Greater Endothelial Apoptosis and Oxidative Stress in Patients with Peripheral Artery Disease

Abstract

We compared apoptosis, cellular oxidative stress, and inflammation of cultured endothelial cells treated with sera from 156 subjects with peripheral artery disease (PAD) and 16 healthy control subjects. Furthermore, we compared circulating inflammatory, antioxidant capacity, and vascular biomarkers between the two groups. The PAD group had a 164% higher value for endothelial cell apoptosis (P < 0.001) and a 62% higher value for endothelial cellular reactive oxygen species production (P < 0.001) than the control group. Furthermore, the PAD group had lower systemic antioxidant capacity measured by hydroxyl radical antioxidant capacity activity (P < 0.001), higher inflammatory and vascular measures of high-sensitivity C-reactive protein (P < 0.001), interleukin-8 (P < 0.001), serum amyloid A (P < 0.001), vascular cell adhesion molecule-1 (P < 0.001), adiponectin (P < 0.001), apolipoprotein B (P = 0.013), apolipoprotein CIII (P = 0.035), lower vascular endothelial growth factor-A (P < 0.001), and hepatocyte growth factor (P < 0.001) than the control group. Subjects with PAD have greater endothelial apoptosis and oxidative stress than control subjects with low burden of comorbid conditions and cardiovascular risk factors. Furthermore, subjects with PAD have lower systemic antioxidant capacity and angiogenic measures and higher circulating inflammatory parameters.

Association between gait characteristics and endothelial oxidative stress and inflammation in patients with symptomatic peripheral artery disease

“…In brief, endothelial cells (purchased from Cell Applications, Inc., San Diego, CA, after passage 4; age of the donors is unknown) were initially cultured in MesoEndo Endothelial Cell Growth Medium (Cell Applications, Inc.) followed by Endothelial Basal Medium supplemented with 10 % fetal calf serum until the time of serum treatment, as described (Gardner et al 2014;Ungvari et al 2009). Cells were 80-90 % confluent before treatment with sera.…”

“…Inter-individual variance is unlikely to contribute to observed differences because detector cells used for each in vitro study were from the same donor. For treatment, fetal calf serum was replaced with serum (10 %; for 24-48 h) collected from our patients (Gardner et al 2014). Cells cultured in Endothelial Basal Medium supplemented with 10 % fetal calf serum served as an additional control.…”

“…Apoptosis assay Cultured endothelial cells were treated with sera from patients for 24 h. Caspase activities using Caspase-Glo 3/7 assay kit (Promega, Madison, WI) were measured to assess apoptotic cell death, as previously reported (Gardner et al 2014). …”

“…Cellular ROS production Hydrogen peroxide (H 2 O 2 ) production in detector endothelial cells was measured fluorometrically by a Tecan Inifinite M200 (Tecan Group Ag, Mannedorf, Switzerland) using the Amplex Red/horseradish peroxidase assay (Life Technologies, Grand Island, NY) to determine cellular ROS induced by factors present in the sera (Gardner et al 2014). …”

“…Transient transfection, nuclear factor K-light-chainenhancer of activated B cells reporter gene assay Transcriptional activity of nuclear factor Klight-chain-enhancer of activated B cells (NF-κB) was tested in serum-treated detector endothelial cells by a reporter gene assay, to determine cellular proinflammatory effects induced by factors in the sera (Gardner et al 2014). Transfections in endothelial cells were performed using the Amaxa Nucleofector technology (Amaxa, Gaithersburg, MD), as we have previously reported (Gardner et al 2014).…”