Fig. 2
Pharmacological knockdown of HSPG mitigates Aβ1-40-induced mitochondrial and cytosolic ROS production in VSMC. Primary human cerebral VSMC were pre-treated with heparin (15 U/mL), heparinase I (HpnI; 5 Sigma U/mL), or heparinase III (HpnIII; 2 Sigma U/mL) for 2 h, washed, loaded with Mitotracker Red CM-H2XRos (MTR; 5 μM; panels a, b) or the cytosolic superoxide-sensitive dye dihydroethidium (10 μM; panel c), washed, and treated with Aβ1-40. In some cases, cells were pre-treated with heat-inactivated (HI) enzyme (at the same concentration of active enzyme) and washed prior to MTR loading and Aβ treatment. Fluorescence was measured after 30 minutes. To determine if HSPG directly interact with Aβ1-40, human VSMC cells were treated with Aβ1-40 for 30 minutes and cell lysates were immunoprecipitated with anti-HSPG antibody and immunoblotted with anti-Aβ antibody (Panel d). Results are representative of 3 independent experiments performed in triplicate. *p < 0.05 vs. vehicle-treated control. #p < 0.05 vs. comparison group