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Volume 1, Number 4, December 2000
Transforming growth factor-β1 induces angiotensin-converting enzyme synthesis in rat cardiac fibroblasts during their differentiation to myofibroblasts
Victor V Petrov, Robert H Fagard, Paul J Lijnen Objectives: Appearance of angiotensin-converting enzyme (ACE)
in fibrotic tissue can be the result of the action either of one particular
growth factor or of cross-talk between multiple factors. Transforming growth
factor-β1 (TGF-β1)
is an effective inducor of the differentiation of cultured fibroblasts to
myofibroblasts, which are heterogeneous cells with different phenotypes.
The present study investigated whether TGF-β1
is able to induce, in vitro, the differentiation of cultured fibroblasts to
myofibroblasts with a phenotype containing ACE.
Design: Adult rat cardiac ventricular fibroblasts were obtained
from hearts perfused with collagenase. Cells from second passage were always used.
Rat cardiac ventricular fibroblasts were incubated with TGF-β1
(10 ng/ml) for seven days.
Cell characterisation was performed
using light microscopy and indirect immunostaining. Presence of vimentin, desmin,
factor VIII, α-smooth muscle actin, and ACE was checked with both immunostaining
and Western blotting. ACE activity was measured fluorometrically with
hippuryl-histidyl-leucine as substrate. Synthesis of DNA was measured
as 3H-thymidine incorporation.
Results: Fibroblasts contained two types of activity of
hip-his-leu degradation, namely a lisinopril-dependent activity (ACE activity)
and a lisinopril-independent activity ('ACE-like' activity) which is performed by
peptidase(s) other than ACE. The ACE activity constituted approximately 30%
of the total activity. TGF-β1 induced
an increase in both ACE activity and ACE protein (detected by immunoblotting) by
4.5 ± 0.9- and 6.9 ± 2.0-fold, respectively (p<0.05). This induction
of ACE was accompanied by a profound modification of the fibroblasts phenotype,
which consisted of a change in cell morphology, an enlargement of cell volume
and an increase in cell protein content. TGF-β1
profoundly inhibited 3H-thymidine incorporation and the number of cells
in growing cultures. The induction of α-smooth muscle actin by TGF-β1
(6.8 ± 2.8-fold increase, p<0.05) simultaneously with these modifications
in cell morphology and proliferation indicates the appearance of myofibroblasts.
These myofibroblasts did not contain desmin.
Conclusion: TGF-β1
is able to induce the appearance of ACE in cultures of adult rat cardiac ventricular
fibroblasts. The appearance of the enzyme is accompanied by the differentiation
of fibroblasts to myofibroblasts. JRAAS 2000;1:342-352. View full PDF article (open in new window)
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