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. 2001 Mar;107(6):727-35.
doi: 10.1172/JCI10720.

The discoidin domain receptor tyrosine kinase DDR1 in arterial wound repair

G Hou  1 , W Vogel, M P Bendeck
Affiliations

The discoidin domain receptor tyrosine kinase DDR1 in arterial wound repair

G Hou et al. J Clin Invest. 2001 Mar.

Abstract

Collagens act as important signaling molecules regulating vascular smooth muscle cell responses during arterial wound repair. Discoidin domain receptors (DDRs) are a novel class of receptor tyrosine kinases that bind to several collagens and stimulate matrix metalloproteinase (MMP) production, but little is known about their expression and function in the vasculature. We posited a critical role for the DDRs controlling smooth muscle cell migration and proliferation and thus repair following arterial injury. Smooth muscle cells were isolated from the aortas of mice with a targeted deletion of the DDR1 gene (DDR1-null) and studied in culture using models that mimic critical steps in neointimal thickening. Our studies suggest that DDR1 plays an important role in regulating attachment to collagen, chemotaxis, proliferation, and MMP production in smooth muscle cells. Following mechanical injury to the carotid arteries, cross-sectional area of the neointima was significantly lower in DDR1-null mice than in wild-type mice. There was also a significant decrease in collagen deposition in the injured arteries of the DDR1-null mice. Our results support the hypothesis that DDR1 plays an important role as a collagen receptor, mediating intimal thickening after vascular injury.

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Figures

Figure 1
Figure 1
(a) DDR1 was activated by type I and type VIII collagen. HEK 293 cells were transfected with vector alone or DDR1 cDNA. The cells were incubated with plain media (C), 10 μg/ml type I collagen (I), or 10 μg/ml type VIII collagen (VIII) for 90 minutes, then cell lysates were collected and subjected to Western blotting. The same blots were sequentially probed with anti-phosphotyrosine (anti-P-Tyr) Ab (upper) and with DDR1 Ab (lower). (b) DDR1 mRNA expression was increased after injury of the rat carotid artery. Northern blot with RNA extracted from control carotids (C) and from carotids at various days after injury, then probed with a cDNA against mouse DDR1. Lower photograph is the methylene-blue–stained Northern blot showing 28s and 18s ribosomal RNA bands demonstrating equal loading in the lanes. (c) A Western blot, with arterial protein extracts from control carotids (C) and carotids taken at various times after injury, was probed with anti-human DDR1 Ab.
Figure 2
Figure 2
DDR1 immunostaining was increased after injury of the rat carotid artery. Vessel cross-sections from uninjured control carotid arteries (a) and carotid arteries at 2 days (b), 4 days (c), and 14 days (d) after balloon-catheter injury were stained with anti-DDR1 Ab. (e) A cross-section was stained in the absence of the anti-DDR1 Ab. ×600.
Figure 3
Figure 3
SMC attachment to collagen was reduced in the absence of DDR1. SMCs from DDR1+/+ mice (filled bars) or DDR1–/– mice (open bars) were allowed to attach for 4 hours to plates coated with 10 nM fibronectin or vitronectin or 100 nM type I collagen or type VIII collagen. Attachment was assessed by staining the cells with toluidine blue and measuring optical density at 595 nm. Experiments were done in triplicate and repeated three times. AValue from DDR1–/– SMCs was significantly less than the value from wild-type SMCs.
Figure 4
Figure 4
Cell proliferation on collagen was less in DDR1–/– SMCs compared with DDR1+/+ SMCs. (a) SMCs from DDR1+/+ mice (filled bars) or DDR1–/– mice (open bars) were stimulated to grow with 10% FCS and incubated with [3H]thymidine on wells coated with 100 nM type I collagen (left) or type VIII collagen (right). AValue from DDR1–/– SMCs was significantly less than value from wild-type SMCs. (b) Number of SMCs per well after plating on 100 nM type I collagen and growing in DMEM containing 10% FCS for 1–4 days. Squares, DDR1+/+ SMCs; circles, DDR1–/– SMCs. Experiments were done in triplicate and repeated three times. AValue from DDR1–/– SMCs was significantly less than value from wild-type SMCs.
Figure 5
Figure 5
SMC migration was reduced in the absence of DDR1. SMCs from DDR1+/+ mice (filled bars) or DDR1–/– mice (open bars) were stimulated to migrate for 4 hours in chemotaxis chambers with 200 nM type I collagen (left) or type VIII collagen (right) added to DMEM in the bottom of the chamber. Migration was quantified by staining and counting the number of cells that migrated to the bottom of the filter. Experiments were done in triplicate and repeated three times. AValues from DDR1–/– SMCs were significantly less than values from wild-type SMCs.
Figure 6
Figure 6
DDR1-null SMCs produced no MMP-9 activity and less MMP-2 activity compared with wild-type cells. Gelatin zymogram containing conditioned media from wild-type (DDR1+/+) or DDR1-null SMCs (DDR1–/–) plated on wells precoated with 0, 100, or 1000 nM type I collagen. MMP-9A, MMP-9 active; MMP-2L, MMP-2 zymogen; MMP-2A, MMP-2 active.
Figure 7
Figure 7
Intimal area was significantly decreased after injury of the DDR1-null mouse carotid compared with wild-type. (a) Cross-sectional area of intima measured 14 days after carotid injury. (b) Cross-sectional area of media measured 14 days after carotid injury. AIntimal area in DDR1–/– mice was significantly less than intimal area in wild-type mice. (n = 6 and 8 for DDR1+/+ and DDR1–/– groups, respectively.) (c) There was less collagen deposition in the carotid arterial intima of the DDR1-null mouse. Picrosirius red staining and polarized light microscopy were used to assess the deposition of birefringent collagen in the vessel wall. ×400.
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