Elsevier

Regulatory Peptides

Volume 107, Issues 1–3, 15 July 2002, Pages 105-113
Regulatory Peptides

Vasodilator effect of angiotensin-(1–7) in mature and sponge-induced neovasculature

https://doi.org/10.1016/S0167-0115(02)00070-8Get rights and content

Abstract

Angiotensin-(1–7) (Ang-(1–7)), a peptide constituent of the renin–angiotensin system, has been shown to act as a vasodilator mediator in pre-existing (skin) and newly formed vasculatures (14-day-old sponge implants). Blood flow was determined by the outflow rate of sodium fluorescein applied intradermally or intraimplant and the results were expressed in t1/2 values (time taken for the fluorescence to reach 50% of the peak in the systemic circulation). We showed that the t1/2 value was significantly lower (4.1±0.46) in the implants compared with the cutaneous vasculature (5.7±0.5). Ang-(1–7) 20 ng was able to decrease t1/2 values in both vasculatures. The specific receptor antagonist, d-Ala7-Ang-(1–7) (A-779), prevented Ang-(1–7)-induced vasodilation and altered the basal vascular tone of the implants. The vasodilator effect was also abolished by nitric oxide (NO) synthase inhibitors in both vasculatures and by indomethacin in the implant. Selective AT1 and AT2 receptor antagonists did not alter the vasodilation induced by the peptide. These results establish the vasodilator effect of Ang-(1–7) in the cutaneous and implant vasculature and that the peptide is produced endogenously by the fibrovascular tissue, and suggest that this peptide contributes for the vasodilation found in newly formed vascular beds (wound healing, chronic inflammatory processes and tumors).

Introduction

Angiogenesis is one of the main components in tumor development, chronic inflammation and healing processes. The neovasculature induced in these conditions differs from the mature vasculature regarding morphological, biochemical and functional aspects. Newly formed blood vessels are highly permeable, vasodilated and have scarce basement membrane [1], [2]. A redundancy of vasomediators (bradykinin, angiotensin II, histamine, prostaglandins (PGs), substance P, nitric oxide) produced by activated endothelial and inflammatory cells have been identified in tissues undergoing neovascularization as seen during wound healing, chronic inflammation and tumor growth. These vasoactive substances are also able to modulate angiogenesis in several experimental models [3], [4], [5], [6], [7], [8].

Angiotensin-(1–7) (Ang-(1–7)), an endogenous bioactive peptide constituent of the renin–angiotensin system (RAS), has been shown to possess antiproliferative and antiangiogenic effect in vitro and in vivo [9], [10]. Besides, vasodilator and vasorelaxing effects have been described in several vascular beds [11], [12], [13], [14], [15], [16], [17].These effects contrast with the proliferative, angiogenic and vasoconstrictor effects of angiotensin II [8], [9], [10], [18], [19], [20]. The subcutaneous sponge implant model in the mouse provides a useful tool for investigating the development of vasoactive regulatory systems and the pharmacological response of the neovasculature to vasoactive substances [2], [21], [22]. Because the predominant state of the neovasculature is vasodilation and the fact that components of the angiotensin system have been shown to modulate angiogenesis in vivo and in vitro, we reasoned that Ang-(1–7) might be involved in the vasodilation found in the neovasculature in sponge implants.

We have previously shown that sponge-induced angiogenesis in mice and the pharmacological reactivity of the neovasculature could be detected by a fluorimetric method [22], [10]. The principle underlying this technique is that measurement of fluorochrome-generated emission in the bloodstream following the application of a fluorescent dye in the sponge implant compartment reflects the local blood flow. Thus, exogenous application of vasoactive substances and/or interference with their endogenous production would alter the fluorescence intensity in the systemic circulation. We have utilized this approach to characterize the involvement of Ang-(1–7) in a pre-existing (skin) and in a newly formed vascular bed (implant).

Section snippets

Animals

Male Swiss mice weighing 25–30 g were used for the experiments.

Preparation and surgical implantation of cannulated sponge discs

Polyether polyurethane sponge discs, 4-mm thick×8-mm diameter (Vitafoam), were used as the matrix for fibrovascular tissue growth [10], [22], [23]. One end of a polythene tubing, 12-mm length×1.2-mm internal diameter (Portex), was secured with three 5.0 silk sutures (Ethicon) to the center of each disc in such a way that the tube was perpendicular to the disc face. The cannulated sponge discs were soaked overnight in 70% v/v ethanol

Determination of the vasodilator effect of Ang-(1–7) in the skin vasculature

In this set of experiments, different doses of Ang-(1–7) [Sigma] (2, 10, 20, 100, 200 ng/10 μl; n=6–9 for each dose) were applied intradermally to determine the most effective dose of the peptide. On the basis of this study, the dose of 20 ng/10 μl was chosen for all the experiments.

Modulation of Ang-(1–7)-induced vasodilatation

Modulators of the Ang-(1–7) action (inhibitors or antagonists) were administered intradermally or intraimplant 3 min before the injection of the agonist—Ang-(1–7)—or vehicle. The following compounds were assessed in

Pattern of fluorescein diffusion in the cutaneous and implant neovasculature

Fig. 1a shows representative time-course curves for the fluorescent dye to peak in the systemic circulation following intradermal and intraimplant injection. The progressive increase in the intensity of the fluorescence detected in the blood samples reflected the pattern of diffusion in both vasculatures. The t1/2 values were achieved by finding the highest value, or peak, during the collection time using the upslope of the curves. The t1/2 values for the skin and implant vasculatures were 6.70

Discussion

We have previously used a combination of the techniques of sponge implantation and the outflow rate of fluorescein to study neovascularization and the pharmacological response of the neovasculature to vasoactive mediators in mice [22], [24]. In the present study, we have used these techniques to characterize the vasodilator effect of Ang-(1–7) in a pre-existing (skin) and sponge-induced vasculatures.

Comparing the pattern of diffusion between skin (pre-existing vascular bed) and implant (newly

Acknowledgements

This work was supported by CNPq and FINEP-PRONEX, Brazil. We wish to thank Soraya S. Silva and Elizabeth Bontempo for technical assistance.

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