Effect of native low density lipoproteins on nitric oxide and superoxide production by endothelial cells

Pathological modifications in biological systems induce acute and chronic alterations in the structural and fanctinal proper&s of the endothelium and modify the interaction between endothelial cells and the other molecules and cell ties involved in the vascular tone homeostasis. Endogenous nitric oxide (NO) is the I&& factor involved in cardiovascular tone regulation, inhibition of smooth muscle proliferation, platelet aggregation and monocyte-macrophage activation. Low density lipoproteins (LDL) play a key role in endothelial cells injury and participate to the atherogenetic process also by affecting the endothelial-dependent vasodilation. The aim of this study was to evaluate in sita the effect of increasing n-LDL concentration on endothelial NO and superoxide (0~~) production and clarify the precise extracellular concentration of LDL that leads to a modified or reduced activity of the L-arginine-NO system in the endothelial cell. Endothelial human aorta cells were used for all the steps of the study and were grown to confluence in Dulbecco’s modified medium (DMEM) containing 100/o FBS and 1% antibiotic/antimyc&ic. LDL fraction was isolated by density gradient ultmcentri@ation and dialysis against phosphate-buffer saline. NO levels were measured in situ by using an electrochemical method based on the oxidation of NO on a oombvxinic micmsemor and measurement of the current generated fimn this process. 02; washetected by chemihaninescence. Endotbelial cells were olaced in the DMEM contain& 5% liooomtein demived semm ILPDS) for 1418 hours and l&r incubated for one hour wi& incr&iig LDL &ncenbatio~~~ 0 to 240 mg chol/dl) with and without Largbdne supplementation. In the next step, the endotheIial cells have bxn tested in the same experimental conditioos (inwzdng LDLconwntmtions) but pretreated with GNAME ( 2 X IO-4 moVL) for 30 minutes and soperoxide desmotsse (SOD, 100 U/mL) before NO sad C$- were measured NO production already decreased sigmficantly at LDL concentration of 70-80 mg cbol/dl, from 280 nM in LDL-free medium to 150 nM at the LDL concentration of 40 mg cbol/dl, to 80 nM at the LDL concentration of 80 mg chol/dl, Larginine pretreatment (10-s Mom) did not totally block the inhibitory LDL effect on NO production but significantly increased NO levels at all the LDL concentrations. Furthermore. the SOD treatment did not modify the LDL effect on NO release. The LDL treatment induced a sharp increase of 02- production in a dose-dependent fashion (from 10 nM at O-30 me cbol/dL LDL to 90 nM at 80 ma cbol/dL LDU. L-aminine sup&nentation did not in&se the basal level Of 02- but p&doced a smal& &= production at higher LDL concentrations. SOD supplementation did not cause a significant reduction of 02- production at every LDL concentration. These results con&m the inhibim role of LDL on NO prod&ion and suggest that the alteration of the Gargjnine-NO system may be already present at normal LDL conceo!mtion, The amount of L arginiw seems to be one of the main imp&mt steps for the optimal filnction of the NO system. We can hypothesize that hypewholestemlemia may damage the endothelial cdl’s membrane integrity and alter the t3amnmnbrane transport of L-arginine decreasing the substrate for the regular enzymatic activity of the constitutivc nitric oxide synthase (cNOS).