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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 18, 2003 as doi:10.1096/fj.02-1193fje. |
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B1
,2
,3
* Institute of Pharmacology, Christian-Albrechts-University Kiel, D-24105 Kiel, Germany;
Institute of Anatomy, Christian-Albrechts-University Kiel, D-24098 Kiel, Germany; and
Institute of Pharmacology and Toxicology, Charité-Hospital, Humboldt University at Berlin, D-10117 Berlin; and German Institute for High Blood Pressure Research, Heidelberg, Germany
3Correspondence: Center for Cardiovascular Research, Institute of Pharmacology and Toxicology, Campus Charité Mitte, Charité-Universitätsmedizin Berlin, Hessische Str. 3-4, 10115 Berlin, Germany. E-mail: thomas.unger{at}charite.de
SPECIFIC AIMS
It has been reported that the AT2 receptor regulates several functions of nerve cells (e.g., ionic fluxes, cell differentiation, and axonal regeneration) but also modulates programmed cell death. In the present study we tested the hypothesis that angiotensin II (ANG II) via its AT2 receptor not only promotes regeneration, but also functional recovery after sciatic nerve crush in adult rats.
PRINCIPAL FINDINGS
1. ANG II-treated animals demonstrated an increase of the toe spread distance and an increase of functional recovery
On day 20 following nerve crush, ANG II-treated animals demonstrated an increase of the toe spread distance that was most visible at a concentration of 10-11 M. At 10-9 M, ANG II also enhanced toe spread distance although to a slightly lesser extend, whereas at 10-7 M ANG II had no effect on nerve regeneration. The NGF group as positive control also showed significantly enhanced toe spread distances compared with the saline-treated group. The effects of exogenous applied ANG II were abolished by coadministration of the AT2 receptor antagonist PD 123319 (10-7 M), reducing the regenerative response to that of the saline control group, while the AT1 receptor antagonist losartan (10-7 M) did not reduce the regenerative response. The combination of both receptor antagonists at the described concentration plus ANG II (10-11 M) had no accelerating effect on nerve regeneration (data not shown). Single treatment with the AT2 receptor antagonist PD 123319 (10-7 M) antagonized nerve regeneration whereas the AT1 receptor antagonist losartan (10-7 M) had no effect. Treatment with ANG II (10-11 M) significantly accelerated recovery of function. The response to local electrical stimulation was reduced to 14.6 days vs. 17.9 days in the control group. The two higher ANG II-concentrations (10-9 M and 10-7 M) were not effective. The NGF group also showed a significantly accelerated response. Cotreatment with the AT1 receptor antagonist losartan resulted in a significantly earlier positive foot withdrawal reflex comparable to the group treated with ANG II (10-11 M) alone, whereas rats cotreated with the AT2 receptor antagonist PD 123319 responded to the electric stimulus like those treated with saline. The combination of both antagonists plus ANG II had no effect on the return of sensorimotor function.
Again, the results for losartan or PD 123319 given alone corresponded to what was found for their combination with exogenously administered ANG II.
2. Light and electron microscopic as well as morphometric examination of the sections revealed markedly enhanced axonal regeneration and remyelination in ANG II-treated animals
Differences in functional recovery were reflected in the morphological appearance of the sciatic nerves 10 mm distal to the lesion site on day 20 after nerve crush. The effect of ANG II was lost in the presence of PD 123319, but not of losartan (Fig. 1
), focusing on an AT2 receptor-mediated effect. This data could be confirmed at the electron microscopic level and by morphometric analysis of axonal diameters in the sciatic nerve on day 20 after nerve crush.
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In ANG II (10-11 M) -treated animals, axonal caliber were significantly greater than with saline-treated controls. The axon caliber of animals cotreated with ANG II plus losartan were also significantly increased whereas ANG II combined with PD 123319 showed no difference compared with saline-treated controls.
Myelination was enhanced in ANG II (10-11 M) -treated animals. Animals cotreated with losartan plus ANG II showed increased mean areas whereas ANG II in combination with PD 123319 had no effect on myelination compared with saline treatment.
3. The enhanced remyelination in ANG II-treated animals could be mediated via the AT2 receptor and NF-
B signaling in Schwann cells
Finally, we determined whether NF-B is involved in the ANG II-mediated remyelination by Schwann cells, using Schwannoma cell cultures stimulated with ANG II in the presence or absence of selective receptor antagonists. Immunohistochemical analysis revealed that ANG II treatment induced activation of NF-B with subsequent translocation from the cytoplasm into the nucleus. To verify these results, we performed gel-shift analysis (Fig. 2
). ANG II induced an increase in NF-B binding activity. The intensity of the ANG II-induced NF-B complex was diminished in the presence of PD 123317, but not of losartan. Furthermore, cotreatment with ANG II plus losartan significantly increased the NF-B complex while combination of ANG II with PD 123319 showed no difference compared with saline-treated controls.
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CONCLUSIONS
In the present study, we noted that ANG II counteracts lesion-induced loss of function after sciatic nerve transection. The results extend our previous ex vivo observations that ANG II acts as a neurotrophic factor through its AT2 receptor and provide the first functional evidence for the regenerative ability of ANG II through its AT2 receptor.
Pharmaceutical application of ANG II to the crushed sciatic nerve increased the rate of axonal regeneration, the morphological equivalent being the presence of large, more myelinated regenerating axons. On the molecular level, AT2 receptor stimulation activates the transcription factor NF-B in Schwann cells, which could result in enhanced remyelination. Functional regeneration was also promoted: the animals demonstrated a faster return of function in the affected hind limb. The dose dependence studies revealed that the most effective ANG II concentration to promote axonal regeneration of sensory fibers was 10-11 M. This low concentration was consistently found to be the most effective regardless of the method used to assess nerve regeneration. The regeneration-accelerating effects of ANG II at the concentration of 10-11 M in the pumping fluid were comparable to those of NFG (positive control) applied at the concentration of 0.22 g/mL via the same route. Higher doses (10-7 and 10-9 M) of ANG II were less effective or ineffective in promoting nerve regeneration in all analyses. Thus, ANG II at appropriate concentrations might engender apoptosis or at least antagonize neuroregeneration, possibly through AT1 receptor activation. It is conceivable that the AT1 and AT2 receptor oppose each other with respect to their effects in cell differentiation and tissue repair and that the balance between their actions determines the net influence of ANG II in a given disease situation. Although exogenously applied ANG II mediates regenerative effects on lesioned peripheral nerves, suggestions about physiological relevance are worthless without knowledge of the expression and possible regulation of this molecule in the intact and regenerating peripheral nervous system.
Inhibition of the AT1 receptor per se enhances regeneration. The findings on the neurotrophic actions of the AT2 receptor may provide a basis for the design of new, receptor-directed, therapeutic concepts for the treatment of human peripheral nerve injuries or for preventing secondary damage in chronic disease, e.g., diabetic neuropathy. The clinical relevance of this approach is already apparent with the increasing use of AT1 receptor antagonists in treating hypertension, stroke, and diabetic nephropathy. Since AT2 receptors are unmasked and ANG II levels increased by AT1 receptor antagonists, part of the organ-protective actions of these drugs might be ascribed to an agonistic action of ANG II at the AT2 receptor site.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1193fje; doi: 10.1096/fj.02-1193fje 
2 K.R. and R.L. contributed equally to this work.
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