Adeno-associated virus-mediated expression of acid sphingomyelinase decreases atherosclerotic lesion formation in apolipoprotein E(-/-) mice.

BACKGROUND: The secretory form of acid sphingomyelinase (ASM) is postulated to play a key role in the retention and aggregation of lipoproteins in the subendothelial space of the arterial wall by converting sphingomyelin in lipoproteins into ceramide. The present study aimed to determine whether the level of circulating ASM activity affects lesion development in mouse model of atherosclerosis. METHODS: Apolipoprotein E deficient (ApoE(-/-) ) mice were injected intravenously with a recombinant adeno-associated virus (AAV8-ASM) that constitutively expressed high levels of human ASM in liver and plasma. RESULTS: Plasma sphingomyelin levels were reduced at early but not later time points after the administration of AAV8-ASM despite persistently elevated circulating ASM. No change in serum lipoprotein levels was observed. Thirteen or 17 weeks after the administration of AAV8-ASM, the amount of plaque formation in the aortic sinus was comparable to that of mice treated with a control AAV. CONCLUSIONS: Unexpectedly, the lesion area of the entire aorta was reduced significantly in the AAV8-ASM virus-treated group. Hepatic expression and secretion of ASM into the circulation did not accelerate or exacerbate, but rather decreased, lesion formation in ApoE(-/-) mice. Thus, plasma ASM activity does not appear to be rate limiting for plaque formation during atherogenesis.

Inhibition of osteoclastogenesis by prolyl hydroxylase inhibitor dimethyloxallyl glycine.

Studies examining the effects of hypoxia upon osteoclast biology have consistently revealed a stimulatory effect; both osteoclast differentiation and resorption activity have been shown to be enhanced in the presence of hypoxia. In the present study we examined the effects of the hypoxia mimetics dimethyloxallyl glycine (DMOG) and desferrioxamine (DFO) upon osteoclastogenesis. In contrast to hypoxia, our studies revealed a dose-dependent inhibition of osteoclast formation from macrophages treated with DMOG and DFO. Moreover, expression of a constitutively active form of hypoxia-inducible factor 1alpha (HIF-1alpha) did not enhance osteoclastogenesis and actually attenuated the differentiation process. DMOG did not affect cell viability or receptor activator of nuclear factor kappaB ligand (RANKL)-dependent phosphorylation of mitogen-activated protein (MAP) kinases. However, RANKL-dependent transcription of tartrate-resistant acid phosphatase (TRAP) was reduced in the presence of DMOG. Additionally, DMOG promoted transcription of the pro-apoptotic mediator B-Nip3. These studies suggest that a hypoxia-responsive factor other than HIF-1alpha is necessary for enhancing the formation of osteoclasts in hypoxic settings.

Systemic delivery of a Peptide-linked morpholino oligonucleotide neutralizes mutant RNA toxicity in a mouse model of myotonic dystrophy.

Expansions of CUG trinucleotide sequences in RNA transcripts provide the basis for toxic RNA gain-of-function that leads to detrimental changes in RNA metabolism. A CTG repeat element normally resides in the 3' untranslated region of the dystrophia myotonica-protein kinase (DMPK) gene, but when expanded it is the genetic lesion of myotonic dystrophy type 1 (DM1), a hereditary neuromuscular disease. The pathogenic DMPK transcript containing the CUG expansion is retained in ribonuclear foci as part of a complex with RNA-binding proteins such as muscleblind-like 1 (MBNL1), resulting in aberrant splicing of numerous RNA transcripts and consequent physiological abnormalities including myotonia. Herein, we demonstrate molecular and physiological amelioration of the toxic effects of mutant RNA in the HSA(LR) mouse model of DM1 by systemic administration of peptide-linked morpholino (PPMO) antisense oligonucleotides bearing a CAG repeat sequence. Intravenous administration of PPMO conjugates to HSA(LR) mice led to redistribution of Mbnl1 protein in myonuclei and corrections in abnormal RNA splicing. Additionally, myotonia was completely eliminated in PPMO-treated HSA(LR) mice. These studies provide proof of concept that neutralization of RNA toxicity by systemic delivery of antisense oligonucleotides that target the CUG repeat is an effective therapeutic approach for treating the skeletal muscle aspects of DM1 pathology.