Date Published: January 1, 2019
Publisher: American Physiological Society
Author(s): Nicholas J. Hunt, Glen P. Lockwood, Alessandra Warren, Hong Mao, Peter A. G. McCourt, David G. Le Couteur, Victoria C. Cogger.
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.
Liver sinusoidal endothelial cells (LSECs) have a unique morphology that promotes bidirectional exchange of substrates between the lumen of the hepatic sinusoid and the surrounding hepatocytes (9). This transfer function is thought to be predominantly performed by transcellular pores called fenestrations, located within the cytoplasmic extensions of LSECs. Fenestrations are 30–300 nm in diameter, and the majority of them are arranged in groups of 10–100 called liver sieve plates (9, 18). We have previously demonstrated that fenestrations act as conduits for lipoproteins (20), pharmacological agents (26), and insulin (27). In health, size and number of fenestrations are dynamic and responsive to various stimuli, such as fasting, alcohol, and various other chemicals (9), but it is also recognized that the overall size and number of fenestrations are reduced in chronic disease and aging (22). Loss of fenestrations in these and other experimental settings is mechanistically linked to impairment of the transfer of substrates and contributes to hyperlipidemia and insulin resistance (21, 27).
Male C57/BL6 mice, 3–4 and 18–24 mo old, were obtained from the Animal Resource Centre in Perth, Western Australia. Animals were housed at the ANZAC Research Institute animal house on a 12-h:12-h light-dark cycle and provided with ad libitum access to food and water. Mice were not fasted before euthanasia by a single intraperitoneal injection with 100 mg/kg ketamine and 10 mg/kg xylazine in saline. The study was approved by the Animal Welfare Committee of the Sydney Local Health District and was performed in accordance with the Australian Code of Practice for the care and use of animals for scientific research (AWC 2016/009). All information provided accords with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.
The morphology of fenestrations in LSECs is responsive to a variety of pharmacological interventions, and this responsiveness is mostly maintained into older age. LSECs isolated from old mice in this study had reduced porosity and frequency of fenestrations, consistent with previous studies in mice as well as rats, humans, and nonhuman primates (9, 23, 25). NMN, sildenafil, and 7-ketocholesterol increased fenestration porosity and frequency in young mice, with similar or greater effects seen in LSECs from old mice (summary data provided in Table 4). This indicates that age-related defenestration can be reversed in vitro and may be a valid therapeutic target for in vivo studies. Moreover, the optimal concentrations of these refenestrating agents were identified in LSECs from old mice, providing a potential target dose for in vivo studies. The results of the dSTORM studies showed that refenestration was associated with significant actin reorganization. Increased NOS protein expression was also seen in LSECs treated with amlodipine, sildenafil, and TRAIL, while sildenafil was the only agent associated with increased phosphorylation of NOS. Overall, our study indicates that agents that increased fenestrations are associated with an alteration of the actin cytoskeleton and in some cases, release of NO; importantly, this responsiveness is maintained in old age.
The study was supported by Australian National Health and Medical Research Council Project no. 1141234 and the Aging and Alzheimer’s Research Foundation (a Division of the Medical Foundation of the University of Sydney).
No conflicts of interest, financial or otherwise, are declared by the authors.
N.J.H., G.P.L., A.W., H.M., and P.A.G.M. performed experiments; N.J.H., G.P.L., and A.W. analyzed data; N.J.H., D.G.L.C., and V.C.C. interpreted results of experiments; N.J.H. prepared figures; N.J.H. drafted manuscript; N.J.H., D.G.L.C., and V.C.C. edited and revised manuscript; N.J.H., D.G.L.C., and V.C.C. approved final version of manuscript.