High-throughput chemical modification of oligonucleotides for systematic structure-activity relationship evaluation.

Chemical modification of siRNA is achieved in a high-throughput manner (96-well plate format) by copper catalyzed azide-alkyne cycloadditions. This transformation can be performed in one synthetic operation at up to four positions with complete specificity, good yield, and acceptable purity. As demonstrated here, this approach extends the current synthetic options for oligonucleotide modifications and simultaneously facilitates the systematic, rapid biological evaluation of modified siRNA.

Molecular umbrella conjugate for the ocular delivery of siRNA.

The synthesis, computer modeling, and biological activity of an octawalled molecular umbrella short interfacing RNA (siRNA) conjugate is described. This molecular umbrella-siRNA conjugate exhibited mRNA knockdown activity in vitro in the absence of a transfection reagent. Evaluation of this molecular umbrella conjugate in vivo, using the rat eye via intravitreal injection, resulted in sequence specific mRNA knockdown in the retina with no obvious signs of toxicity, as judged by ophthalmic examination.

In vivo activation of peroxisome proliferator-activated receptor-delta protects the heart from ischemia/reperfusion injury in Zucker fatty rats.

Peroxisome proliferator-activated receptor (PPAR)-delta is a transcription factor that belongs to the PPAR family. PPAR-delta is abundantly expressed in the heart, and its role in the heart is largely unknown. We tested whether pharmacological activation of PPAR-delta protects the heart from ischemia/reperfusion (I/R) injury in male Zucker fatty rats, a rodent model of obesity and dyslipidemia. A highly selective PPAR-delta agonist, [4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl] thio]-2-methylphenoxy]acetic acid (GW0742), was administered for 7 days at 10 mg/kg/day (p.o., once a day). Ischemic injury was produced by occlusion of the left anterior descending artery for 30 min followed by reperfusion for up to 24 h. Treatment with GW0742 reduced serum levels of cardiac troponin-I and infarct size by 63% (p < 0.01) and 32% (p < 0.01), respectively, and improved left ventricular function. Treatment with GW0742 up-regulated gene expression involved in cardiac fatty acid oxidation, increased fat use in the heart, and reduced serum levels of free fatty acids. The enhanced cardiac expression of interleukin (IL)-6, IL-8, intercellular adhesion molecule-1, and monocyte chemoattractant protein-1 induced by I/R were significantly attenuated by GW0742. Treatment with GW0742 also reduced apoptotic cardiomyocytes by 34% and cardiac caspase-3 activity by 61% (both p < 0.01 versus vehicle). GW0742 differentially regulated Bcl family members, favoring cell survival, and attenuated I/R-induced cardiac mitochondrial damage. In addition, GW0742 treatment augmented the cardiac Akt signaling pathway, as reflected by enhanced phospho-3-phosphoinositide-dependent kinase-1 and p-Akt. The results indicate that activation of PPAR-delta protected the heart from I/R injury in Zucker fatty rats, and multiple mechanisms including amelioration of lipotoxicity, anti-inflammation, and up-regulation of prosurvival signaling contribute together to the cardioprotection.

Effects of p38 MAPK Inhibitor on angiotensin II-dependent hypertension, organ damage, and superoxide anion production.

Angiotensin II (Ang II) activates p38 mitogen-activated protein kinase (p38 MAPK) and increases reactive oxygen species (ROS), but the nature of the relationship in vivo is not fully understood. We assess the effect of SB239063AN, a highly selective, orally active, p38 MAPK inhibitor, on Ang II-dependent hypertension, target-organ damage and ROS production. Sprague-Dawley rats and MAPKAP kinase-2 knockout mice were infused with Ang II. Ang II infusion increased the levels of phosphorylated p38 MAPK in the heart and aorta. Production of superoxide anion and expression of NAD(P)H oxidase subunit gp91 in the aorta were increased 4- and 5-fold, respectively. In addition, Ang II infusion led to endothelial dysfunction, progressive and sustained hypertension, and cardiac hypertrophy. Treatment with SB239063AN (800 ppm in the diet) significantly attenuated the levels of phosphorylated p38 MAPK in the heart and aorta, reduced superoxide anion generation by 57% (P < 0.01), markedly suppressed gp91 mRNA expression, prevented endothelial dysfunction, and blunted both the hypertension and cardiac hypertrophy. Ang II-dependent hypertension was also significantly attenuated in MAPKAP kinase-2 knockout mice. The results suggest that Ang II induced hypertension, organ damage, and ROS production are possibly mediated by p38 MAPK and inhibition of p38 MAPK may offer a therapeutic approach for cardiovascular disease.

p38 MAPK inhibitors ameliorate target organ damage in hypertension: Part 2. Improved renal function as assessed by dynamic contrast-enhanced magnetic resonance imaging.

Recent evidence suggests p38 mitogen-activated protein kinase (MAPK) signal transduction plays an important role in the pathogenesis of progressive renal disease. Using dynamic contrast enhanced magnetic resonance imaging (MRI), we evaluated chronic treatment with a p38 MAPK inhibitor, trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl-methoxypyridimidin-4-yl)imidazole (SB-239063), on renal function in a hypertension model of progressing renal dysfunction. Spontaneously hypertensive-stroke prone rats were placed on a high salt/fat diet (SFD) or maintained on normal chow diet (ND). SFD animals with albuminuria at 4 to 8 weeks (> or =10 mg/day inclusion criteria), were randomized into p38 MAPK inhibitor treatment (SB-239063, 1200 ppm in diet) or vehicle groups. The progression of blood pressure and albuminuria during the treatment period (approximately 6 weeks) was decreased by 12 and 60%, respectively, in the SFD + SB-239063 versus SFD control group. Renal perfusion and filtration were assessed by in vivo MRI at the end of the study. Relative cortical perfusion was increased in the SFD + SB-239063 group compared with the SFD control group as reflected by a 29% decrease in time to peak of contrast agent in the cortex. Additionally, the regional renal glomerular filtration rate index (Kcl) was increased by 39% in the SFD + SB-239063 versus SFD control group and was normalized to the ND control group. Greater functional heterogeneity was observed in the SFD control versus SFD + SB-239063 or ND control group. All alterations of renal function were supported by histopathological findings. In conclusion, chronic treatment with a p38 MAPK inhibitor, SB-239063, attenuates functional and structural renal degeneration in a hypertensive model of established renal dysfunction.