A cell culture model using rat coronary artery adventitial fibroblasts to measure collagen production

Abstract

Background: We have developed a rat cell model for studying collagen type I production in coronary artery adventitial fibroblasts. Increased deposition of adventitial collagen type I leads to stiffening of the blood vessel, increased blood pressure, arteriosclerosis and coronary heart disease. Although the source and mechanism of collagen deposition is yet unknown, the adventitia appears to play a significant role. To demonstrate the application of our cell model, cultured adventitial fibroblasts were treated with sex hormones and the effect on collagen production measured.

Methods: Hearts (10-12 weeks) were harvested and the left anterior descending coronary artery (LAD) was isolated and removed. Tissue explants were cultured and cells (passages 2-4) were confirmed as fibroblasts using immunohistochemistry. Optimal conditions were determined for cell tissue harvest, timing, proliferation and culture conditions. Fibroblasts were exposed to 10-7 M testosterone or 10-7 M estrogen for 24 hours and either immunostained for collagen type I or subjected to ELISA.

Results: Results showed increased collagen staining in fibroblasts treated with testosterone compared to control and decreased staining with estrogen. ELISA results showed that testosterone increased collagen I by 20% whereas estrogen decreased collagen I by 15%.

Conclusion: Data demonstrates the usefulness of our cell model in studying the specific role of the adventitia apart from other blood vessel tissue in rat coronary arteries. Results suggest opposite effects of testosterone and estrogen on collagen synthesis in the rat coronary artery adventitial fibroblasts.

Figure 1

Removal of left anterior descending coronary artery (LAD) from SHR male rat. A) intact LAD, B) heart with LAD removed, C) isolated LAD, D) LAD length, E) LAD diameter, and F) fibroblasts migrating from LAD explant.

Figure 2

Cell Characterization. Cultured rat coronary artery adventitia fibroblasts (LAD) and aortic vascular smooth cells (VSM) (positive control) stained for desmin, α-smooth muscle actin (α-SMA) and vimentin. LAD stained negative for desmin and αSMA and positive for vimentin, a fibroblast marker. VSM stained positive for all 3 proteins. Desmin and α-SMA are specific VSM markers.

Figure 3

Cultured fibroblasts immunostained for collagen type I after 24 h exposure to 10^-7M testosterone or 10^-7M estrogen. A) Testosterone increased the amount of collagen present compared to control (C) as indicated by the increased number of secretory vesicles and the intensity of staining. B) Estrogen did not have any significant effect on collagen production.

Figure 4

Collagen Type I (μg) measured by ELISA in LAD fibroblasts treated with 10^-7M testosterone or 10^-7M estrogen. Testosterone significantly increased the amount of collagen deposited (2.912 ± 0.247 μg) compared to control values (2.475 ± 0.211 μg) (* p < 0.05, 1 tailed t-test). Estrogen decreased the amount of collagen (2.103 ± 0.262 μg) compared to control (p = 0.058, 1 tailed t-test). Collagen was measured in wells with a 0.32 cm² growth area from a 96 well culture plate.