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 ß receptor (TGFßR) 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)