Background: Extracellular vesicle (EV)-derived microcalcifications formed in collagen-poor fibrous caps contribute to plaque rupture. Collagen accumulation and calcification are major determinants of plaque stability, although the mechanisms linking fibrotic and calcific responses are unknown. The collagen receptor discoidin domain receptor-1 (DDR-1) regulates plaque calcification in vivo; however, its role in the release of calcifying EVs remains unclear. We hypothesize that DDR-1 regulates the processes of fibrosis and EV-induced calcification in atherosclerotic plaques. Methods and results: Smooth muscle cells (SMC) from the carotid arteries of wild type and DDR-1 knockout (DDR-1-/-) mice (n=5 per group) were cultured in control or calcifying media. At days 14 and 21, cells were harvested and EVs isolated for analysis. Compared to wild type cells, DDR-1-/- SMCs exhibited a 3.5-fold increase in EV release (p<0.001) as well as elevated EV calcifying potential determined by EV-bound alkaline phosphatase (ALP) activity (470.4+/-30.0 vs. 19.1+/-2.3 ng/well/mg protein). DDR-1-/- SMCs showed an increase in ALP activity (220.3+/-31.2 vs. 6.9+/-1.2 ng/well/mg protein), calcification measured by alizarin red S absorbance (3.56+/-0.11 vs. 0.10+/-0.0008), and collagen type I accumulation (p=0.04). Transforming growth factor-[beta] (TGF[beta]) signaling has been implicated in both fibrotic and calcific responses. DDR-1-/- cells released significantly more TGF[beta] in calcifying medium (253.4+/-12.4 vs. 167.2+/-8.8 pg/ml, p<0.001), suggesting a connection between DDR-1 and TGF[beta] signaling. A selective TGF[beta] receptor I inhibitor mitigated the osteogenic potential of the DDR-1-/- phenotype with a significant decrease in EV release, ALP activity in SMCs and EVs, extracellular matrix calcification and collagen production. Analysis of TGF[beta]-associated pathways in DDR-1-/- SMCs revealed significantly increased p38 phosphorylation while phosphorylated Smad3 and JNK were inhibited.
|Status||Gepubliceerd - 10 nov 2015|