Hepatic stellate cells retain retinoid-laden lipid droplets after cellular transdifferentiation into activated myofibroblasts

Abstract

<p class="first" id="d568532e212">Loss of retinyl ester (RE)-rich lipid droplets (LDs) from hepatic stellate cells (HSCs) is cited as a key event in their cellular transdifferentiation to activated, pro-fibrotic myofibroblasts; however, it remains unclear if changes in LD morphology or RE content are causal for transdifferentiation. To better understand LD dynamics in vitro within a common model of HSC activation, we used novel approaches preserving LD morphology and allowing for quantitation of RE. The size and quantity of LDs within in vitro and in vivo bile duct ligation (BDL)-activated HSCs were quantitated using adipocyte differentiation-related protein (ADRP) labeling and oil red o (ORO) staining (gold standard), and RE content was determined using fluorescence microscopy. We found during HSC activation in vitro that LD number differed significantly when measured by ADRP and ORO, respectively ( <i>day 1</i>: 56 vs. 5, <i>P</i> = 0.03; <i>day 4</i>: 101 vs. 39, <i>P</i> = 0.03; <i>day 14</i>: 241 vs. 12, <i>P</i> = 0.02). Ex vivo HSCs activated in vivo contained the same number of LDs as <i>day 4</i> in vitro activated HSCs (118 vs. 101, <i>P</i> = 0.54). Decline in LD RE occurred beyond <i>day 4</i> in vitro and <i>day 1</i> ex vivo <i>,</i> after HSC transdifferentiation was underway. Lastly, in situ HSCs examined using electron microscopy show LDs tend to be smaller but are ultimately retained in BDL injured livers. Therefore, we conclude that during HSC transdifferentiation, LDs are not lost but are retained, decreasing in size. Additionally, RE content declines after transdifferentiation is underway. These data suggest that these LD changes are not causal for HSC transdifferentiation. </p><p id="d568532e248"> <b>NEW &amp; NOTEWORTHY</b> Loss of retinoid-laden lipid droplets from hepatic stellate cells has long been held as a hallmark of their transdifferentiation into activated myofibroblasts, the dominant cells that drive hepatic fibrosis. This study demonstrates that stellate cells activated in culture and after liver injury in vivo retain their lipid droplets and that these droplets become smaller and more numerous, with decreases in droplet retinoid concentration occurring only after cellular transdifferentiation is underway. </p>