Discoidin Domain Receptor-1 Regulates Calcific Extracellular Vesicle Release in Vascular Smooth Muscle Cell Fibrocalcific Response via Transforming Growth Factor-β Signaling

Abstract

Objective: Collagen accumulation and calcification are major determinants of atherosclerotic plaque stability. Extracellular vesicle (EV)-derived microcalcifications in the collagen-poor fibrous cap may promote plaque rupture. In this study, we hypothesize that the collagen receptor discoidin domain receptor-1 (DDR-1) regulates collagen deposition and release of calcifying EVs by vascular smooth muscle cells (SMCs) through the transforming growth factor-β (TGF-β) pathway.

Approach and results: SMCs from the carotid arteries of DDR-1(-/-) mice and wild-type littermates (n=5-10 per group) were cultured in normal or calcifying media. At days 14 and 21, SMCs were harvested and EVs isolated for analysis. Compared with wild-type, DDR-1(-/-) SMCs exhibited a 4-fold increase in EV release (P<0.001) with concomitantly elevated alkaline phosphatase activity (P<0.0001) as a hallmark of EV calcifying potential. The DDR-1(-/-) phenotype was characterized by increased mineralization (Alizarin Red S and Osteosense, P<0.001 and P=0.002, respectively) and amorphous collagen deposition (P<0.001). We further identified a novel link between DDR-1 and the TGF-β pathway previously implicated in both fibrotic and calcific responses. An increase in TGF-β1 release by DDR-1(-/-) SMCs in calcifying media (P<0.001) stimulated p38 phosphorylation (P=0.02) and suppressed activation of Smad3. Inhibition of either TGF-β receptor-I or phospho-p38 reversed the fibrocalcific DDR-1(-/-) phenotype, corroborating a causal relationship between DDR-1 and TGF-β in EV-mediated vascular calcification.

Conclusions: DDR-1 interacts with the TGF-β pathway to restrict calcifying EV-mediated mineralization and fibrosis by SMCs. We therefore establish a novel mechanism of cell-matrix homeostasis in atherosclerotic plaque formation.

Keywords: extracellular matrix; extracellular vesicles; fibrosis; smooth muscle; transforming growth factors.

Fig.1

Nanoparticle Tracking Analysis (NTA) of EVs released from wild type (WT) and DDR-1^−/− vSMCs cultured for 14 and 21 days in normal (NM) and calcifying media supplemented with 10mM β-Glycerophosphate (β-GP). A, Size distribution of EVs at days 14 and 21 shows a characteristic uniform size between 100 and 300nm. B, Concentration of EVs released by DDR-1^−/− vSMCs is significantly increased compared to WT vSMCs in normal and calcifying media at 14 and 21 days of culture. C, TEM imaging of wild type (left) and DDR-1^−/− (right) vSMCs after 21 days of culture. EVs in the ECM of DDR-1^−/− vSMCs (white arrowheads). Representative images at 11,000× magnification, bar: 500nm. D, ALP activity assay in purified EVs reveals increased calcification potential in EVs released from DDR-1^−/− vSMCs. Results shown as mean±SD, n=5 for each group, **=p≤0.01, ***=p≤0.001, ****=p≤0.0001.

Fig.2

ALP expression and calcification in wildtype and DDR-1^−/− vSMCs. A, Representative images of ALP activity staining. B, ALP activity assay in cell lysates shows increased activity of cytoplasmic and cell membrane-bound ALP. C, Representative images of Alizarin Red S staining in wild type (top) and DDR-1^−/− vSMCs (bottom). D, Quantification of mineralization measured by Alizarin Red S absorbance at 550nm. Mean±SD, n=5 per group, representative results of 3 independent experiments. E, ALP mRNA levels at 14 days of culture relative to wild type normal media (WT NM) and normalized to rplp0, n=10 per group. Statistical differences labeled as follows: *=p≤0.05, **=p≤0.01, ***=p≤0.001, ****=p≤0.0001.

Fig.3

DDR-1 knockout and TGF-β signaling. A, concentration of TGF-β1 detected by ELISA in cell media supernatant after 21 days of culture. B, TGF-β1 mRNA at 14 days of culture relative to wild type normal media (WT NM) and normalized to rplp0. Representative results of 3 independent experiments in triplicates. C, Western Blot analysis for Smad3 (top panel) and p38 (bottom panel) phosphorylation under native conditions (left) and with addition of 2µmol/L SB431542 (right) after 21 days of culture. D, quantification of Western Blot band intensities normalized to GAPDH using NIH ImageJ v1.48. Mean±SD, n=10 per group, *=p≤0.05, ***=p≤0.001, ****=p≤0.0001.

Fig.4

SB431542 and SB203580 diminish the osteogenic potential of DDR-1^−/− vSMCs. A, Alizarin Red S staining after 21 days of culture in native culture media (left), with addition of 2µmol/L SB431542 (middle) or 10µmol/L SB203580 (right). N=5 per group. B, Alizarin Red S absorbance of the stained wells of A, normalized to wild type vSMCs cultured in native media. C, Relative EV concentration, normalized to wild type. D, Relative ALP activity in EVs, normalized to wild type. Mean±SD, n=5 per group, **=p≤0.01, ***=p≤0.001, ****=p≤0.0001.

Fig.5

DDR-1 acts as a feedback regulator on vascular fibrocalcific response. A, Fluorescent labeling of collagen by CNA probe (green) and microcalcific deposits by osteosense (red) produced by wild type (WT) and DDR-1^−/− vSMCs in native media (top panel) and with addition of 2µmol/L SB431542 (bottom panel) after 21 days of culture. Bar represents 60µm. Representative images of 4 independent experiments. B, % positive area for collagen (green) and microcalcifications (red). Mean±SD, n=5 per group, **=p≤0.01, ***=p≤0.001. C, Top panel: Picrosirius Red staining of LDL-R^−/− and DDR-1^−/− LDL-R^−/− aortic arches under polarized microscope at 4× magnification, bar: 200µm. Bottom panel: Immunohistochemical staining for TGF-β1 at 2× magnification. Images correspond to Picrosirius Red stained areas of B. Bar: 100µm. N=6 for each group. For each n, 2 sections were stained for evaluation. D, Quantification of relative frequency of thin, loose and thick, densely packed collagen fibers represented by green and orange birefringence using ImageJ v1.48. N=6 per group. E, DDC-SEM of plaque areas from LDL-R^−/− and DDR-1^−/− LDL-R^−/− aortic arches, with a human atheroma for comparison. Mineral is presented in orange and ECM is presented in green. Magnification 10,000×, bar: 1µm, n=6 per group.

Fig.6

Ways of DDR-1 interaction with TGF-β signaling through direct suppression of TGF-β1 (1), phosphorylation of p38 (2) or activation of Smad3 (3), leading to downregulation of calcifying EV release and EV-bound ALP by vSMCs. Inverse regulation of Smad3 and p38 activation through possible downstream interaction (4).