Manipulating fenestrations in young and old liver sinusoidal endothelial cells.

Nicholas J Hunt,Glen P Lockwood,Victoria C Cogger,Hong Mao,Alessandra Warren,David G Le Couteur,Peter A G Mccourt

doi:10.1152/ajpgi.00179.2018

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.

Highlights

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]
We studied drugs that act on the pathways that influence nitric oxide (NO) [sildenafil [2, 14], amlodipine [24, 46], and simvastatin [35, 38]], serotonergic pathway/phospholipase C (DOI) [31], endothelin receptor [34], death receptor (TNF-related apoptosis-inducing ligand, TRAIL) [7], and NADϩ [5] in mice using scanning electron microscopy (SEM) and Direct stochastic optical reconstruction microscopy (dSTORM)
The morphology of fenestrations in LSECs is responsive to a variety of pharmacological interventions, and this responsiveness is mostly maintained into older age

Summary

Introduction

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]. 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]

Methods

Results

Conclusion