Abstract
Fenestrations are pores within liver sinusoidal endothelial cells (LSECs) that enable the transfer of substrates (particularly insulin and lipoproteins) between blood and hepatocytes. With increasing age, there are marked reductions in fenestrations, referred to as pseudocapillarization. Currently, fenestrations are thought to be regulated by vascular endothelial growth factor and nitric oxide (NO) pathways promoting remodeling of the actin cytoskeleton and cell membrane lipid rafts. We investigated the effects of drugs that act on these pathways on fenestrations in old (18-24 mo) and young mice (3-4 mo). Isolated LSECs were incubated with either cytochalasin 7-ketocholesterol, sildenafil, amlodipine, simvastatin, 2, 5-dimethoxy-4-iodoamphetamine (DOI), bosentan, TNF-related apoptosis-inducing ligand (TRAIL) or nicotinamide mononucleotide (NMN). LSECs were visualized under scanning electron microscopy to quantify fenestration porosity, diameter, and frequency, as well as direct stochastic optical reconstruction microscopy to examine actin and NO synthase. In young and old LSECs, fenestration porosity, diameter and frequency were increased by 7-ketocholesterol, while porosity and/or frequency were increased with NMN, sildenafil, amlodipine, TRAIL, and cytochalasin D. In old mice only, bosentan and DOI increased fenestration porosity and/or frequency. Modification of the actin cytoskeleton was observed with all agents that increased fenestrations, while NO synthase was only increased by sildenafil, amlodipine, and TRAIL. In conclusion, agents that target NO, actin, or lipid rafts promote changes in fenestrations in mice LSECs. Regulation of fenestrations occurs via both NO-dependent and independent pathways. This work indicates that age-related defenestration can be reversed pharmacologically, which has potential translational relevance for dyslipidemia and insulin resistance. NEW & NOTEWORTHY We demonstrate the effects of multiple nitric oxide-dependent and -independent pharmaceutical agents on fenestrations of the liver sinusoidal endothelium. Fenestrations are reorganized in response to nicotinamide mononucleotide, sildenafil, amlodipine, and TNF-related apoptosis-inducing ligand. This work indicates that age-related defenestration can be reversed pharmacologically, which has potential translational relevance for dyslipidemia and insulin resistance in old age.