Collagen production in cardiac fibroblasts during inhibition of aminopeptidase B.
OBJECTIVE
To determine whether the aminopeptidase B inhibitor, arphamenine A, could affect collagen production and expression in control and TGF-ss1-treated cardiac fibroblasts.


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.
Department of Cardiovascular Diseases, Katholoke Universiteit Leuven (K.U.Leuven) Belgium. paul.lijnen@med.kuleuven.ac.be
Paul J
PJ
Lijnen
Victor V
VV
Petrov
Marisa
M
Turner
Robert H
RH
Fagard
D000409:Alanine / Q000378:metabolism; D000626:Aminopeptidases / Q000037:antagonists & inhibitors*; D000818:Animals; D001120:Arginine / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis*; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D006146:Guanidines / Q000494:pharmacology; D006321:Heart / Q000187:drug effects; D008297:Male; D020782:Matrix Metalloproteinases / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology*; D008249:Protein-Lysine 6-Oxidase / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1
D006146:Guanidines; D011480:Protease Inhibitors; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C040038:arphamenine A; D003094:Collagen; D004247:DNA; D001120:Arginine; D008249:Protein-Lysine 6-Oxidase; D000626:Aminopeptidases; C019721:aminopeptidase B; D020782:Matrix Metalloproteinases; D000409:Alanine