In the Gage Lab we are focused on understanding what makes endothelial cells specialized for the organ they support and reside within. It is remarkable that nearly every organ has capillary endothelial cells that perform unique functions tuned to the needs of that organ. Examples of this include: the blood-brain-barrier with specialized solute transport, the alveolus with thinned endothelium for gas exchange, the bone marrow and spleens endothelial gaps for red blood cell generation and recycling, and the liver with fenestrated, scavenging, and secretory sinusoidal capillaries. Unfortunately, in disease, these functions are routinely lost. We aim to understand the cell-cell interactions and signals that impart these special functions so that we can return diseased endothelium to a normal fully functional organ specific state. In this way, we apply our gained knowledge of regulatory signals of vascular functions to design new therapeutic strategies to acquired and inherited vascular diseases. These therapies range from genetic and pharmacologic interventions to durable cell replacement strategies with stem cell derived endothelium. 

Most of our work focuses on the liver. In Canada, liver disease is on the rise and 1 in 4 Canadians of all ages are affected. Whether acute (eg. drug overdose), chronic (eg. NAFLD, NASH, and cirrhosis), congenital (eg. Hemophilia A), or cancer (eg. liver metastasis), what unifies many liver diseases is that liver endothelium specifically lose normal functions as the disease progresses. In the liver, capillaries known as Liver Sinusoidal Endothelial Cells (LSECs) reside among many other cell types in the liver sinusoid (please see below).  In this location, LSECs are functionally specialized for transcellular transport (eg. LDL) biomolecule scavenging (eg. hyaluronan, immunoglobulin, bacterial debris, damaged blood proteins), protein secretion (eg. coagulation factor FVIII) and sustaining liver regeneration via paracrine signalling. 

The Liver and the Sinusoid: (MacParland et al. 2018. Article)

The Gage Lab's research centers around three project themes that leverage a common humanized mouse model. Here we use human pluripotent stem cells to model early human development and the formation and specification of endothelial progenitors and venous endothelial cells (VEC) (see below). This work is done in the lab with cell culture technologies that give us access to cells at very early stages of development so we can explore the genetics of early development. With purified VECs generated, we surgically deliver these cells into mouse models where they engraft and grow in the liver of the mice while responding to the liver niche signals of the sinusoid that drive maturation and maintenance of function. Remarkably these human VECs replace over 90% of the mouse liver vasculature leading to near complete humanization of the organs blood vessels. The hPSC-LSECs that reside in the mouse show functions of human LSEC specific fenestration patterns, rapid scavenging, FVIII secretion and extensive transcriptional matching to healthy primary human LSECs.



Based on this human developmental and in vivo maturation model, we now focus on three project themes: 

1)    Regulators of human LSEC functional maturation and maintenance
What signals drive function? What signals sustain these functions lost to disease? We are currently using both candidate and unbiased transcriptomics approaches to identify signalling pathways that control specific LSEC functions. Linking genotype to LSEC phenotype, we use lentiviral vectors, CRISPR-Cas9, and extensive molecular phenotyping (scRNA-seq, flow cytometry, RT-qPCR, histology, and electron microscopy) to reveal direct and indirect functional regulation. 

2)    Designing endothelial function restorative therapeutics
As we build an understanding of signals that impart and sustain LSEC functions, we reveal therapeutic targets to fight liver disease. For example, acquired liver diseases (drug induced liver injury, NASH, NAFLD, and liver metastasis) all reduce LSEC scavenging and transport functions. We hypothesize that therapeutic rescue of these functions may indirectly rescue other liver functions by supporting other liver cell types (eg. hepatocytes). This work models disease in our humanized mouse model to ensure designed therapeutics provide predicated therapeutics benefits directly or indirectly through human LSECs in vivo. 

3)    Cell therapy for Hemophilia A and liver disease
Some liver vascular disease are of genetic origin leading to a loss of function in liver endothelium. An example of this is Hemophilia A where all the body’s cells fail to produce sufficient FVIII protein to regulate blood clotting. Since LSECs are numerous and naturally make high levels of FVIII, we previously applied our hPSC-based VEC cell therapy model to Hemophilia A mice collaboratively with Dr. Antonia Follenzi (Università, del Piemonte Orientale, Italy). Mice receiving hPSC-VEC cell therapy corrected their severe bleeding to a mild form due to the bioactive hPSC-LSEC produced FVIII. We are currently working to advance this approach to become free from immune rejection constraints and leverage the other natural and engineered proteins that hPSC-LSECs make to fight liver disease from within the organ.