Epidermal growth factor prevents APOE4-induced cognitive and cerebrovascular deficits in female mice.

Cerebrovascular dysfunction is re-emerging as a major component of aging, and may contribute to the risk of developing Alzheimer's disease (AD). Two important risk factors for cerebrovascular dysfunction are APOE and female sex, which are primarily researched in the context of high amyloid-ß (Aß) levels as found in AD. However, APOE4 and sex modulate Aß-independent pathways that may induce cerebrovascular dysfunction as a downstream consequence. Therefore, testing the activity of factors that target cerebrovascular dysfunction in Aß-independent models that incorporate APOE4 and female sex is crucial. We have previously demonstrated that peripheral administration of the epidermal growth factor (EGF) prevents cognitive dysfunction, cerebrovascular leakiness, and cerebrovascular coverage deficits in female mice that express APOE4 and overproduce Aß, without affecting Aß levels. These data raise the question of whether EGF protects the cerebrovasculature from general stress-induced damage. Therefore, the goal of this study was to determine whether EGF prevents Aß-independent cerebrovascular dysfunction. In eight-month old mice that express human APOE, the interaction of APOE4 and female sex induced cognitive dysfunction, increased cerebrovascular leakiness and lowered vessel coverage. Importantly, in a prevention paradigm (from six to eight and a half months of age), EGF ameliorated cognitive decline and cerebrovascular deficits in female mice that express APOE4. Thus, developing treatment strategies based on EGF signaling could provide alternative therapeutic options for age-related cerebrovascular dysfunction and reduce AD risk.

Vascular basement membrane alterations and ß-amyloid accumulations in an animal model of cerebral small vessel disease.

Non-amyloid cerebral small vessel disease (CSVD) and cerebral amyloid angiopathy (CAA) may be interrelated through the damaged basement membranes (BMs) and extracellular matrix changes of small vessels, resulting in a failure of ß-amyloid (Aß) transport and degradation. We analyzed BM changes and the pattern of deposition of Aß in the walls of blood vessels in spontaneously hypertensive stroke-prone rats (SHRSP), a non-transgenic CSVD model. In 45 SHRSP and 38 Wistar rats aged 18 to 32 weeks: (i) the percentage area immunostained for vascular collagen IV and laminin was quantified; (ii) the capillary BM thickness as well as endothelial and pericyte pathological changes were analysed using transmission electron microscopy (TEM); and (iii) the presence of vascular Aß was assessed. Compared with controls, SHRSP exhibited a significantly higher percentage area immunostained with collagen IV in the striatum and thalamus. SHRSP also revealed an age-dependent increase of the capillary BM thickness and of endothelial vacuoles (caveolae) within subcortical regions. Endogenous Aß deposits in the walls of small blood vessels were observed in the cortex (with the highest incidence found within fronto-parietal areas), striatum, thalamus and hippocampus. Vascular ß-amyloid accumulations were frequently detected at sites of small vessel wall damage. Our data demonstrate changes in the expression of collagen IV and of the ultrastructure of BMs in the small vessels of SHRSP. Alterations are accompanied by vascular deposits of endogenous Aß. Impaired ß-amyloid clearance along perivascular and endothelial pathways and failure of extracellular Aß degradation may be the key mechanisms connecting non-amyloid CSVD and CAA.

Epidermal growth factor prevents APOE4 and amyloid-beta-induced cognitive and cerebrovascular deficits in female mice.

Cerebrovascular (CV) dysfunction is emerging as a critical component of Alzheimer's disease (AD), including altered CV coverage. Angiogenic growth factors (AGFs) are key for controlling CV coverage, especially during disease pathology. Therefore, evaluating the effects of AGFs in vivo can provide important information on the role of CV coverage in AD. We recently demonstrated that epidermal growth factor (EGF) prevents amyloid-beta (Aß)-induced damage to brain endothelial cells in vitro. Here, our goal was to assess the protective effects of EGF on cognition, CV coverage and Aß levels using an AD-Tg model that incorporates CV relevant AD risk factors. APOE4 is the greatest genetic risk factor for sporadic AD especially in women and is associated with CV dysfunction. EFAD mice express human APOE3 (E3FAD) or APOE4 (E4FAD), overproduce human Aß42 and are a well characterized model of APOE pathology. Thus, initially the role of APOE and sex in cognitive and CV dysfunction was assessed in EFAD mice in order to identify a group for EGF treatment. At 8 months E4FAD female mice were cognitively impaired, had low CV coverage, high microbleeds and low plasma EGF levels. Therefore, E4FAD female mice were selected for an EGF prevention paradigm (300 µg/kg/wk, 6 to 8.5 months). EGF prevented cognitive decline and was associated with lower microbleeds and higher CV coverage, but not changes in Aß levels. Collectively, these data suggest that EGF can prevent Aß-induced damage to the CV. Developing therapeutic strategies based on AGFs may be particularly efficacious for APOE4-induced AD risk.

Epidermal growth factor prevents oligomeric amyloid-ß induced angiogenesis deficits in vitro.

Cerebrovascular dysfunction is a critical component of Alzheimer's disease (AD) pathogenesis. Oligomeric amyloid-ß42 (oAß42) is considered a major contributor to AD progression. However, data are limited on the role of oAß42 in brain endothelial cell vessel degeneration/angiogenesis, including the interaction with angiogenic mediators. Thus, the current study determined the effect of oAß42 on angiogenesis in vitro, utilizing single brain endothelial cell cultures and triple cultures mimicking the microvascular unit (MVU: brain endothelial cells, astrocytes, and pericytes). oAß42 dose-dependently reduced angiogenesis and induced vessel disruption. Critically, epidermal growth factor prevented oAß42-induced deficits, implicating angiogenic pathways as potential therapeutics for AD.

Lymphatic Clearance of the Brain: Perivascular, Paravascular and Significance for Neurodegenerative Diseases.

The lymphatic clearance pathways of the brain are different compared to the other organs of the body and have been the subject of heated debates. Drainage of brain extracellular fluids, particularly interstitial fluid (ISF) and cerebrospinal fluid (CSF), is not only important for volume regulation, but also for removal of waste products such as amyloid beta (Aß). CSF plays a special role in clinical medicine, as it is available for analysis of biomarkers for Alzheimer's disease. Despite the lack of a complete anatomical and physiological picture of the communications between the subarachnoid space (SAS) and the brain parenchyma, it is often assumed that Aß is cleared from the cerebral ISF into the CSF. Recent work suggests that clearance of the brain mainly occurs during sleep, with a specific role for peri- and para-vascular spaces as drainage pathways from the brain parenchyma. However, the direction of flow, the anatomical structures involved and the driving forces remain elusive, with partially conflicting data in literature. The presence of Aß in the glia limitans in Alzheimer's disease suggests a direct communication of ISF with CSF. Nonetheless, there is also the well-described pathology of cerebral amyloid angiopathy associated with the failure of perivascular drainage of Aß. Herein, we review the role of the vasculature and the impact of vascular pathology on the peri- and para-vascular clearance pathways of the brain. The different views on the possible routes for ISF drainage of the brain are discussed in the context of pathological significance.

Vascular basement membranes as pathways for the passage of fluid into and out of the brain.

In the absence of conventional lymphatics, drainage of interstitial fluid and solutes from the brain parenchyma to cervical lymph nodes is along basement membranes in the walls of cerebral capillaries and tunica media of arteries. Perivascular pathways are also involved in the entry of CSF into the brain by the convective influx/glymphatic system. The objective of this study is to differentiate the cerebral vascular basement membrane pathways by which fluid passes out of the brain from the pathway by which CSF enters the brain. Experiment 1: 0.5 µl of soluble biotinylated or fluorescent Aß, or 1 µl 15 nm gold nanoparticles was injected into the mouse hippocampus and their distributions determined at 5 min by transmission electron microscopy. Aß was distributed within the extracellular spaces of the hippocampus and within basement membranes of capillaries and tunica media of arteries. Nanoparticles did not enter capillary basement membranes from the extracellular spaces. Experiment 2: 2 µl of 15 nm nanoparticles were injected into mouse CSF. Within 5 min, groups of nanoparticles were present in the pial-glial basement membrane on the outer aspect of cortical arteries between the investing layer of pia mater and the glia limitans. The results of this study and previous research suggest that cerebral vascular basement membranes form the pathways by which fluid passes into and out of the brain but that different basement membrane layers are involved. The significance of these findings for neuroimmunology, Alzheimer's disease, drug delivery to the brain and the concept of the Virchow-Robin space are discussed.

A Simulation Model of Periarterial Clearance of Amyloid-ß from the Brain.

The accumulation of soluble and insoluble amyloid-ß (Aß) in the brain indicates failure of elimination of Aß from the brain with age and Alzheimer's disease (AD). There is a variety of mechanisms for elimination of Aß from the brain. They include the action of microglia and enzymes together with receptor-mediated absorption of Aß into the blood and periarterial lymphatic drainage of Aß. Although the brain possesses no conventional lymphatics, experimental studies have shown that fluid and solutes, such as Aß, are eliminated from the brain along 100 nm wide basement membranes in the walls of cerebral capillaries and arteries. This lymphatic drainage pathway is reflected in the deposition of Aß in the walls of human arteries with age and AD as cerebral amyloid angiopathy (CAA). Initially, Aß diffuses through the extracellular spaces of gray matter in the brain and then enters basement membranes in capillaries and arteries to flow out of the brain. Although diffusion through the extracellular spaces of the brain has been well characterized, the exact mechanism whereby perivascular elimination of Aß occurs has not been resolved. Here we use a computational model to describe the process of periarterial drainage in the context of diffusion in the brain, demonstrating that periarterial drainage along basement membranes is very rapid compared with diffusion. Our results are a validation of experimental data and are significant in the context of failure of periarterial drainage as a mechanism underlying the pathogenesis of AD as well as complications associated with its immunotherapy.

The role of APOE in cerebrovascular dysfunction.

The ε4 allele of the apolipoprotein E gene (APOE4) is associated with cognitive decline during aging, is the greatest genetic risk factor for Alzheimer's disease and has links to other neurodegenerative conditions that affect cognition. Increasing evidence indicates that APOE genotypes differentially modulate the function of the cerebrovasculature (CV), with apoE and its receptors expressed by different cell types at the CV interface (astrocytes, pericytes, smooth muscle cells, brain endothelial cells). However, research on the role of apoE in CV dysfunction has not advanced as quickly as other apoE-modulated pathways. This review will assess what aspects of the CV are modulated by APOE genotypes during aging and under disease states, discuss potential mechanisms, and summarize the therapeutic significance of the topic. We propose that APOE4 induces CV dysfunction through direct signaling at the CV, and indirectly via modulation of peripheral and central pathways. Further, that APOE4 predisposes the CV to damage by, and exacerbates the effects of, additional risk factors (such as sex, hypertension, and diabetes). ApoE4-induced detrimental CV changes include reduced cerebral blood flow (CBF), modified neuron-CBF coupling, increased blood-brain barrier leakiness, cerebral amyloid angiopathy, hemorrhages and disrupted transport of nutrients and toxins. The apoE4-induced detrimental changes may be linked to pericyte migration/activation, astrocyte activation, smooth muscle cell damage, basement membrane degradation and alterations in brain endothelial cells.

The Cerebrovascular Basement Membrane: Role in the Clearance of ß-amyloid and Cerebral Amyloid Angiopathy.

Cerebral amyloid angiopathy (CAA), the accumulation of ß-amyloid (Aß) peptides in the walls of cerebral blood vessels, is observed in the majority of Alzheimer's disease (AD) brains and is thought to be due to a failure of the aging brain to clear Aß. Perivascular drainage of Aß along cerebrovascular basement membranes (CVBMs) is one of the mechanisms by which Aß is removed from the brain. CVBMs are specialized sheets of extracellular matrix that provide structural and functional support for cerebral blood vessels. Changes in CVBM composition and structure are observed in the aged and AD brain and may contribute to the development and progression of CAA. This review summarizes the properties of the CVBM, its role in mediating clearance of interstitial fluids and solutes from the brain, and evidence supporting a role for CVBM in the etiology of CAA.