Progressive Improvement in Endothelial Nitric Oxide Release due to Synthase Dimer Formation with LDL Reductions below 70 mg/dL during Hyperglycemia

In diabetes, endothelial cell (EC) dysfunction is characterized by reduced nitric oxide (NO) release and increased nitroxidative stress due to NO synthase (eNOS) dimer uncoupling. Reductions in LDL improve EC function but the effect is not well understood at levels below the target of 70 mg/dL. We measured NO and peroxynitrite (ONOO⁻) release as well as eNOS dimer formation in ECs under hyperglycemic conditions following progressive reductions of LDL in vitro. Human umbilical vein endothelial cells (HUVECs) were incubated with LDL at concentrations ranging from 150 to <10 mg/dL with high glucose (250 mg/dL). Cells were stimulated with calcium ionophore, and assayed for NO and ONOO⁻ release using porphyrinic sensors. eNOS dimer levels were measured using immunochemical approaches. Results showed that reductions in LDL levels had a disproportionate benefit on endothelial NO and ONOO⁻ release below 70 mg/dL. A reduction in LDL levels from 70 to <10 mg/dL produced a 181% increase in NO release (163 ± 18 to 458 ± 34 nM; p<0.0001) and 116% decrease in ONOO⁻ release (393 ± 27 to 182 ± 23 nM; p<0.0001), corresponding to a 513% increase in the NO-to-ONOO⁻ release ratio (0.42 ± 0.04 to 2.56 ± 0.40; p<0.0001), a comprehensive indicator of EC function. These changes also correlated with increased eNOS dimer formation by 819%. By contrast, only a modest 27% change in EC function was observed as LDL levels were reduced from 150 to 70 mg/dL with regard to the NO-to-ONOO⁻ release ratio. In the absence of hyperglycemia, the benefits of LDL lowering on EC function and eNOS dimerization were also very evident, but to a lesser extent. Thus, progressive improvements in endothelial NO bioavailability and eNOS dimer formation were observed at LDL levels below 70 mg/dL to <10 mg/dL. These in vitro data suggest that reductions in LDL to very low levels may be effective in reversing EC dysfunction for the diabetic patient.