Hemolysis is a key characteristic of sickle cell disease (SCD) that contributes to the disease pathogenesis and clinical heterogeneity. In SCD, cell free hemoglobin and its byproducts (heme and iron) cause vasculopathy as well as a slew of clinical consequences, including liver failure. Although liver injury affects up to ~40% of hospitalized SCD patients, therapeutic approaches to prevent liver injury in SCD are limited. In the Pradhan-Sundd lab we are interested in understanding the molecular (signalling) mechanism of hemolysis induced acute and chronic liver injury. We are also investigating the processes of hepatic hemoglobin, heme, and iron clearance in SCD.

Hemophilia A is an X-linked recessive bleeding disorder caused by the absence of coagulation factor- VIII (FVIII). Individuals who are affected are at risk of spontaneous bleeding into joints which can be life threatening. Recent advances in liver-directed gene transfer suggest that gene therapy can successfully treat hemophilia A. FVIII is produced in the liver sinusoidal endothelial cells. Thus, liver sinusoidal endothelial cell viability and functioning are important for successful liver directed gene transfer in Hemophilia. We have recently shown liver sinusoidal endothelial maladaptive structural changes in FVIII-deficient mice and their deleterious impact on the efficacy of liver-directed gene transfer. Abnormal endothelial function was recently recognized in patients with hemophilia A. Our current research focuses on investigating the molecular mechanisms that contribute to the loss of endothelial fenestration in individuals with FVIII deficiency, and how this loss impacts liver-directed gene therapy in patients with hemophilia.

Hemolysis is a key characteristic of sickle cell disease (SCD) that contributes to the disease pathogenesis and clinical heterogeneity. In SCD, cell-free hemoglobin and its byproducts (heme and iron) cause vasculopathy as well as a slew of clinical consequences, including liver failure. Although liver injury affects up to 40% of hospitalized SCD patients, therapeutic approaches to prevent liver injury in SCD are limited.

In the Pradhan-Sundd lab, we are interested in understanding the molecular (signaling) mechanism of hemolysis-induced acute and chronic liver injury. We are also investigating the processes of hepatic hemoglobin, heme and iron clearance in SCD. Some parts of this research are K01 funded.

P-selectin inhibition has been shown to prevent vaso-occlusive events in SCD patients. However, the chronic effect of P-selectin inhibition in SCD remains to be determined. We used quantitative liver intravital microscopy, molecular biology and biochemical techniques in our recently generated P-selectin deficient SCD mice to evaluate the effect of chronic P-selectin deficiency in the liver and spleen.

Using quantitative liver intravital microscopy, we recently showed that chronic P-selectin deficiency attenuates liver ischemia, but fails to prevent hepatobiliary injury (Blood, 2021). Remarkably, we found that this failure in resolution of hepatobiliary injury in P-selectin deficient SCD mice is associated with an increase in cellular senescence and reduced epithelial cell proliferation in the liver. These findings highlight the importance of investigating the long-term effects of chronic P-selectin inhibition therapy on liver pathophysiology in SCD patients. Some parts of this project are funded by the ASH Junior Faculty Scholar Award.

Chronically transfused SCD patients develop severe iron overload in liver, heart, spleen and endocrine organs with increased expression of inflammatory markers and mortality. Similarly increased hemolysis due to hemoglobin polymerization leads to continuous accumulation of iron particles in SCD. However, baseline changes in hepatic iron metabolism and homeostasis due to ongoing hemolysis are less understood in SCD.

Under normal physiological conditions, iron is predominantly stored in hepatocytes as ferritin form. Apart from hepatocytes, Kupffer cells (hepatic macrophages) are also involved in iron metabolism through engulfing and phagocytosing iron particles and promoting iron homeostasis in the liver. Previous studies have shown that iron accumulation from increased hemolysis associated with SCD activated macrophages to an M1-like proinflammatory phenotype via ROS and TLR4-controlled signaling, which was preventable by heme scavengers or iron chelators.

Although macrophage activation is a known phenotype of SCD, the role of tissue-specific macrophages in SCD-induced chronic organ injury initiation, progression or maintenance is not well understood. We are interested in understanding the role of Kupffer cells in SCD iron homeostasis. Additionally, we are interested in identifying novel biomarkers of hepatic iron overload using mouse models, basic biochemistry, molecular biology and imaging techniques.

Hemophilia A is an X-linked, recessive bleeding disorder caused by the absence of coagulation factor VIII (FVIII). Individuals who are affected are at risk of spontaneous bleeding into joints, which can be life-threatening. Recent advances in liver-directed gene transfer suggest that gene therapy can successfully treat hemophilia A. FVIII is produced in the liver sinusoidal endothelial cells. Thus, liver sinusoidal endothelial cell viability and functioning are important for successful, liver-directed gene transfer in hemophilia. We have recently shown liver sinusoidal, endothelial, maladaptive structural changes in FVIII-deficient mice and their deleterious impact on the efficacy of liver-directed gene transfer. Abnormal endothelial function was recently recognized in patients with hemophilia A. Our current research focuses on investigating the molecular mechanisms that contribute to the loss of endothelial fenestration in individuals with FVIII deficiency, and how this loss impacts liver-directed gene therapy in patients with hemophilia.

Sickled red blood cells (RBCs) undergo untimely senescence and need to be cleared from circulation by reticuloendothelial macrophages to prevent persistent organ damage. However, it’s unclear how senescent RBCs are eliminated. Current research suggests that in under-stressed conditions, the liver is the primary site of RBC clearance. Recent reports have suggested that liver sinusoidal endothelial cells (LSECs) aid in the tethering of aged RBCs within hepatic sinusoids, thus facilitating their engulfment by liver macrophages called Kupffer cells.

Recently, we showed that SCD mice manifest exacerbated liver senescence and progressive liver injury under baseline conditions. Here, we will use state-of-the-art imaging, molecular biology, biochemistry and advanced omics approaches to identify the mechanism of RBC clearance in SCD liver and the effect of RBC senescence in chronic liver injury.