Complement Upregulates Runx-2 to Induce Profibrogenic Change in Aortic Valve Interstitial Cells

Abstract

Background: Calcium accumulation and fibrotic activities are principal mechanisms for calcific aortic valve disease (CAVD). Active complement products are observed in human stenotic aortic valves. Runt-related transcription factor 2 (Runx-2) is involved in tissue calcification. We hypothesized that complement upregulates Runx-2 to induce profibrogenic change in human aortic valve interstitial cells (AVICs).

Methods: AVICs were isolated from 6 normal and 6 CAVD donor valves. Cells were treated with complement cocktails. Profibrogenic activities and associated signaling molecules were analyzed by Western blot assay and collagen staining.

Results: Complement time and dose dependently enhanced profibrogenic activities in AVICs, and complement exposure also induced total collagen deposition in AVICs. Complement-induced profibrogenic responses were associated with increased Runx-2 expression and phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Genetic silencing of Runx-2 decreased both matrix metalloproteinase 9 (MMP-9) and collagen I levels. Pharmacological inhibition of ERK1/2 decreased complement-mediated MMP-9, collagen I, and Runx-2 expression as well as total collagen deposition in human AVICs. Further, treating AVICs with heat-deactivated complement resulted in reduced MMP-9, collagen I, and Runx-2 levels compared with active complement treatment.

Conclusions: Complement induced profibrogenic activities in AVICs by activation of ERK1/2-mediated Runx-2 signaling pathways. This study demonstrates a potential role for complement-mediated CAVD pathogenesis, establishing a possible therapeutic target to limit CAVD progression.

Figure 1.

Complement enhances profibrogenic activities in AVICs. A. Normal human AVICs were treated with pooled human complement serum cocktail (220 μg/ml) for different days (24h, 48h and 72h) or for different doses of (110–440 μg/ml, 48 h). Representative Immunoblot and densitometry data show that complement cocktail time and dose dependently enhance profibrogenic activities in AVICs. Data are presented as means ± SD of 6 experiments using different cell isolates from distinct donor valves. *P < 0.05 vs. control; ^$P < 0.05 vs. control. B. Complement up-regulates collagen production in human AVICs. Normal AVICs were treated with complement cocktail (220 μg/ml) for 14 days. Picro Sirius Red (PSR) staining was applied to stain collagens. Representative images (scale bar = 150 μm) and spectrophotometric analysis of eluted stain show that cells exposed to complement cocktail formed a greater amount of collagen deposits. Data are presented as means ± SD of 6 experiments using different cell isolates from distinct donor valves. *P < 0.05 vs. control. C. Complements induce Runx-2 expression in AVICs. Normal human AVICs were treated with pooled human complement serum cocktail (220 μg/ml) for different days (24h, 48h and 72h) or for different doses of (110–440 μg/ml, 48 h). Complements time and dose dependently induce Runx-2 expression in AVICs. Data are presented as means ± SD of 6 experiments using different cell isolates from distinct donor valves. *P < 0.05 vs. control

Figure 2.

Runx-2 is an essential transcription factor for profibrogenic activities in AVICs. A. diseased human AVICs were treated with Runx-2 shRNA for 48h. Knock down Runx-2 suppresses MMP9 and collagen I expression in diseased human AVICs. B. Normal human AVICs were treated with complement cocktail (220 μg/ml) for 48h with or without genetically silencing Runx-2. Knock down Runx-2 abrogates complement induced Runx-2, MMP9 and collagen I expression in normal human AVICs. Data are presented as means ± SD of 6 experiments using different cell isolates from distinct donor valves. *P < 0.05 vs. control shRNA; ^#P<0.05 vs. complement treatment.

Figure 3.

Complement up-regulates profibrogenic activities through ERK1/2 pathway. A. Normal AVICs are treated with complement cocktail (220μg/ml) for 5 min up to 24h. Representative immunoblot result shows that complement induces ERK1/2 activation in human AVICs. B. Normal human AVICs were treated with complement cocktail (220 μg/ml) for 48h with or without ERK1/2 inhibitor UO126 at three different doses. Inhibition of ERK1/2 dose dependently reduces complement mediated ERK1/2 activation in AVICs. Data are presented as means ± SD of 6 experiments using different cell isolates from distinct donor valves. *P < 0.05 vs. DMSO; ^#P<0.05 vs. complement treatment. C. Normal human AVICs were treated with complement cocktail (220μg/ml) for 48h with or without ERK1/2 inhibitor UO126 at three different doses. Inhibition of ERK1/2 reduces complement mediated Runx-2, MMP-9 and collagen I expression in AVICs. Data are presented as means ± SD of 6 experiments using different cell isolates from distinct donor valves. *P < 0.05 vs. DMSO; ^#P<0.05 vs. complement treatment.

Figure 4.

Treating cells with inactivated complement reduces profibrogenic activities in AVICs. A. Normal human AVICs were treated with active or heat deactivated complement (220μg/ml) for 48h. Heat inactivation resulted in reduced MMP-9, collagen I and Runx-2 levels compared to active complement. Data are presented as means ± SD of 6 experiments using different cell isolates from distinct donor valves. *P < 0.05 vs. control; ^#P<0.05 vs. active complement treatment. B. Complement was heated up to 56 °C or 100 °C for 30 min. Western blot result shows that heat inactivation degrades most of the components of the complement.

Figure 5.

Single complement component did not induce profibrogenic activities in AVICs. Normal human AVICs were treated with recombinant anaphylatoxin C3a or C5a for 48h. Neither C3a nor C5a can induce MMP-9, collagen I and Runx-2 expressions in AVICs. Data are presented as means ± SD of 6 experiments using different cell isolates from distinct donor valves. *P < 0.05 vs. control; ^#P<0.05 vs. active complement treatment.