Endothelium protects smooth muscle from oxidative stress in skeletal muscle resistance arteries

Abstract

Vascular dysfunction associated with oxidative stress is characteristic of cardiovascular disease. Advanced age can protect microvascular endothelium from oxidative stress (PMC4422569) however it is unknown whether endothelial cells (ECs) or smooth muscle cells (SMCs) of intact microvessels are more susceptible to damage during oxidative stress. Using the superior epigastric artery freshly dissected from anesthetized male C57BL/6 mice (3–4 months old), we tested the hypothesis that, compared to SMCs, ECs are more susceptible to damage during acute oxidative stress. Individual vessels (length, ~2 mm; internal diameter, ~150 μm) were cannulated at both ends and pressurized to 100 cmH2O with physiological salt solution (PSS), then superfused (2 ml/min) with PSS containing 200 μM H2O2 at 37°C (pH=7.36). At defined durations, the lumen was perfused (0.1 ml/min) with PSS containing Hoechst 33342 (turquoise, 1 μM) and propidium iodide (red, 2 μM) to stain nuclei of all cells and nuclei of cells with damaged membranes, respectively. Staining was quantified in the vessel mid‐region (~500 μm × 150 μm; n=5) which contained nuclei of 93±1 ECs (parallel to vessel axis) and 114±2 SMCs (perpendicular to vessel axis). Within 20–30 min of H2O2 superfusion, membrane disruption (red nuclei) occurred in ECs but not SMCs. Following 50 min of H2O2, 60±2% of SMC and 37±2% of EC nuclei stained red (n=5; P<0.05). Neither EC nor SMC nuclei stained red following equivalent superfusion with control PSS. In vessels denuded of endothelium (50 μm diameter tungsten wire), 50 min superfusion with H2O2 stained 90±2% of SMC nuclei red (n=5; P<0.05, denuded vs intact endothelium). In contrast, SMC nuclei of denuded vessels did not stain red following equivalent superfusion with control PSS. Thus, with H2O2 delivered abluminally, membrane damage occurred sooner for ECs than for SMCs and denuding the endothelium exacerbated SMC damage. We suggest that the endothelium of resistance arteries is more vulnerable to oxidative stress while protecting its surrounding smooth muscle layer.Support or Funding InformationNIH R37HL041026