Nicholas J. Hunt [1, 2, 3], Glen Lockwood [1, 2, 3], Sophie Sun Woo Kang [1, 2, 3], Alessandra Warren , Hong Mao , Peter McCourt [3, 4], David G. Le Couteur [1, 2, 3], Victoria C. Cogger [1, 2, 3]
 Centre for Education and Research on Ageing, Concord Repatriation General Hospital, Sydney, Australia
 Biogerontology Group, ANZAC Research Institute, Concord Repatriation General Hospital, Sydney, Australia
 Nutritional Ecology Group, Charles Perkins Centre, University of Sydney, Sydney, Australia
 Department of Medical Biology, University of Tromsø, Norway
Fenestrations are pores within liver sinusoidal endothelial cells (LSECs) that enable the transfer of substrates (such as insulin and some lipoproteins) between blood and hepatocytes. With increasing age there are marked changes in the LSEC, referred to as pseudocapillarization, which include reduction in the frequency and diameter of fenestrations. Currently it is thought that fenestrations are regulated by pathways, particularly vascular endothelial growth factor and nitric oxide (NO), that lead to remodelling of the actin cytoskeleton and cell membrane lipid rafts. Here we investigated the effects of drugs that act on these pathways on fenestration porosity, diameter and frequency in old (18-24m) and young mice (3-4m). Isolated LSECs were incubated for 30 mins with either: cytochalasin D, 7-ketocholesterol, sildenafil, amlodipine, metformin, TNF-related apoptosis-inducing ligand (TRAIL) or nicotinamide mononucleotide (NMN). To extend on this work we examined NO mechanistic pathways promoted via these drugs using in vitro cell cultures and the SK-HEP1 cell line. 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, metformin and cytochalasin D. 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 re-fenestration of LSECs from old mice. Regulation of fenestrations occurs via both NO-dependent and -independent pathways. Age-related defenestration can be reversed pharmacologically which has potential translational relevance for dyslipidemia and insulin resistance in old age.