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Our purpose was to determine whether the action of oxidative free radicals released by endothelial cells and vascular smooth muscle cells grown in culture could be responsible for certain modifications to low density lipoprotein (LDL). In these experiments we showed that after a 48-hour incubation with human umbilical vein endothelial cells or bovine aortic smooth muscle cells, human LDL: 1) became oxidized, as evidenced by reactivity to thiobarbituric acid; 2) lost variable amounts of sterol relative to protein (up to 20%); 3) had an increased relative electrophoretic mobility (by 30% to 70%); and 4) became toxic to proliferating fibroblasts. None of these changes occurred after a 48-hour incubation with confluent fibroblasts or bovine aortic endo- S everal investigators have reported that low density lipoprotein (LDL) is toxic to cultured vascular cells under certain conditions. 1 " 3 Our more recent results indicated that toxic LDL is formed by oxidation of a lipid component of the lipoprotein during its isolation from plasma if antioxidants are insufficient. 4 We have also demonstrated that this oxidation occurs by a free radical mechanism that involves superoxide anion and/or hydrogen peroxide. 5 Although oxygen-free radicals such as these have been implicated as effectors of tissue damage, 6 ' 7 the toxic action of oxidized LDL is effected by an oxidized lipid producad by free radicals rather than by free radicals generated during propagation of the oxidation reaction. 5 From Henriksen, et al. 8 " 10 have reported that cultured human umbilical vein endothelial cells (EC) and vascular smooth muscle cells (SMC), but not human fibroblasts or bovine aortic EC, can modify LDL. The changes in LDL produced by incubation with human umbilical vein EC and SMC include increased anodic electrophoretic mobility, a reduced ratio of total cholesterol to protein, and increased degradation by macrophages. 8 " 10 Bowman et al. 11 have reported that pulmonary artery EC, but not lung fibroblasts, produce superoxide anion in vitro. Since superoxide anion appeared to be involved in spontaneous oxidation of LDL during its isolation, 5 we chose to determine whether cultured EC and SMC could oxidize LDL and render it cytotoxic and to identify the relationship between oxidation and certain other EC modifications of LDL, including the changes in electrophoretic mobility and the decreased steroi content reported by Henriksen et al. Methods Cell PreparationThe methods for preparation of human umbilical vein EC, bovine aortic EC, and bovine aortic SMC have been described by DiCorleto and BowenPope. 12 Briefly, primary cultures of bovine aortic EC were isolated by previously used methods in 5% bovine cell-free plasma-derived serum, 13 which was 357 by guest on

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Although angiotensin II (Ang II) and the heptapeptide Ang-(1-7) differ by only one amino acid, the two peptides produce different responses in vascular smooth muscle cells. We previously showed that Ang II stimulated phosphoinositide hydrolysis, whereas Ang II and Ang-(1-7) released prostaglandins. We now report that Ang II and Ang-(1-7) differentially modulate rat aortic vascular smooth muscle cell growth. Ang-(1-7) inhibited [3H]thymidine incorporation in response to stimulation by fetal bovine serum, platelet-derived growth factor, or Ang II. The reduction in serum-stimulated thymidine incorporation by Ang-(1-7) depended on the concentration of the heptapeptide over the range of 1 nmol/L to 1 mumol/L, with a maximal inhibition of 60% by 1 mumol/L Ang-(1-7). Ang-(1-7) also inhibited the serum-stimulated increase in cell number to a maximum of 77% by 1 mumol/L Ang-(1-7). The attenuation of serum-stimulated thymidine incorporation by Ang-(1-7) was unaffected by antagonists selective for angiotensin type 1 (AT1) or type 2 (AT2) receptors; however, [Sar1,Ile1]Ang II and [Sar1,Thr2]Ang II were effective antagonists, indicating that growth inhibition by Ang-(1-7) was a result of angiotensin receptor activation. In contrast, Ang II stimulated [3H]thymidine incorporation in cultured vascular smooth muscle cells over the same concentration range, with a maximal stimulation of 314% at 1 mumol/L Ang II. Ang II also increased the total number of cells (to 145% of control), suggesting that enhanced thymidine incorporation was associated with vascular smooth muscle cell proliferation. The AT1 antagonist losartan or L-158,809 but not AT2 antagonists blocked [3H]thymidine incorporation by Ang II. These results suggest that Ang-(1-7) and Ang II exhibit opposite effects on the regulation of vascular smooth muscle cell growth. The inhibition of proliferation by Ang-(1-7) appears to be mediated by a novel angiotensin receptor that is not inhibited by AT1 or AT2 receptor antagonists.