Senescent cells secrete bioactive molecules, including reactive oxygen species (ROS) and pro-inflammatory cytokines. Conversely, pro-inflammatory signalling stabilizes the senescent phenotype. Cell ...senescence and chronic low-level inflammation increase with age have been proposed as causal factors in ageing. In this thesis I demonstrate that senescent cells accumulate with age in mouse liver and are temporally and spatially associated with markers of oxidative stress and inflammation, including activation of hepatic stellate cells which is generally associated with fibrosis. Moreover, I show that hepatocyte senescence is driven by DNA damage at the level of telomeres as shown by an age-dependent exponential Increase in the number of TIF, occurring independently of telomere length. In order to test experimentally, a potential link between inflammation and cellular senescence, I analyzed markers of cell senescence and features of age-related functional decline in several tissues from mice lacking the transcription factor nfkb1-1. I found that compromised signalling through NF-KB led to a pro-inflammatory phenotype and stabilized cell senescence in vitro as well as in multiple tissues in vivo. Accordingly, regenerative capacity in the liver, absorptive area in the intestine, epidermal thickness of the skin and accumulated fat mass in various depots were all reduced and both healthspan and lifespan of nfkb 1-1- mice were shortened. This data shows for the first time that widespread cell senescence resulting from persistent pro- inflammatory signalling can seriously accelerate mammalian ageing.
Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor ...delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.
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