The role of Sphingosine 1-phosphate in the Recruitment and Retention of Haematopoietic Stem Cells in the Injured Liver
Lead Research Organisation:
University of Birmingham
Department Name: Immunity and Infection
Abstract
Chronic liver disease is an increasing cause of morbidity & mortality in the UK, for which there are limited treatment options. Stem cells within our bone marrow have the ability to migrate to the liver during injury and promote repair, and this has led to the use of stem cell infusions in clinical trials. However, stem cells only enter the liver in small numbers, so if we can better understand the factors that control their entry and retention within the liver, we will be able to improve their use as a therapy for liver disease.
We propose that S1P, a naturally occurring chemical within the body, is responsible for regulating the number of stem cells within the liver.
We will study the changes of S1P levels that occur in liver disease, and then conduct a series of experiments in which we alter the levels of S1P within the liver to determine what effect this has on the number of stem cells within the liver and also on liver repair.
This work will determine whether increasing levels of S1P in the liver increases the number of stem cells in the liver and in turn improves liver repair.
We propose that S1P, a naturally occurring chemical within the body, is responsible for regulating the number of stem cells within the liver.
We will study the changes of S1P levels that occur in liver disease, and then conduct a series of experiments in which we alter the levels of S1P within the liver to determine what effect this has on the number of stem cells within the liver and also on liver repair.
This work will determine whether increasing levels of S1P in the liver increases the number of stem cells in the liver and in turn improves liver repair.
Technical Summary
Recent studies have demonstrated that Haematopoietic Stem Cells (HSCs) promote hepatic repair and regeneration in rodents, with supportive early clinical trial data. As recruitment and delivery of HSCs to the liver is inefficient, there is a need to better understand the mechanisms regulating their hepatic recruitment and retention.
Sphingosine 1-phosphate (S1P), a bioactive lipid, plays a key role in regulating the recirculation of HSCs from tissue to bone marrow via an S1P-gradient. Egress of HSCs from non-lymphoid tissue (low S1P concentration) into lymph vessels (high S1P concentration) is governed by their expression of S1P1-receptors.
We hypothesise that modulation of hepatic S1P levels alters HSC recruitment and retention within the injured liver, and that enhanced HSC retention will lead to accelerated resolution of fibrosis and increased hepatocyte proliferation.
To determine the effect of liver injury on S1P gradients, we will quantify S1P levels over time in the liver and lymph in normal & carbon tetrachloride (CCl4) liver-injured mice allowing us to establish if the S1P gradient from liver to lymph is reversed or reduced following injury. This is important, as given the increase in hepatic S1P levels following liver injury, it allows us to determine the relative contribution of S1P to HSC recruitment (no change in gradient), or retention within the liver (reduction/reversal in gradient).
We will then determine the functional contribution hepatic S1P has in the hepatic recruitment (rather than retention) of HSCs by use of in vitro flow based assays, which accurately model the in vivo interaction of HSCs with hepatic sinusoidal endothelial cells. We will determine the effect of antagonism of S1P receptors on recruitment and trans-migration of HSCs in this model as well as in the CCl4 injured mouse liver by intra-vital microscopy.
Finally, using mice that have undergone CD45 mis-matched BM transplant, we will deliver shRNA using an adenoviral vector to knockdown the key enzymes regulating the synthesis (SphK1/2) and degradation (SGPL) of S1P within the liver, thus altering the hepatic S1P concentration. By analysing single-cell liver digests we will quantify numbers of BM-derived HSCs (by CD45 mis-match) within the liver, thus determining the effect of S1P modulation on the recruitment/retention of HSCs within the CCl4 injured liver and the subsequent effect on liver fibrosis and regeneration.
This series of studies will define the role of S1P in the hepatic recruitment and retention of HSCs, as well as the impact of this on liver repair.
Sphingosine 1-phosphate (S1P), a bioactive lipid, plays a key role in regulating the recirculation of HSCs from tissue to bone marrow via an S1P-gradient. Egress of HSCs from non-lymphoid tissue (low S1P concentration) into lymph vessels (high S1P concentration) is governed by their expression of S1P1-receptors.
We hypothesise that modulation of hepatic S1P levels alters HSC recruitment and retention within the injured liver, and that enhanced HSC retention will lead to accelerated resolution of fibrosis and increased hepatocyte proliferation.
To determine the effect of liver injury on S1P gradients, we will quantify S1P levels over time in the liver and lymph in normal & carbon tetrachloride (CCl4) liver-injured mice allowing us to establish if the S1P gradient from liver to lymph is reversed or reduced following injury. This is important, as given the increase in hepatic S1P levels following liver injury, it allows us to determine the relative contribution of S1P to HSC recruitment (no change in gradient), or retention within the liver (reduction/reversal in gradient).
We will then determine the functional contribution hepatic S1P has in the hepatic recruitment (rather than retention) of HSCs by use of in vitro flow based assays, which accurately model the in vivo interaction of HSCs with hepatic sinusoidal endothelial cells. We will determine the effect of antagonism of S1P receptors on recruitment and trans-migration of HSCs in this model as well as in the CCl4 injured mouse liver by intra-vital microscopy.
Finally, using mice that have undergone CD45 mis-matched BM transplant, we will deliver shRNA using an adenoviral vector to knockdown the key enzymes regulating the synthesis (SphK1/2) and degradation (SGPL) of S1P within the liver, thus altering the hepatic S1P concentration. By analysing single-cell liver digests we will quantify numbers of BM-derived HSCs (by CD45 mis-match) within the liver, thus determining the effect of S1P modulation on the recruitment/retention of HSCs within the CCl4 injured liver and the subsequent effect on liver fibrosis and regeneration.
This series of studies will define the role of S1P in the hepatic recruitment and retention of HSCs, as well as the impact of this on liver repair.