—Oxidative processes play an important role in atherogenesis. Because superoxide anion and nitric oxide (NO) are important mediators in vascular pathology, we studied the expression of extracellular superoxide dismutase (EC-SOD) and inducible nitric oxide synthase (iNOS) in human and rabbit atherosclerotic lesions by using simultaneous in situ hybridization and immunocytochemistry and EC-SOD enzyme activity measurements. We also analyzed the presence in the arterial wall of oxidized lipoproteins and peroxynitrite-modified proteins as indicators of oxidative damage and possible mediators in vascular pathology. EC-SOD and iNOS mRNA and protein were expressed in smooth muscle cells and macrophages in early and advanced lesions. The expression of both enzymes was especially prominent in macrophages. As measured by enzyme activity, EC-SOD was the major SOD isoenzyme in the arterial wall. EC-SOD activity was higher in highly cellular rabbit lesions but lower in advanced, connective tissue–rich human lesions. Despite the abundant expression of EC-SOD, malondialdehyde-lysine and hydroxynonenal-lysine epitopes characteristic of oxidized lipoproteins and nitrotyrosine residues characteristic of peroxynitrite-modified proteins were detected in iNOS-positive, macrophage-rich lesions, thus implying that malondialdehyde, hydroxynonenal, and peroxynitrite are important mediators of oxidative damage. We conclude that EC-SOD, iNOS, and the balance between NO and superoxide anion play important roles in atherogenesis. EC-SOD and iNOS are highly expressed in lesion macrophages. High EC-SOD expression in the arterial wall may be required not only to prevent deleterious effects of superoxide anion but also to preserve NO activity and prevent peroxynitrite formation. Modulation of arterial EC-SOD and iNOS activities could provide means to protect arteries against atherosclerotic vascular disease.

Background —Atherosclerotic lesions contain foam cells that arise from monocyte-macrophages and smooth muscle cells (SMCs) by excessive uptake of lipoproteins. There are many candidate receptors for the lipid accumulation, such as LDL receptor (LDLR), VLDL receptor (VLDLR), LDL receptor-related protein (LRP), and scavenger receptors (SRs). However, little quantitative information exists on the expression of these receptors in normal and atherosclerotic arteries. Methods and Results —Competitive reverse transcription–polymerase chain reaction and in situ hybridization were used for the studies in New Zealand White (NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbit aortic intima-medias. NZW rabbits were fed a 1% cholesterol diet for 0 (control group), 3, 6, or 14 weeks. LDLR mRNA expression was low in aortic intima-medias of all groups. Of the analyzed receptors, LRP had the highest expression in the control group, and its mRNA was induced threefold in the 14-week group, the aortas of which had extensive lesions. SR expression was low and VLDLR expression moderate in the control group. Both receptors were highly induced during cholesterol feeding (SRs, 3-fold and 270-fold induction; VLDLR, 15-fold and 100-fold induction in the 3-week and 14-week groups, respectively). Comparable results were obtained from WHHL rabbits: high basal LRP mRNA in normal intima-medias; moderate induction of LRP and marked induction of SRs and VLDLR in fatty streaks and fatty plaques. In situ hybridization indicated that LRP and VLDLR were expressed in SMCs and macrophages. VLDLR expression was also observed in endothelial cells. SR expression was detected only in macrophages. Conclusions —SR and VLDLR mRNAs were highly induced in atherosclerotic lesions. VLDLR and LRP may be involved in the formation of both SMC–and macrophage-derived foam cells, whereas SRs play an important role in lipid uptake in macrophages.

Macrophage- and smooth muscle cell (SMC)-derived foam cells are typical constituents of human atherosclerotic lesions. At least three receptor systems have been characterized that could be involved in the development of foam cells: alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2 MR/LRP), scavenger receptor, and LDL receptor. We studied the expression of these receptors in human atherosclerotic lesions with in situ hybridization and immunocytochemistry. An abundant expression of alpha 2MR/LRP mRNA and protein was found in SMC and macrophages in both early and advanced lesions in human aortas. alpha 2MR/LRP was also present in SMC in normal aortas. Scavenger receptor mRNA and protein were expressed in lesion macrophages but no expression was found in lesion SMC. LDL receptor was absent from the lesion area but was expressed in some aortas in medial SMC located near the adventitial border. The results demonstrate that (a) alpha 2MR/LRP is, so far, the only lipoprotein receptor expressed in lesions SMC in vivo; (b) scavenger receptors are expressed only in lesion macrophages; and (c) both receptors may play important roles in the development of human atherosclerotic lesions.

Oxidized low density lipoprotein (LDL) possesses several atherogenic properties. The mechanisms by which LDL becomes oxidized in vivo remain unknown, but previous studies have suggested that 15-lipoxygenase may be one of the factors involved in the initiation of LDL oxidation in the arterial wall. 3 wk after a retrovirus-mediated 15-lipoxygenase gene transfer into iliac arteries of normocholesterolemic rabbits there was a threefold increase in 15-lipoxygenase activity but no signs of LDL oxidation. However, when animals were made moderately hypercholesterolemic by feeding a 0.13% cholesterol diet for 2-3 wk starting from day 4 after the gene transfer, oxidation-specific lipid-protein adducts characteristic of oxidized LDL were detected in 15-lipoxygenase-transduced arteries. Control experiments in which contralateral iliac arteries were transduced with beta-galactosidase-containing retroviruses showed only occasional signs of the presence of oxidation-specific adducts. The results support the hypothesis that products derived from the 15-lipoxygenase activity are involved in the induction of LDL oxidation within the arterial wall, provided that sufficient concentrations of lipoproteins are present in the artery.

To identify novel genes influencing blood pressure response to thiazide diuretic therapy for hypertension, we conducted genome-wide association meta-analyses of ≈1.1 million single-nucleotide polymorphisms in a combined sample of 424 European Americans with primary hypertension treated with hydrochlorothiazide from the Pharmacogenomic Evaluation of Antihypertensive Responses study (n=228) and the Genetic Epidemiology of Responses to Antihypertensive study (n=196). Polymorphisms associated with blood pressure response at P 4/4 mm Hg) across study samples. The rs2273359 polymorphism in the GNAS-EDN3 region also replicated for same-direction association with systolic blood pressure response in the Nordic Diltiazem study, and the combined 3-study meta-analysis P value approached genome-wide significance ( P =5.5×10 ⁻⁸ ). The findings document clinically important effects of genetic variation at novel loci on blood pressure response to a thiazide diuretic, which may be a basis for individualization of antihypertensive drug therapy and identification of new drug targets.

Background: Identification of genetic markers of antihypertensive drug responses could assist in individualization of hypertension treatment. Methods and Results: We conducted a genome‐wide association study to identify gene loci influencing the responsiveness of 228 male patients to 4 classes of antihypertensive drugs. The Genetics of Drug Responsiveness in Essential Hypertension ( GENRES ) study is a double‐blind, placebo‐controlled cross‐over study where each subject received amlodipine, bisoprolol, hydrochlorothiazide, and losartan, each as a monotherapy, in a randomized order. Replication analyses were performed in 4 studies with patients of European ancestry ( PEAR Study, N=386; GERA I and II Studies, N=196 and N=198; SOPHIA Study, N=372). We identified 3 single‐nucleotide polymorphisms within the ACY 3 gene that showed associations with bisoprolol response reaching genome‐wide significance ( P <5×10 ⁻⁸ ); however, this could not be replicated in the PEAR Study using atenolol. In addition, 39 single‐nucleotide polymorphisms showed P values of 10 ⁻⁵ to 10 ⁻⁷ . The 20 top‐associated single‐nucleotide polymorphisms were different for each antihypertensive drug. None of these top single‐nucleotide polymorphisms co‐localized with the panel of >40 genes identified in genome‐wide association studies of hypertension. Replication analyses of GENRES results provided suggestive evidence for a missense variant (rs3814995) in the NPHS 1 (nephrin) gene influencing losartan response, and for 2 variants influencing hydrochlorothiazide response, located within or close to the ALDH 1A3 (rs3825926) and CLIC 5 (rs321329) genes. Conclusions: These data provide some evidence for a link between biology of the glomerular protein nephrin and antihypertensive action of angiotensin receptor antagonists and encourage additional studies on aldehyde dehydrogenase–mediated reactions in antihypertensive drug action.