissue extracts is AngII.Figure 2


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

Anand U, Yiangou Y, Sinisi M, Fox M, MacQuillan A, Quick T, Korchev YE, Bountra C, McCarthy T, Anand P - Mol Pain (2015)

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).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4482278&req=5

Fig2: 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).
Mentions: Serial sections of human peripheral nerve immunostained with antibodies to AngII (Figure 2a), and TRPV1 (Figure 2c) showed positive nerve fibres. AngII was present in injured human nerve fibres (Figure 2b), also shown with the structural nerve marker neurofilaments (Figure 2d). Image analysis (% area) of nerves immunostained with AngII antibodies showed no significant difference between control (n = 12) and injured (n = 13) human nerves (Figure 2e). AngII levels by ELISA were also similar in control human nerves (4.09 ± 0.36 pmol/g, n = 31), injured nerve trunks (3.99 ± 0.79 pmol/g, n = 7), and painful neuromas (3.43 ± 0.73 pmol/g, n = 12) (Figure 2f). AngIII-specific and Ang-(1-7) ELISA levels were undetectable (below <0.03 and <0.05 pmol/g respectively), indicating that the major angiotensin analogue in these human nerve tissue extracts is AngII.Figure 2

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Peripheral Neuropathy Unit, Centre for Clinical Translation, Hammersmith Hospital, Imperial College London, Area A, Ground Floor, Du Cane Rd, London, W12 ONN, UK. u.anand@imperial.ac.uk.

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.

No MeSH data available.


Related in: MedlinePlus