Fig 1: The extracellular domain of syndecan-4 is shed and cleavage of osteopontin is increased during advanced stages of extracellular matrix remodeling after pressure overload. A, Left ventricular syndecan-4 mRNA at different time points after aortic banding (AB). The line indicates sham levels. The 24-hour, 7-day, and 21-day time points were previously published.40 B, Syndecan-4 mRNA in myocardial biopsies from patients with aortic stenosis. C, Immunoblot (bottom panel) and quantification (top panel) of the 15-kDa extracellular syndecan-4 fragment representing the shed ectodomain and full-length syndecan-4 (20–25 kDa) in myocardium after 24 hours, 7 days, and 21 days of AB. D and E, Immunoblot and quantification of full-length osteopontin (FL-OPN) and thrombin-cleaved osteopontin (cl-OPN) after 24 hours (D) and 21 days (E) of AB. Collagen 1a2 (F) and collagen 3a1 (G) mRNA expression in left ventricular tissue lysate from wild-type (WT) and syndecan-4 knockout mice subjected to 21 days of AB. mRNA levels were normalized to GAPDH for mouse samples and to Rpl32 for human samples. Student t test was used to determine significant differences in A through E. Two-way ANOVA with Tukey's multiple comparisons test was used to determine significant differences in F and G. Numbers are indicated in graphs. n.s. indicates not significant. *P<0.05, **P<0.01, ***P<0.005.
Fig 2: Syndecan-4 protects osteopontin from cleavage by thrombin in the presence of Ca2+. Quantification and representative Western blot of recombinant full-length osteopontin (FL-OPN) protein levels after incubation with recombinant extracellular syndecan-4 (1–146), 2 mmol/L Ca2+, and thrombin. One-way ANOVA with Holm-Sidak's multiple comparisons test, as indicated, was used to determine significant differences. Numbers are indicated in graphs. n.s. indicates not significant. *P<0.05, ***P<0.005.
Fig 3: Syndecan‐4 protects osteopontin from cleavage by thrombin, thereby preventing osteopontin‐induced profibrotic collagen production. Osteopontin is immediately upregulated in left ventricular tissue after inducing pressure overload by aortic banding. In this early remodeling phase, syndecan‐4 is also highly upregulated. Osteopontin binds to the extracellular part of syndecan‐4, thereby becoming protected from cleavage by thrombin that enters the myocardial tissue from the circulation. At later phases of remodeling, the extracellular part of syndecan‐4 is shed, and the protective effect on osteopontin cleavage is lost. The resulting cleaved osteopontin fragment induces profibrotic collagen expression via transforming growth factor β receptor (TGFβR) signaling, thereby promoting fibrosis. cl‐OPN indicates thrombin‐cleaved osteopontin; FL‐OPN, full‐length osteopontin.
Fig 4: Syndecan-4 binds osteopontin in left ventricular tissue and cardiac fibroblasts. A, Detection of a syndecan-4–osteopontin high-molecular-weight complex in left ventricular homogenates from wild-type (WT) mice on native PAGE using anti–syndecan-4. Stripping and reprobing with anti-osteopontin confirmed that the 2 antibodies recognized the same protein complex in WT homogenates. The opposite probing order (anti-osteopontin, stripped and reprobed with anti–syndecan-4) is shown in Figure S5A. Left ventricle homogenate from syndecan-4 knockout mice was used as negative control (lanes 1–4). Vinculin was used as loading control. Immunoprecipitation of syndecan-4–osteopontin complex in cell lysates from NIH 3T3 fibroblasts (B) and primary neonatal cardiac fibroblasts from rat (C). Recombinant syndecan-4 (amino acids 1–146) and osteopontin proteins were used as positive controls for the immunoblotting.
Supplier Page from Sino Biological, Inc. for Mouse Syndecan-4 / SDC4 Protein (Fc Tag)