The Effect of a Novel Heparan Sulfate Mimetic on Inflammatory Migration Across the Blood-Brain Barrier Endothelium

The brain is one of the most delicate organs in the body, therefore it requires protection from both endogenous and exogenous insults that may cause injury. To protect from damage from inside the body, a range of cells, termed the blood-brain barrier (BBB), work synergistically to prevent large molecules and other cells from crossing from the blood into the brain. One of the most important cell types in this network is the endothelial cells that line the lumen of the vasculature. These endothelial cells have functionally enhanced barrier properties compared to other endothelial surfaces in the body by reducing pro-migratory functions and increasing tight cell contacts. This prevents excessive migration of circulating immune cells into the brain and stops potentially damaging larger molecules crossing into the brain. However, this all changes in the face of neuroinflammation.

Multiple sclerosis (MS) is an autoimmune neuroinflammatory disease that is characterised by demyelination in the central nervous system (CNS). This damaging process is carried out by autoreactive immune cells at the dysregulated blood-brain barrier. Once inside the brain, these cells attack the myelin sheaths that surround neuronal axons, leading to progressive neurological decline. The most successful treatments are ones that broadly prevent the trafficking of leukocytes into the brain. While these treatments significantly improve the quality of life for people living with MS, side effects of wide immunosuppression can have detrimental effects on their lives. These side effects can be life-threatening and identify the need for continued treatment development.

Heparanase, and its substrate heparan sulfate, have been implicated in migratory functions of immune cells in MS. Heparanase is a ubiquitously expressed enzyme that is responsible for degrading the extracellular matrix component heparan sulfate that covers individual cells. Leukocytes use heparanase to destabilise the extracellular matrix at the blood-brain barrier to cause the breakdown of the tightly regulated barrier function. Previous studies have shown that inhibiting heparanase in a murine model of MS, experimental autoimmune encephalomyelitis EAE, reduced disease, and migration of leukocytes into the CNS. This heparanase inhibition can be achieved by using heparan sulfate mimetics, compounds that mimic endogenous heparan sulfate but have inhibitory properties. While these have shown promise in preventing heparanase-mediated cancer metastasis and reducing experimental autoimmune encephalomyelitis, many of the current heparan sulfate mimetics require expensive and
complex synthesis with a range of off-target effects, reducing their ability to be clinically useful.

Tet-29 is a novel heparan sulfate mimetic developed by the Ferrier Institute. The structure consists of a dendritic core with four sulphated oligosaccharide arms. Due to its compact size and cheaper starting materials the synthesis of Tet-29 is faster and more economical. This thesis proposes that Tet-29 is protective in EAE by regulating the migration of immune cells by inhibiting lymphocyte-derived heparanase. This regulation of immune cell trafficking was investigated using histological analysis of brains from treated mice, and by developing an in vitro model of migration across the brain endothelium.

Immunohistochemical analysis of brain sections from EAE mice showed the integrity of the blood-brain barrier was rescued with treatment. Using staining methods to determine the integrity of the BBB under EAE conditions showed a rescue to BBB integrity. Integrity was measured by identifying the amount of albumin, a blood plasma protein, leaking out of the blood vessels. This was significantly increased in EAE, but almost non-existent in healthy mice with EAE treated therapeutically with Tet-29. Alongside this, reduced expression of important migratory proteins, ICAM-1 and VCAM-1, were also reduced with treatment.

To further investigate the mechanism of how Tet-29 regulates trafficking into the CNS, an in vitro model of the BBB endothelium was established. It was first shown that ConA and CCL21 can drive the trafficking of splenocytes across the endothelial barrier. Further migration assays containing Tet-29 showed a reduction in splenocyte migration in response to ConA and CCL21-driven migration.

Overall, this work shows that Tet-29 may be capable of rescuing the BBB integrity by inhibiting migratory responses exhibited by inflammatory immune cells. While the exact mechanism of the migratory regulation requires further research, this work furthers our understanding of Tet-29 in neuroinflammation.