Abstract
Introduction: Sickle cell disease (SCD) is an autosomal recessive genetic disorder that affects ~100,000 African Americans and over 8 million people worldwide. Sickling of red blood cells (RBCs) promotes hemolysis, leading to the release of cell-free hemoglobin (Hb), which acts as a damage-associated molecular pattern (DAMP) molecule and promotes tissue damage. Research over the last decade has documented a cascade of events associated with cell-free Hb accumulation in SCD, including increased adhesion of leukocytes and red blood cells (RBCs) to the endothelium, nitric oxide scavenging, Hb-heme-induced TLR4 pathway activation, elevated oxidative stress and sterile inflammation-all of which correlate with disrupted blood flow, exacerbate vaso-occlusive pain crises, and lead to end-organ damage and increased mortality. In the absence of a functional spleen, the liver plays a predominant role in cell-free Hb clearance in SCD. Recently, we have shown that, along with hepatic Kupffer cells, liver sinusoidal endothelial cells (LSECs) also contribute to Hb clearance in both SCD mice and patient liver tissue. We have also shown that cell-free Hb accumulation leads to LSEC senescence and dysfunction. However, the exact role of Hb accumulation-driven LSEC dysfunction in promoting SCD pathophysiology is not well understood.
Method: In this study, we sought to understand the effect of LSEC-specific Hb accumulation on regulating vWF expression, homeostasis, and promoting SCD disease pathophysiology utilizing two different acute models (oxyhemoglobin (oxy-Hb) and LPS treatment in SCD mice) of LSEC-specific Hb accumulation.
Results: Consistent with previous reports, SCD mice at baseline exhibited an enhanced expression in total hepatic vWF protein as seen by western blot analysis. We next examined vWF expression in acute models of LSEC Hb accumulation. Remarkably, oxy-Hb or LPS caused a significant increase in vWF expression as well as enhanced cleavage in the liver. As LSECs promote vWF synthesis, secretion, and clearance, we next examined these processes in SCD mice liver at baseline and post-acute Hb challenge. Remarkably, whereas vWF synthesis, multimerization, and secretion appeared unaffected in SCD mice post-acute Hb challenge, we found delayed vWF clearance and increased vWF accumulation in hepatic Kupffer cells and LSECs. Co-immunoprecipitation and proximity ligation assays confirmed a direct binding of Hb with vWF in LSECs of SCD mice. Mechanistically, we found that LSEC Hb accumulation significantly reduces the expression of the scavenger receptors stabilin 1 and 2, which then inhibits hepatic vWF clearance in SCD mice post-Hb challenge.
Conslusion: Our study identifies LSECs as key regulators of vWF homeostasis in sickle cell disease. These findings reveal endothelial Hb accumulation as a previously unrecognized driver of SCD pathophysiology and suggest that targeting hepatic Hb clearance pathways could offer new therapeutic strategies to reduce vascular complications and improve patient outcomes.
