Systematic review of shared risk factors in the prevention and treatment of stroke and AD.
We performed a systematic review, evaluating the current evidence in the literature relating to two related questions:
- Does treatment of vascular risk factors prevent AD?
- Are vascular risk factors associated with Alzheimer’s disease?
The systematic review on treatment of vascular risk factors and prevention of dementia in Alzheimer’s disease has been published in Journal of Alzheimer’s Disease. This provides a definitive analysis of data on the Impact of treatment of vascular risks factors on both all dementia, and Alzheimer’s disease, incidence. In brief this study showed that antihypertensive treatment was associated with a non-significant reduced risk of dementia in RCTs and with reduced AD risk in prospective studies. In prospective studies, treatment of hyperlipidemia with statins, but not nonstatin lipid-lowering agents, was associated with reduced risk of dementia. Data from one RCT and six prospective studies did not support a beneficial impact of antidiabetic drugs or insulin therapy on dementia risk. The conclusion was that current evidence indicates that antihypertensives and statins might reduce the incidence of dementia and AD, but that further trials to determine the effect of VRF on AD are needed.
Mendelian Randomization and polygenic risk score analysis of shared conventional and novel risk factors between stroke and AD
The main objective of this task was to evaluate the causal association of biomarkers and risk factors with stroke/AD. Many such associations have been reported in the literature but often lack convincing evidence of causality. In our analysis, we tried to overcome this by using a two-sample Mendelian Randomization approach. In this approach, the odds ratio for association of genetic variants with trait A are used in combination with the odds ratio (and standard errors) for the same variants with trait B to evaluate the causal impact of trait A on trait B. The approach has the benefit on being susceptible to reverse confounding, which can affect epidemiological studies, and can be used to make claims of causality in the absence of data from randomization control trials.
We completed a Mendelian randomization study to assess whether potentially modifiable risk factors, including socioeconomic, lifestyle/dietary, cardiometabolic, and inflammatory factors, are associated with Alzheimer’s disease. We found that genetically predicted higher educational attainment is associated with reduced risk of Alzheimer’s disease. A paper on this work was published in the BMJ.
Further, we examined further the role of homocysteine as a potential risk factor for stroke and dementia. We conducted a Mendelian randomization study to evaluate the association of circulating total homocysteine and vitamin B levels with stroke subtypes. We found that genetically predicted total blood homocysteine was associated with increased risk of small vessel stroke but not with other ischaemic stroke subtypes. This has important implications for the design of homocysteine-lowering trials in stroke and dementia prevention. A paper on this work was published in Annals of Neurology.
We also conducted a Mendelian randomization study to assess whether modifiable lifestyle factors, including educational attainment, smoking, and body mass index are associated with stroke and its subtypes. We found that genetically predicted years of education was inversely associated with ischaemic, large artery, and small vessel stroke, and intracerebral haemorrhage. We also found that genetically predicted smoking, body mass index, and waist-hip ratio were associated with ischaemic and large artery stroke. A paper on this work was published in Stroke.
Lastly, we conducted a Mendelian randomization study to explore the effects of genetic predisposition to type 2 diabetes, hyperglycaemia, insulin resistance, and pancreatic β-cell dysfunction on risk of stroke subtypes and related cerebrovascular phenotypes. We found that genetic predisposition to type 2 diabetes and higher HbA1c levels were associated with higher risk of ischaemic, large artery, and small vessel stroke. We also found associations of genetically predicted insulin resistance and β-cell dysfunction with large artery and small vessel stroke that might have implications for antidiabetic treatments targeting these mechanisms. A paper on this work was published in Neurology.
Identification of novel disease pathways which may play a role in shared risk of stroke and AD
We investigated the hypothesis that endothelial dysfunction might play a role in small vessel stroke and AD, further by trying to identify novel determinants of endothelial dysfunction. To do this we have performed GWAS analysis on datasets of individuals who have had endothelial function measured non-invasively by forearm ultrasound measurement of flow-mediated vasodilation.
We identified a variant in PDE3A, encoding phosphodiesterase 3A which impairs endothelial function in adolescents and confers risk of ischaemic stroke. The same variant was associated with FMD in a second study in young adults, but not in older adults. Our interpretation was that a genetic variant in PDE3A influences endothelial function in early life and leads to subsequent increased risk of ischaemic stroke. Subtle, measurable changes to the vasculature in early life due to genetics influence subsequent risk of ischaemic stroke.
We have also performed a comprehensive analysis of the role of a wide range of haematological traits in the risk of stroke using Mendelian Randomisation techniques. Several factors on the intrinsic clotting pathway were significantly associated with CES and LAS, but not with SVS. On the common pathway, increased gamma fibrinogen was significantly associated with AIS/CES. Furthermore, elevated plateletcrit was significantly associated with AIS/CES, eosinophil percentage of white cells with LAS, and thrombin-activatable fibrinolysis inhibitor activation peptide antigen with AIS. These results implicate components of the intrinsic and common pathways of the clotting cascade, as well as several other haematological traits, in the pathogenesis of CES and possibly LAS, but not SVS. The lack of associations with SVS suggests thrombosis may be less important for this stroke subtype. Plateletcrit and factor XI are potentially tractable new targets for secondary prevention of ischaemic stroke, while factor VIII and γ' fibrinogen require further population-based studies to ascertain their possible aetiological roles. A paper on this work was published in Brain.
Finally, we conducted a metabolomics study to evaluate the association of metabolites with vascular cognitive impairment and dementia in patients with cerebral small vessel disease. We found multiple distinct metabolic signatures associated with imaging markers of small vessel disease, cognition, and conversion to dementia. Further research should assess causality and the use of metabolomic screening to improve the ability to predict future disease severity and dementia risk in SVD. The metabolomic profiles may also provide novel insights into disease pathogenesis and help identify novel treatment approaches. A preprint of this paper has been published on medRxiv.
Organ-on-a-Chip model of neurovascular system to validate biomarkers and test treatment options
MIMETAS developed an organ-on-a-chip model of the neurovascular unit (NVU) that consists of human neurons, astrocytes, and endothelial cells. We assessed cell morphology, endothelial barrier integrity, expression of relevant markers, and neuronal activity. After the model was established, a procedure to model ischemic stroke was developed and the effect of ischemic stroke on the NVU on-a-chip model was characterized.
Development of an NVU on-a-chip
The OrganoPlate 3-lane allows parallel culture of 40 miniaturized tissues in microfluidic chips. In each chip, a three-dimensional human NVU triculture model was grown under medium perfusion. The NVU cultures were characterized by immunostaining. The culture comprises a vessel of brain endothelial cells, in direct contact with collagen-embedded astrocytes, and complex networks of neurons. Cultures remained viable for a minimum of two weeks.
The vessel of primary brain endothelial cells (BECs) shows expression of adherens junction proteins vascular endothelial cadherin and platelet endothelial cell adhesion molecule 1 as well as tight junction proteins claudin-5 and zona occludens 1. The presence of these markers and their localization at the cell-cell contacts is indicative of barrier formation. In addition, the endothelial cells produce basement membrane proteins collagen-IV, fibronectin, and laminin, which make up essential components of the NVU’s basement membrane. The vessel of brain endothelial cells is grown against a collagen-gel containing iPSC-derived astrocytes, which express astrocytic marker S100 calcium-binding protein B. In direct contact with the collagen-embedded astrocytes are networks of iPSC-derived neurons and astrocytes, positive for neuronal marker beta-III-tubulin and astrocytic marker s100β, respectively. To ensure neuronal functionality, electrophysiological activity was detected by means of calcium imaging, confirming spontaneous neuronal firing in the NVU cultures.
Lastly, the expression of important BBB transporters was confirmed using PCR. These transporters included GLUT1, Pg-p, BCRP1, MRP1, and TfR. For GLUT1 and P-gp, functional assays were performed, in which these transporters showed lowered activity in presence of well-known inhibitors.
Mimicking ischemic stroke
Ischemic stroke was mimicked in the NVU on-a-chip model using a 3-fold approach: (1) glucose depletion was modeled using glucose-free medium, (2) hypoxia was mimicked using antimycin-A, an inhibitor of complex III of the electron transport chain, and (3) perfusion was stopped by removing the OrganoPlate from a rocker platform. Each approach was assessed separately and in combination with the other approaches and compared to control chips.
Results showed that when perfusion is continued, no-glucose alone or antimycin-A alone do not alter NVU barrier integrity, but the combination of no-glucose and antimycin-A induces a significant disruption of the endothelial barrier, causing leakage of sodium fluorescein out of the models’ lumen. In contrast, in absence of perfusion, no-glucose alone or antimycin-A alone both did cause a decrease in barrier integrity compared to control. A combination of no-glucose and antimycin-A again showed a further disruption of barrier integrity.
Similar results were observed when studying the mitochondrial membrane potential in the NVU on-a-chip exposed to stroke-mimicking conditions (data not shown). Under perfused conditions, no-glucose alone or antimycin A alone did not significantly alter the model’s mitochondrial membrane potential, while a combination of no-glucose + antimycin-A resulted in a strong reduction in mitochondrial membrane potential. Under static conditions, no-glucose alone and antimycin-A alone resulted in a decrease in mitochondrial membrane potential. When no-glucose, antimycin-A, and static culture (no perfusion) were combined, the strongest decrease was observed.
Lastly, a third assay, which studied adenosine triphosphate (ATP) levels, supported the findings of the other two assays. Under stroke mimicking conditions (no-glucose + antimycin-A + static culture), the NVU on-a-chip model showed lowered ATP.
Conclusion
The human NVU on-a-chip model described here consists of a microvessel of primary brain endothelial cells, in direct contact with iPSC-derived astrocytes and in co-culture with iPSC-derived neurons. The model presents with a tight barrier, relevant BBB transporters, and spontaneously active neurons. To mimic ischemic stroke, an approach was taken that combines hypoglycemia, chemical hypoxia, and interrupted flow. The resulting phenotype includes impaired barrier function and mitochondrial membrane potential of the endothelial vessel, as well as reduced ATP in both blood- and brain compartments. The NVU on-a-chip model can be used for fundamental studies of NVU function in disease and investigation of potential restorative therapies. Due to the platform’s relatively high throughput and compatibility with automation, the NVU on-a-chip model opens up possibilities for drug compound screening.
Significant results
During CoSTREAM, Work Package 6 achieved several significant results:
- Antihypertensives and statins might reduce the incidence of dementia and Alzheimer’s disease
- Genetically predicted total blood homocysteine was associated with increased risk of small vessel stroke, but not with other ischaemic stroke subtypes. This is important for the design of trials in stroke and dementia prevention
- Genetically predicted years of education was inversely associated with ischaemic, large artery, and small vessel stroke, and intracerebral haemorrhage.
- Genetically predicted smoking, body mass index, and waist-hip ratio were associated with ischaemic and large artery stroke
- Genetic predisposition to type 2 diabetes and higher HbA1c levels were associated with higher risk of ischaemic, large artery, and small vessel stroke.
- Identified associations of genetically predicted insulin resistance and β-cell dysfunction with large artery and small vessel stroke that might have implications for antidiabetic treatments targeting these mechanisms
- Identified a variant in PDE3A, which was associated with flow-mediated dilatation in adolescents and confers risk of ischaemic stroke.
- We concluded that a genetic variant in PDE3A influences endothelial function in early life and leads to increased risk of ischemic stroke, and that subtle, measurable changes to the vasculature that are influenced by genetics also influence risk of ischemic stroke.
- Implicated components of the clotting cascade, as well as several other haematological traits, in the pathogenesis of cardioembolic stroke and possibly large artery stroke, but not small vessel stroke.
- Identified plateletcrit and factor XI as potentially tractable new targets for secondary prevention of ischaemic stroke, while factor VIII and γ' fibrinogen require further population-based studies to ascertain their possible aetiological roles.
- Discovered multiple distinct metabolic signatures associated with imaging markers of small vessel disease, cognition, and conversion to dementia.
- Developed a novel in vitro model system of the neurovascular unit
