Extracellular superoxide dismutase protects against matrix degradation of heparan sulfate in the lung

Abstract

Asbestosis is a form of interstitial lung disease caused by the inhalation of asbestos fibers, leading to inflammation and pulmonary fibrosis. Inflammation and oxidant/antioxidant imbalances are known to contribute to the disease pathogenesis. Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that has been shown to protect the lung from oxidant-mediated damage, inflammation, and interstitial fibrosis. Extracellular matrix (ECM) components, such as collagen and glycosaminoglycans, are known to be sensitive to oxidative fragmentation. Heparan sulfate, a glycosaminoglycan, is highly abundant in the ECM and tightly binds EC-SOD. We investigated the protective role of EC-SOD by evaluating the interaction of EC-SOD with heparan sulfate in the presence of reactive oxygen species (ROS). We found that ROS-induced heparin and heparan sulfate fragments induced neutrophil chemotaxis across a modified Boyden chamber, which was inhibited by the presence of EC-SOD by scavenging oxygen radicals. Chemotaxis in response to oxidatively fragmented heparin was mediated by Toll-like receptor-4. In vivo, bronchoalveolar lavage fluid from EC-SOD knockout mice at 1, 14, and 28 days after asbestos exposure showed increased heparan sulfate shedding from the lung parenchyma. We demonstrate that one mechanism through which EC-SOD inhibits lung inflammation and fibrosis in asbestosis is by protecting heparin/heparan sulfate from oxidative fragmentation.

FIG. 1. Proposed interactions of EC-SOD, ROS, and heparan sulfate

(1) Reactive oxygen species (ROS) are generated near the lung epithelium by asbestos fibers; (2) heparan sulfate side chains are fragmented and released into the airspace; (3) EC-SOD has a high affinity for heparan sulfate and binds via a heparin-binding domain. EC-SOD is localized at the cell surface and may prevent heparan sulfate fragmentation by scavenging ROS generated by asbestos. (4) If EC-SOD is insufficient, HS will be cleaved by attacking ROS while the remaining EC-SOD, as well as other bound growth factors and cytokines, will be released from the epithelium. Loss of HS and EC-SOD could increase tissue susceptibility to oxidative injury from asbestos fibers, resulting in inflammation and fibrosis.

FIG. 2. Superoxide is produced in a Fenton-like chemical reaction, and EC-SOD inhibits this production

WST-1 reagent was used in a colorimetric assay to determine the relative amount of superoxide produced in our ROS reaction system. Absorbance was measured at 450 nm, and the optical density (O.D.) reported is proportional to superoxide production. Similar ROS production was seen with heparan sulfate as well.

FIG. 3. EC-SOD inhibits neutrophil chemotaxis induced by preventing oxidative fragmentation of heparin and heparan sulfate

Chemotaxis was assessed in a modified Boyden chamber, and ROS, from Fenton-type reactions. EC-SOD significantly inhibited neutrophil chemotaxis induced by ROS-fragmented (A) heparin (*p < 0.001) and (B) heparan sulfate (t, p < 0.05 vs. heparan sulfate without ROS). ROS was used at a Cu(II) concentration of 1.25 μM and 1.5 mM H₂O₂. Heparin or heparan sulfate without ROS contained only CuSO₄ solution and no H₂O₂. 100 Units of EC-SOD is depicted and functions by scavenging superoxide radicals and preventing further hydroxyl radical formation. Statistical analysis completed by ANOVA and Bonferonni's posttest.

FIG. 3. EC-SOD inhibits neutrophil chemotaxis induced by preventing oxidative fragmentation of heparin and heparan sulfate

Chemotaxis was assessed in a modified Boyden chamber, and ROS, from Fenton-type reactions. EC-SOD significantly inhibited neutrophil chemotaxis induced by ROS-fragmented (A) heparin (*p < 0.001) and (B) heparan sulfate (t, p < 0.05 vs. heparan sulfate without ROS). ROS was used at a Cu(II) concentration of 1.25 μM and 1.5 mM H₂O₂. Heparin or heparan sulfate without ROS contained only CuSO₄ solution and no H₂O₂. 100 Units of EC-SOD is depicted and functions by scavenging superoxide radicals and preventing further hydroxyl radical formation. Statistical analysis completed by ANOVA and Bonferonni's posttest.

FIG. 4. Toll-like receptor 4 mediates heparin fragment-induced PMN chemotaxis

Anti-TLR4 antibody inhibits PMN chemotaxis induced by ROS-fragmented heparin. *p < 0.05. Data analysis used ANOVA and Bonferonni's posttest.

FIG. 5. EC-SOD prevents HS shedding from the ECM after asbestos-induced injury

HS shedding increases in the BALF in EC-SOD knockout mice at (A) 1 day (*p < 0.001), (B) 14 days, and (C) 28 days after asbestos exposure compared with wild-type mice. Ten micrograms of protein was loaded for each sample. Significant increases were seen in three HS species (HS1, HS2, and HS3), detected by Western blot with antibody MAB2040 (normalized to protein loading by Ponceau red staining of the membrane). HS1, 2, and 3 are predicted to be syndecans by molecular mass, 35–80 kDa.

FIG. 5. EC-SOD prevents HS shedding from the ECM after asbestos-induced injury

HS shedding increases in the BALF in EC-SOD knockout mice at (A) 1 day (*p < 0.001), (B) 14 days, and (C) 28 days after asbestos exposure compared with wild-type mice. Ten micrograms of protein was loaded for each sample. Significant increases were seen in three HS species (HS1, HS2, and HS3), detected by Western blot with antibody MAB2040 (normalized to protein loading by Ponceau red staining of the membrane). HS1, 2, and 3 are predicted to be syndecans by molecular mass, 35–80 kDa.

FIG. 5. EC-SOD prevents HS shedding from the ECM after asbestos-induced injury

HS shedding increases in the BALF in EC-SOD knockout mice at (A) 1 day (*p < 0.001), (B) 14 days, and (C) 28 days after asbestos exposure compared with wild-type mice. Ten micrograms of protein was loaded for each sample. Significant increases were seen in three HS species (HS1, HS2, and HS3), detected by Western blot with antibody MAB2040 (normalized to protein loading by Ponceau red staining of the membrane). HS1, 2, and 3 are predicted to be syndecans by molecular mass, 35–80 kDa.