Volume 6, Number 2, September 2005

Collagen Production in Cardiac Fibroblasts During Inhibition of Aminopeptidase B
Paul J Lijnen, Victor V Petrov, Marisa Turner, Robert H Fagard

Objective. To determine whether the aminopeptidase B inhibitor, arphamenine A, could affect collagen production and expression in control and TGF-ß1-treated cardiac fibroblasts.
Design and Methods. Cardiac fibroblasts from passage 2 from normal male adult rats were cultured to confluency and incubated with and without 600 pmol/l TGF-ß1 for 2 days in serum-free Dulbecco’s modified Eagle’s medium and then incubated with 100 ?mol/l arphamenine A for 1 day in this medium with added ascorbic acid, ß-aminopropionitrile and titriated proline. Soluble collagen was measured in the conditioned medium and non-soluble collagen in the cell layer. Aminopeptidase activity was estimated by spectrophotometric determination of the liberation of p-nitroaniline from alanine- or arginine-p-nitroanilide. Matrix metalloproteinase (MMP) and lysyl oxidase activity were assayed in the conditioned medium. A semi-quantitative reverse transcriptase- polymerase chain reaction was used to examine the expression of lysyl oxidase and collagen type I and III.
Results. Arphamenine A dose-dependently inhibited basal and TGF-ß1-stimulated aminopeptidase activity. Arphamenine A reduced soluble and non-soluble collagen production in control and TGF-ß1-treated cardiac fibroblasts, while it decreased collagen type I and III expression only in TGF-ß1-treated fibroblasts. Lysyl oxidase, MMP-1 and MMP-2 activity were inhibited by arphamenine A in the conditioned media of control and TGF-ß1-treated cardiac fibroblasts.
Conclusions. Our data show that the specific aminopeptidase B inhibitor, arphamenine A, reduces collagen production in cardiac fibroblasts and that this reduction is accompanied by a pronounced inhibition of lysyl oxidase.

JRAAS 2005;6:69-77.

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