Announcement
Thank you for attending THSNA 2026. The virtual meeting is now closed.
Thank you for attending THSNA 2026. The virtual meeting is now closed.
Presentation Details
| Imanixil-mediated disruption of lipid trafficking impairs hepatic hemoglobin clearance in Sickle cell Disease AYYANAR SIVANANTHAM1, NANDHINE RAJASEKAR2, MARTA WOLOSOWICZ1, NICHOLAS SWENDROWSKI1, TIRTHADIPA PRADHAN-SUNDD2, 3. 1Thrombosis and Hemostasis Program, Versiti Blood Research Institute and Blood Center of Wisconsin, Milwaukee, WI, USA.2Transfusion Medicine, Vascular Biology and Cell Therapy Program, Versiti Blood Research Institute, Milwaukee, WI, USA.3Department of Cell Biology, Neurobiology and Anatomy, Medical college of Wisconsin, Milwaukee, WI, USA |
Abstract
Background: Intrahepatic accumulation of cell-free hemoglobin (Hb) is a major pathological driver of oxidative stress, sterile inflammation and hepatobiliary injury in sickle cell disease (SCD) and other hemolytic disorders. Accumulation of cell-free Hb is associated with several acute and chronic organ dysfunction in SCD. In the absence of a functional spleen, the liver plays a predominant role in Hb clearance in SCD. Our recent findings demonstrated that along with hepatic Kupffer cells, liver sinusoidal endothelial cells (LSECs) also internalize sickle hemoglobin (HbS). Moreover, using a small molecule inhibitor screen we further demonstrated that Hbs internalizes to LSECS through a slow, pH-dependent, lipid-dependent fluid-phase endocytic mechanism, where lipid supplementation significantly enhances Hb uptake and lipid depletion through simvastatin, nystatin, or the cholesterol-lowering agent Imanixil significantly impairs endothelial Hb internalization. As Hbs clearance is predominantly Kupffer cells (KCs) driven, we hypothesized that KC-mediated Hb clearance may also depend on cellular lipid availability in exposure to the sinusoidal milieu. Objectives: To determine whether disruption of lipid trafficking impairs HbS uptake in KCs and identify potential novel regulators of Hb trafficking in SCD. of SCD mice. Methods: Primary murine Kupffer cells were isolated and exposed to fluorescently labeled HbS. Imanixil, a cholesterol-lowering small molecule known to interfere with lipid trafficking and membrane-dependent endocytosis, was used to determine the requirement of lipid availability for KC HbS uptake. KCs were treated with Imanixil for 24 hours, followed by exposure to HbS for 30 minutes. Quantitative fluorescence uptake assays were performed to measure intracellular HbS. Extracellular Hb levels were assessed in cell supernatants, and KC activation was evaluated by measuring pro-inflammatory cytokines. Observations were compared with previously characterized lipid-dependent mechanisms in LSECs to determine mechanistic overlap between the two hepatic cell populations. Results: Pharmacologic disruption of lipid handling with Imanixil significantly impaired KC HbS internalization, demonstrating a critical dependence on cellular lipid availability. Imanixil-treated KCs showed reduced intracellular HbS, elevated extracellular Hb accumulation, and increased release of pro-inflammatory cytokines, indicating functional consequences of impaired Hb uptake. Importantly, these inhibitory effects were consistent with those previously observed in LSECs under lipid-depleted conditions. The magnitude and direction of lipid perturbation effects in KCs support a model in which both hepatocyte-associated phagocytes and endothelial cells rely on lipid-supported vesicular pathways to efficiently clear circulating Hb during hemolysis. Conclusion: These findings demonstrate that both LSECs and Kupffer cells depend on lipid-mediated endocytic pathways for efficient Hb clearance, establishing lipid availability as a major regulator of hepatic Hb regulation. Also, this study identifies lipid trafficking disruption as a mechanistic barrier to Hb detoxification and provides a foundation for future studies aimed at defining molecular lipid–endocytic pathway regulation and developing therapeutic strategies to mitigate hemolysis-associated liver injury in SCD.
No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author.
No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author.