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As FDA-approved small RNA drugs start to enter clinical medicine, ongoing studies for the microRNA (miRNA) class of small RNAs expand its preclinical and clinical research applications. A growing number of reports suggest a significant utility of miRNAs as biomarkers for pathogenic conditions, modulators of drug resistance, and/or as drugs for medical intervention in almost all human health conditions. The pleiotropic nature of this class of nonprotein-coding RNAs makes them particularly attractive drug targets for diseases with a multifactorial origin and no current effective treatments. As candidate miRNAs begin to proceed toward initiation and completion of potential phase 3 and 4 trials in the future, the landscape of both diagnostic and interventional medicine will arguably continue to evolve. In this mini-review, we discuss miRNA drug discovery development and their current status in clinical trials.

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease and accelerates atherosclerosis in apoE ؊/؊ mice. Despite the observations that homocysteine causes endoplasmic reticulum (ER) stress and programmed cell death (PCD) in cultured human vascular endothelial cells, the cellular factors responsible for this effect and their relevance to atherogenesis have not been completely elucidated. We report here that homocysteine induces the expression of T-cell death-associated gene 51 (TDAG51), a member of the pleckstrin homology-related domain family, in cultured human vascular endothelial cells. This effect was observed for other ER stress-inducing agents, including dithiothreitol and tunicamycin. TDAG51 expression was attenuated in homozygous A/A mutant eukaryotic translation initiation factor 2␣ mouse embryonic fibroblasts treated with homocysteine or tunicamycin, suggesting that ER stress-induced phosphorylation of eukaryotic translation initiation factor 2␣ is required for TDAG51 transcriptional activation. Transient overexpression of TDAG51 elicited significant changes in cell morphology, decreased cell adhesion, and promoted detachmentmediated PCD. In support of these in vitro findings, TDAG51 expression was increased and correlated with PCD in the atherosclerotic lesions from apoE ؊/؊ mice fed hyperhomocysteinemic diets, compared with mice fed a control diet. Collectively, these findings provide evidence that TDAG51 is induced by homocysteine, promotes detachment-mediated PCD, and contributes to the development of atherosclerosis observed in hyperhomocysteinemia.

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Background-A causal relation between hyperhomocysteinemia (HHcy) and accelerated atherosclerosis has been established in apolipoprotein E-deficient (apoE Ϫ/Ϫ ) mice. Although several cellular stress mechanisms have been proposed to explain the atherogenic effects of HHcy, including oxidative stress, endoplasmic reticulum (ER) stress, and inflammation, their association with atherogenesis has not been completely elucidated. Methods and Results-ApoEϪ/Ϫ mice were fed a control or a high-methionine (HM) diet for 4 (early lesion group) or 18 (advanced lesion group) weeks to induce HHcy. Total plasma homocysteine levels and atherosclerotic lesion size were significantly increased in early and advanced lesion groups fed the HM diet compared with control groups. Markers of ER stress (GRP78/94, phospho-PERK), oxidative stress (HSP70), and inflammation (phospho-IB-␣) were assessed by immunohistochemical staining of these atherosclerotic lesions. GRP78/94, HSP70, and phospho-IB-␣ immunostaining were significantly increased in the advanced lesion group fed the HM diet compared with the control group. HSP47, an ER-resident molecular chaperone involved in collagen folding and secretion, was also increased in advanced lesions of mice fed the HM diet. GRP78/94 and HSP47 were predominantly localized to the smooth muscle cell-rich fibrous cap, whereas HSP70 and phospho-IB-␣ were observed in the lipid-rich necrotic core. Increased HSP70 and phospho-IB-␣ immunostaining in advanced lesions of mice fed the HM diet are consistent with enhanced carotid artery dihydroethidium staining. Interestingly, GRP78/94 and phospho-PERK were markedly increased in macrophage foam cells from early lesions of mice fed the control or the HM diet.

Objective-Peroxynitrite, a potent oxidant generated by the reaction of NO with superoxide, has been implicated in the promotion of atherosclerosis. We designed this study to determine whether peroxynitrite induces its proatherogenic effects through induction of endoplasmic reticulum (ER) stress. Methods and Results-Human vascular endothelial cells treated with Sin-1, a peroxynitrite generator, induced the expression of the ER chaperones GRP78 and GRP94 and increased eIF2␣ phosphorylation. These effects were inhibited by the peroxynitrite scavenger uric acid. Sin-1 caused the depletion of ER-Ca 2ϩ , an effect known to induce ER stress, resulting in the elevation of cytosolic Ca 2ϩ and programmed cell death (PCD). Sin-1 treatment was also found, via 3-nitrotyrosine and GRP78 colocalization, to act directly on the ER. Adenoviral-mediated overexpression of GRP78 in endothelial cells prevented Sin-1-induced PCD. Consistent with these in vitro findings, 3-nitrotyrosine was observed and colocalized with GRP78 in endothelial cells of early atherosclerotic lesions from apolipoprotein E-deficient mice. Conclusions-Peroxynitrite is an ER stress-inducing agent. Its effects include the depletion of ER-Ca 2ϩ , a known mechanism of ER stress induction. The observation that 3-nitrotyrosine-containing proteins colocalize with markers of ER stress within early atherosclerotic lesions suggests that peroxynitrite contributes to atherogenesis through a mechanism involving ER stress. Key Words: endothelium Ⅲ nitric oxide Ⅲ endoplasmic reticulum Ⅲ atherosclerosis Ⅲ calcium E ndothelial dysfunction/injury represents a key early step in atherogenesis. The majority of risk factors for atherosclerosis, including, hyperlipidemia, hypertension, diabetes, and smoking, are associated with endothelial dysfunction. 1 A major function of the vascular endothelium is the regulation of vascular tone by NO through NO-induced smooth muscle relaxation. 2 Reduced NO bioavailability is a common feature in atherosclerosis and can result from oxidative stress. 3 In the apolipoprotein E-deficient (apoE Ϫ/Ϫ ) mouse model of atherosclerosis, endothelial dysfunction, as shown by decreased NO-mediated vasodilation to acetylcholine, correlates with increased atherosclerotic lesion size. 4 The availability of NO and superoxide (. O 2 -) within the atherosclerotic lesion creates the conditions for peroxynitrite formation because NO and . O 2 -react at physiological pH to form peroxynitrite. 5 A marker of peroxynitrite generation, 3-nitrotyrosine (3-NT) is elevated in human atherosclerotic lesions. 6,7 However, the mechanism by which reduced NO bioavailability and peroxynitrite formation contribute to atherosclerosis remains uncertain. See coverEndoplasmic reticulum (ER) stress, a cellular stress pathway induced by the accumulation of unfolded proteins in the ER, may offer an explanation for the contribution of peroxynitrite to atherosclerosis. ER stress results in an evolutionary conserved cellular response involving the upregulation of a set of genes, including ...

SummaryTo analyze plant mechanisms for resistance to UV radiation, mutants of Arabidopsis that are hypersensitive to UV radiation (designated uvh and uvr) have been isolated. UVR2 and UVR3 products were previously identi®ed as photolyases that remove UV-induced pyrimidine dimers in the presence of visible light. Plants also remove dimers in the absence of light by an as yet unidenti®ed dark repair mechanism and uvh1 mutants are defective in this mechanism. The UVH1 locus was mapped to chromosome 5 and the position of the UVH1 gene was further delineated by Agrobacterium-mediated transformation of the uvh1-1 mutant with cosmids from this location. Cosmid NC23 complemented the UV hypersensitive phenotype and restored dimer removal in the uvh1-1 mutant. The cosmid encodes a protein similar to the S. cerevisiae RAD1 and human XPF products, components of an endonuclease that excises dimers by nucleotide excision repair (NER). The uvh1-1 mutation creates a G to A transition in intron 5 of this gene, resulting in a new 3¢ splice site and introducing an in-frame termination codon. These results provide evidence that the Arabidopsis UVH1/AtRAD1 product is a subunit of a repair endonuclease. The previous discovery in Lilium longi¯orum of a homolog of human ERCC1 protein that comprises the second subunit of the repair endonuclease provides additional evidence for the existence of the repair endonuclease in plants. The UVH1 gene is strongly expressed in¯ower tissue and also in other tissues, suggesting that the repair endonuclease is widely utilized for repair of DNA damage in plant tissues.

Niemann-Pick type C (NPC) is an autosomal recessive disease characterized by impaired cholesterol homeostasis due to a defect in the intracellular transport of unesterified cholesterol. While accumulation of lysosomal cholesterol is the most apparent microscopic finding, cholesterol has also been shown to accumulate in the trans-cisternae of the Golgi apparatus. Caveolin-1, a cholesterol-binding protein that cycles between the Golgi apparatus and the plasma membrane, has been hypothesized to participate in cholesterol transport. Using the BALB/c murine model for NPC disease, we found that the expression of caveolin-1 in total liver homogenates from heterozygous and homozygous affected animals was altered. Immunoblot analysis of liver homogenates from heterozygous mice revealed that caveolin-1 is significantly (p < 0.005) elevated, 4.9 fold, compared to normal mice. Total liver homogenates from homozygous affected mice also had a significant (p < 0.05) increase in caveolin-1 expression, 1.6 fold, compared to normal mice. Immunohistochemical staining of liver cross-sections revealed that the increased caveolin-1 was localized to sinusoidal endothelial cells in heterozygous mice. The Triton insoluble floating fraction (TIFF) was isolated using liver from each genotype and analyzed for caveolin-1 expression. Caveolin-1 in the TIFF from heterozygous mice was significantly (p < 0.01) elevated, 1.8 fold, compared to normal and homozygous affected animals; normal and homozygous affected animals, however, were not significantly different from each other. The TIFF isolated from homozygous affected mice revealed a 15 fold increase in unesterified cholesterol compared to the TIFF isolated from heterozygous and normal mice. In summary, these findings demonstrate an altered expression of caveolin-1 in liver from heterozygous and homozygous NPC mice and increased concentration of cholesterol from TIFF in homozygous affected NPC mice. The identification of these alterations in the TIFF suggests involvement of detergent insoluble membrane structures, possibly caveolae and/or detergent insoluble glycosphingolipid-enriched complexes (DIGs), in the cholesterol trafficking defect in this disorder.