The objective of this study was to determine whether nitric oxide (NO) is responsible for the vascular smooth muscle relaxation elicited by endothelium-derived relaxing factor (EDRF). EDRF is an unstable humoral substance released from artery and vein that mediates the action of endothelium-dependent vasodilators. NO is an unstable endothelium-independent vasodilator that is released from vasodilator drugs such as nitroprusside and glyceryl trinitrate. We have repeatedly observed that the actions of NO on vascular smooth muscle closely resemble those of EDRF. In the present study the vascular effects of EDRF released from perfused bovine intrapulmonary artery and vein were compared with the effects of NO delivered by superfusion over endotheliumdenuded arterial and venous strips arranged in a cascade. EDRF was indistinguishable from NO in that both were labile (1i/2 = 3-5 sec), inactivated by pyrogallol or superoxide anion, stabilized by superoxide dismutase, and inhibited by oxyhemoglobin or potassium. Both EDRF and NO produced comparable increases in cyclic GMP accumulation in artery and vein, and this cyclic GMP accumulation was inhibited by pyrogallol, oxyhemoglobin, potassium, and methylene blue. EDRF was identified chemically as NO, or a labile nitroso species, by two procedures. First, like NO, EDRF released from freshly isolated aortic endothelial cells reacted with hemoglobin to yield nitrosylhemoglobin. Second, EDRF and NO each similarly promoted the diazotization of sulfanilic acid and yielded the same reaction product after coupling with N-(1-naphthyl)-ethylenediamine. Thus, EDRF released from artery and vein possesses identical biological and chemical properties as NO.Both artery and vein are capable of releasing endotheliumderived relaxing factor (EDRF) in response to chemically diverse vasodilators (1-9). Endothelium-dependent relaxation of artery and vein appears to be mediated by increases in tissue cyclic GMP levels (10-13), and such effects are inhibited by methylene blue, hemoglobin, and myoglobin (13-15). Nitroso compounds, organic nitrate and nitrite esters, and inorganic nitrite cause vascular smooth muscle relaxation and cyclic GMP accumulation by endotheliumindependent mechanisms, and these actions are attributed to the release of nitric oxide (NO) (16)(17)(18)(19). NO itself is a labile substance that causes transient relaxation and cyclic GMP accumulation in both artery and vein (16)(17)(18)(19)(20) MATERIALS AND METHODSReagents. Acetylcholine chloride, phenylephrine hydrochloride, A23187, pyrogallol, hemoglobin (human), and superoxide dismutase (bovine liver) were obtained from Sigma. Glyceryl trinitrate (10% wt/wt triturate in lactose) was a gift from Imperial Chemical Industries (Macclesfield, England), and propylbenzylylcholine mustard was provided by the National Institute for Medical Research (Mill Hill, London). NO (99o pure) was obtained from Matheson. A saturated solution of NO (1-2 mM) in oxygen-free water (prepared by vacuum evacuation and nitrogen flushing) was pre...

SUMMARY Alu RNA accumulation due to DICER1 deficiency in the retinal pigmented epithelium (RPE) is implicated in geographic atrophy (GA), an advanced form of age-related macular degeneration that causes blindness in millions of individuals. The mechanism of Alu RNA-induced cytotoxicity is unknown. Here we show that DICER1 deficit or Alu RNA exposure activates the NLRP3 inflammasome and triggers TLR-independent MyD88 signaling via IL-18 in the RPE. Genetic or pharmacological inhibition of inflammasome components (NLRP3, Pycard, Caspase-1), MyD88, or IL-18 prevents RPE degeneration induced by DICER1 loss or Alu RNA exposure. These findings, coupled with our observation that human GA RPE contains elevated amounts of NLRP3, PYCARD and IL-18, and evidence of increased Caspase-1 and MyD88 activation, provide a rationale for targeting this pathway in GA. Our findings also reveal a function of the inflammasome outside the immune system and an immunomodulatory action of mobile elements.

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell death. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.

Estradiol is known to exert a protective effect against the development of atherosclerosis, but the mechanism of this hormonal action is unknown. One of the early events in the development of atherosclerosis is the adhesion of macrophages to endothelial cells, and nitric oxide (NO) inhibits this process. We show that basal release of NO is greater with endothelium-intact aortic rings from female rabbits than those from males. Oophorectomy diminishes both circulating estradiol concentration and basal release of NO to levels seen in male rabbits. These data establish that basal NO release from endothelium-intact aortic rings depends on circulating estradiol concentration and offer an explanation for the protective effect of estradiol against the development of atherosclerosis.

We previously used high-density expression arrays to interrogate a genetic cross between strains C3H/HeJ and C57BL/6J and observed thousands of differences in gene expression between sexes. We now report analyses of the molecular basis of these sex differences and of the effects of sex on gene expression networks. We analyzed liver gene expression of hormone-treated gonadectomized mice as well as XX male and XY female mice. Differences in gene expression resulted in large part from acute effects of gonadal hormones acting in adulthood, and the effects of sex chromosomes, apart from hormones, were modest. We also determined whether there are sex differences in the organization of gene expression networks in adipose, liver, skeletal muscle, and brain tissue. Although coexpression networks of highly correlated genes were largely conserved between sexes, some exhibited striking sex dependence. We observed strong body fat and lipid correlations with sex-specific modules in adipose and liver as well as a sexually dimorphic network enriched for genes affected by gonadal hormones. Finally, our analyses identified chromosomal loci regulating sexually dimorphic networks. This study indicates that gonadal hormones play a strong role in sex differences in gene expression. In addition, it results in the identification of sex-specific gene coexpression networks related to genetic and metabolic traits.