Adventitial fibroblast abormality in thoracic aortic aneurysms and aortic dissections

Abstract

Background: Development of thoracic aortic aneurysms and aortic dissections (TAAD) is attributed to unbearable wall tension superimposed on defective aortic wall integrity and impaired aortic repair mechanisms. Central to this repair mechanisms are well-balanced and adequately functional cellular components of the aortic wall, including endothelial cells, smooth muscle cells (SMCs), inflammatory cells, and adventitial fibroblasts. Adventitial fibroblasts naturally produce aortic extracellular matrix (ECM), and, when aortic wall is injured, they can be transformed into SMCs, which in turn are involved in aortic remodeling. We postulated the hypothesis that adventitial fibroblasts in patients with TAAD may have defects in ECM production and SMC transformation.

Materials and methods: Adventitial fibroblasts were procured from the adventitial layer of fresh aortic tissues of patients with TAAD (Group I) and of multi-organ donors (Group II), and 4-passage cell culture was performed prior to the experiment. To assess ECM production, cells were treated with TNF-α (50 pM) and the expression of MMP-2 / MMP-3 was analyzed using western blot technique. To assess SMC transformation capacity, cells were treated with TGF-β1 and expression of SM α-actin, SM-MHC, Ki-67 and SM calponin was evaluated using western blot technique. Fibroblasts were then treated with TGF-β1 (10 pM) for up to 10 days with TGF-β1 supplementation every 2 days, and the proportion of transformed SMC in the cell line was measured using immunofluorescence assay for fibroblast surface antigen every 2 days.

Results: MMP-3 expression was significantly lower in group I than in group II. TGF-β1-stimulated adventitial fibroblasts in group I expressed less SM α-actin, SM-MHC, and Ki-67 than in group II. SM-calponin expression was not different between the two groups. Presence of fibroblast was observed on immunofluorescence assay after more than 6 days of TGF-β1 treatment in group I, while most fibroblasts were transformed to SMC within 4 days in group II.

Conclusion: ECM production and SMC transformation are compromised in adventitial fibroblasts from patients with TAAD. This result suggests that functional restoration of adventitial fibroblasts could well be a novel approach for the prevention and treatment of TAAD.

Keywords: Aneurysm; Aorta; Aortic dissection; Fibroblast.

Fig. 1

(A) The extracellular matrix metabolism was reduced in adventitial fibroblasts from the patients with TAAD. The MMP-2 expression in the control group was 98.6% before TNFα (50 pM) treatment and 108.9% after treatment, and 24.3% before treatment and 30.4% after treatment in the TAAD group. The MMP-3 expression in the control group enhanced from 99.7% to 140.9% and from 83.9% to 97.5% in the TAAD group. Thus, TNFα treated adventitial fibroblasts from TAAD aortas expressed less MMP-3 than fibroblasts from healthy control group. (B) The smooth muscle cell transformation was reduced in adventitial fibroblasts from the patients with TAAD. The SM α-actin expression before TGFβ1 (10 pM) treatment in the control group was 94.8% and 141.6% after treatment, while 100.0% before treatment and 110.6% after treatment in the TAAD group. The SM-MHC expression in the control group was 51.9% before treatment and 116.8% after treatment while 66.2% before treatment and 68.5% after treatment in the TAAD group. The Ki-67 expression in the control group was 49.7% before treatment and 168.1% after treatment, while 77.1% before treatment and 72.3% after treatment in the TAAD group. The SM calponin expression in the control group was 46.4% and 144.9% after treatment, while 60.9% before treatment and 143.4% after treatment in the TAAD group. Thus, TGF-β1-stimulated adventitial fibroblasts from TAAD aortas expressed less SM α-actin and SM-MHC and Ki-67. Percent of controls were compared with the β actin expression. TNF=Tumor necrosis factor; MMP=Matrix metalloproteinase; C=Control group; TAAD=Thoracic aortic aneurysm and dissection; TGF=Transforming growth factor; SM=Smooth muscle; MHC=Major histocompatibility complex.

Fig. 2

The immunohistochemical staining after fibroblasts were exposed to TGF-β1 (10 pM) for up to 10 days. TGF-β1 was supplemented every 2 days and the expressions of fibroblast specific marker (fibroblast surface protein) and smooth muscle cell specific marker (SM α actin) were examined every 2 days. At 4^th day after treatment, the fibroblast surface protein was not expressed and SM α actin was fully expressed in the control group. In TAAD group, however, the fibroblast surface protein was still expressed at 6^th day after treatment (×100). Control=Normal adventitial fibroblast; TGF=Transforming growth factor; SM=Smooth muscle; TAAD=Thoracic aortic aneurysm and dissection.