Role of endothelial cell dysfunction in cerebral small vessel disease using a novel rodent model

Abstract

Cerebral small vessel disease (SVD) is the most common cause of vascular dementia and can occur in combination with other neurodegenerative diseases, worsening clinical signs. SVD doubles a patient’s risk of developing dementia or stroke. SVD is a disease which affects the blood vessels and surrounding parenchyma, particularly the penetrating vessels that go to the deep white matter regions of the brain.

Previously, it was thought that the main cause of sporadic forms of SVD was hypertension leading to breakdown of the blood-brain barrier (BBB). However, it is now known that 30% of patients are normotensive, in addition to anti-hypertensive clinical trials in SVD being unsuccessful at preventing disease progression. The move away from thinking of hypertension as the primary cause of SVD has been supported by recent work in our lab using an in-bred rodent model of SVD, the Spontaneously hypertensive stroke prone rat (SHRSP).

Instead, the SHRSPs were found to have an underlying dysfunction of the endothelial cells, cells which form a major component of the BBB, which predates hypertension. The dysfunctional endothelial cells were also shown to exert a block on oligodendrocyte maturation, thought to be a contributor to the white matter changes seen in SVD. A homozygous mutation of the Atp11b gene was discovered to be present in the SHRSP leading to loss of ATP11B flippase protein. Loss of ATP11B was shown to be sufficient to replicate the endothelial dysfunction in rat and human cells. Additionally, a single nucleotide polymorphism (SNP) in the human ATP11B gene was associated with the magnetic resonance imaging (MRI) findings of sporadic SVD in a large human dataset (CHARGE consortium).

To understand the role of ATP11B in SVD pathology, a novel transgenic rodent model with a global knockout of ATP11B was created, the Atp11bKO rat. This rodent model is normotensive, unlike the SHRSP, allowing us to dissect the causes of SVD independent of hypertension. This thesis continues the characterisation of the Atp11bKO rat focusing on how endothelial cell dysfunction may affect the BBB allowing us to understand how these early changes may be contributing to disease in humans.

Little is known about ATP11B, its location and function. Therefore, to further understand how this protein may be involved in disease we wanted to elucidate how loss of this flippase may affect the endothelial cells. ATP11B is a P4-ATPase flippase, responsible for maintaining lipid membrane bilayer asymmetry, particularly selective towards phosphatidylserine (PS), on either the plasmalemma or intracellular membranes such as in vesicles. We found that loss of ATP11B does not affect plasmalemma PS asymmetry, however, this does alter cell morphology at the ultrastructural level.

We next examined how the known loss of the endothelial cell tight junction protein Claudin-5 (CLDN5) in the Atp11bKO, (a feature of endothelial cell dysfunction) and these ultrastructural morphological abnormalities affected BBB integrity. There were no changes in endothelial cell ability to form a functional BBB in vitro or in vivo at an early age. However, there was evidence, at a later age, of disruption of the BBB in vivo, consistent with the MRI evidence of disruption of BBB in SVD patients in the later stages of disease. The Atp11bKO rat model also displayed similar characteristics to human SVD with white matter pathology and MRI changes, including brain atrophy.

To investigate further how dysfunctional endothelial cells in the Atp11bKO rat lead to downstream effects on the brain white matter and cause atrophy, we compared the transcriptomes of Atp11bKO and wild type endothelial cells by RNA sequencing. This has revealed several gene changes (e.g., Mical3, Cd9, Ccn1) which both may contribute to the endothelial cell dysfunction and link this with surrounding brain parenchymal changes.

These data further characterise the Atp11bKO rat as a novel normotensive model of SVD, and provide further steps to understanding the mechanism of this pathology. Our findings support a model of human SVD, where genetic vulnerability and early life factors influence SVD risk, manifest as an early intrinsic endothelial cell dysfunction and white matter structural vulnerability, which is later further influenced by environmental events (including possible hypertension) and ageing, leading to BBB breakdown and neurodegeneration.