Optimization of the in vitro Model of Cardiac Fibrosis

Despite the prevalence of cardiac fibrosis, there are currently no effective treatments to reverse it. A major obstacle is the lack of reliable in vitro models. An increase in angiotensin II and collagen occurs in cardiac fibrosis. Therefore, we hypothesized that the combination of angiotensin II with ascorbic acid and dextran sulfate could induce fibrosis in neonatal rat cardiac fibroblasts (nrCFs) and be used as an in vitro model of cardiac fibrosis.

nrCFs were treated with angiotensin II, ascorbic acid, and dextran sulfate. Key features of cardiac fibrosis were evaluated using Alizarin Red, Picrosirius Red, Masson's trichrome staining, quantitative reverse transcription polymerase chain reaction, and immunocytochemistry and scratch assay.

Although dextran sulfate increased the expression of alpha-smooth muscle actin (α-SMA), it did not increase collagen deposition and cell migration. Therefore, it seems that the combination of 500 nM angiotensin II with 100 μM ascorbic acid would be effective in induction of cardiac fibrosis in vitro, which increased (1) the expression of collagen, α-SMA, and vimentin at protein level, (2) the expression of collagen type I alpha 1 chain, collagen type III alpha 1 chain, matrix metalloproteinase 2, and transforming growth factor-beta 1 at ribonucleic acid level, and (3) cell proliferation and migration.

In this study, 72 hour-treatment of nrCFs with 500 nM angiotensin II and 100 μM ascorbic acid was effective in the creation of an in vitro model of cardiac fibrosis. It is hoped that this model will be useful for screening antifibrotic treatments.