Extracellular superoxide dismutase protects cardiovascular syndecan-1 from oxidative shedding

Abstract

The extracellular matrix is a complex system that regulates cell function within a tissue. The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is bound to the matrix, and previous studies show that a lack of EC-SOD results in increased cardiac injury, fibrosis, and loss of cardiac function. This study tests the hypothesis that EC-SOD protects against cardiac fibrosis mechanistically by limiting oxidative stress and oxidant-induced shedding of syndecan-1 in the extracellular matrix. Wild-type and EC-SOD null mice were treated with a single dose of doxorubicin, 15 mg/kg, and evaluated on day 15. Serum and left-ventricle tissue were collected for biochemical assays, including Western blot, mRNA expression, and immunohistochemical staining for syndecan-1. The loss of EC-SOD and doxorubicin-induced oxidative injury led to increases in shed syndecan-1 in the serum, which originates from the endothelium of the vasculature. The shed syndecan-1 ectodomain induces proliferation of primary mouse cardiac fibroblasts. This study suggests that one mechanism by which EC-SOD protects the heart against cardiac fibrosis is the prevention of oxidative shedding of cardiovascular syndecan-1 and its subsequent induction of fibroblast proliferation. This study provides potential new targets for understanding and altering fibrosis progression in the heart.

Figure 1

Loss of EC-SOD results in an increase in lung: body weight ratio after doxorubicin treatment, suggesting pulmonary congestion and cardiac dysfunction. Data are reported as mean lung: body weight ratio ± SEM. n=7, *p<0.05.

Figure 2

Loss of EC-SOD and oxidative stress cause increased shedding of syndecan-1 into the serum and decreased mRNA expression in the left ventricle. Syndecan-1 was detected by western blot in serum samples from control- or doxorubicin-treated wild type and ECSOD KO mice. A) Syndecan-1 shedding into the serum. Data reported as net intensity of syndecan-1 normalized to ponceau red staining, mean ± SEM, n=3. *p<0.05 compared to control WT; **p<0.05 EC-SOD KO strain compared to wild type. 2-way anova statistical analysis was completed, with a Bonferroni post-test. B) Syndecan-1 mRNA expression in left ventricle tissue. Data reported as mean percent relative ddCt expression ± SEM (normalized to GAPDH), n=3-4. *p<0.05 compared to control-treated EC-SOD KO mice.

Figure 3

Syndecan-1 is shed from endothelial cells of the vasculature. Paraffin sections (4 μm) of the left ventricle were stained for syndecan-1. A) Histological scoring of syndecan-1 staining of the LV vasculature. Data are reported as mean score ± SEM, n=6. *p<0.05. Statistical analysis included non-parametric ANOVA. B) Representative images of control and doxorubicin-treated wild type and EC-SOD KO left ventricle sections stained for syndecan-1. Syndecan-1 decreases in the vasculature of doxorubicin-treated wild type mice and in both control and doxorubicin-treated EC-SOD KO mice. C) Left ventricle homogenates were evaluated for syndecan-1 by western blot. Densitometry data are normalized to ponceau red for protein loading and data are reported as mean net normalized intensity ± SEM, n=3. Doxorubicin causes a trend to decreased levels of syndecan-1, however data are not statistically significant.

Figure 4

Shed syndecan-1 ectodomain promotes fibroblast proliferation. Primary adult mouse cardiac fibroblasts were treated with 1 μg/ml syndecan-1 ectodomain core protein for A) 24 hours or B) 48 hours. Cell proliferation was assessed. Optical density is proportional to the number of metabolically active cells. Data are reported as mean score μ SEM, n=12 *p<0.05.

Figure 5

Summary schematic of the role of oxidative stress and syndecan-1 in cardiac fibrosis. Oxidative injury to the heart, which is regulated by EC-SOD, can result in matrix remodeling, fibrosis and loss of function. An increase in oxidative injury and subsequent shedding of syndecan-1 ectodomains may further promote fibrosis in the myocardium.