AGFs play a key role in controlling total vessel coverage, especially during disease pathology. In AD research the effects that AFGs will induce on AD-like pathology in vivo is a controversial topic. Previously we have demonstrated that EGF can prevent oligomeric Aβ-induced disruption of total vessel length in vitro. Here our goal was to determine whether EGF prevents cognitive and CV dysfunction in an AD-Tg model that incorporates human APOE and high Aβ levels, both of which are important for AD and CV function. We demonstrated that E4FADF mice are cognitively impaired, have higher CV leakiness and lower vessel coverage than other groups at 8 months and also had low plasma EGF levels. Critically, we report for the first time that cognitive performance and vessel coverage was higher in E4FADF mice treated with EGF in a prevention paradigm. These data support further research on the development of AGF-based therapies for AD.
Sex and APOE4-induced cognitive and CV dysfunction in EFAD mice
Our data support that sex and APOE4 impact cognition and CV dysfunction in EFAD mice. In E4FADF the CV dysfunction was accompanied by cognitive dysfunction. In E3FADF mice CV leakiness was higher, CV coverage lower and plasma EGF levels lower compared to EFADM mice. As there were no changes in cognitive function in E3FADF mice compared to EFADM mice, the CV effects may represent early changes that later manifest as cognitive decline. These findings raise important questions on the relationship to human and other in vivo data and the mechanisms underlying sex-induced APOE modulated changes in cognition and CV coverage.
In AD patients
APOE4-induced risk is greater in females [
2,
19,
20,
41,
45] consistent with our finding that E4FADF are cognitively impaired compared to all other groups tested at 8 months. In partial contrast a few reports have demonstrated impaired cognition in male
APOE4-TR mice (which do not overproduce human Aβ) compared to
APOE3-TR mice [
46,
56]. However, in a separate detailed analysis of
APOE-TR mice the deficits in memory were primarily observed in female
APOE4-TR mice [
21]. Data on CV coverage is lacking in human patients stratified for sex and
APOE genotype. In 9 and 12 months old
APOE-TR mice lower microvessel coverage has been demonstrated [
1,
4], although the effect of sex is unclear. A detailed comparison of EFAD and
APOE-TR mice may reveal whether Aβ accelerates the
APOE4 and female sex driven cognitive deficits and changes in CV coverage. In AD microbleeds are associated with male sex, higher blood pressure, lower CSF Aβ42 and
APOE4 [
5]. These data contrast with the female bias observed in EFAD mice [
12,
20]. A confounding factor is whether additional AD-risk factors are present in male AD patients assessed for microbleeds. For example, level of exercise, hypertension, hyperglycemia, hyperinsulinemia and insulin resistance observed in type 2 diabetes all modulate AD-risk and the CV. Further, men are at a higher risk of developing cardiovascular events. The controlled laboratory conditions utilized here may be more relevant for studying the interaction among sex,
APOE and Aβ in the absence of additional risk factors. Further research could focus on introducing additional AD-risk factors in vivo.
Females are at a higher risk for AD, which is often attributed to the loss of sex hormones during the menopause [
45]. In the mouse model utilized here, with no ovariectomy, there may be inherent sex-differences that modulate CV and AD-like pathology. Extracellular Aβ levels from highest to lowest were: E4FADF > E4FADM = E3FADF > E3FADM consistent with previous reports [
12,
51,
61]. Thus, higher levels of Aβ in E4FADF mice could induce or accelerate signaling cascades that cause CV degeneration. As described above CV coverage is lower in
APOE4-TR mice which supports an acceleration rather than primary causation of CV deficits caused by Aβ. The low CV coverage and EGF levels in E3FADF mice compared to E4FADM mice are not associated with higher Aβ levels, supporting an Aβ-level independent mechanism. Females prior to menopause are at an increased risk for some autoimmune diseases and there are a number of proposed mechanisms underlying these effects [
28]. For example, females may have a greater immune/inflammatory response in the periphery and the brain [
28]. Although potentially beneficial for fighting infection an enhanced or prolonged immune/inflammatory response in females may predispose, or potentiate Aβ-induced direct and indirect damage to the CV. It is also noteworthy that lower vessel coverage was associated with low plasma EGF levels in EFADF mice. Low EGF plasma appear to increase the conversion of MCI to AD [
33] but a sex bias has not been reported. Early reports in mice have demonstrated lower plasma EGF levels in females compared to males [
42]. Further, plasma EGF levels in female mice are altered by the circadian rhythm, pregnancy, [
53] and are increased with testosterone treatment [
52]. Although 17-β estradiol is protective against brain endothelial damage in vitro [
38], perhaps the male sex hormones exert a greater protective effect. A possibility is that low EGF levels in females result in lower protection of the CV to damage in AD, which is potentiated by
APOE4 and accelerated by Aβ. Speculating further, the low plasma levels of EGF in female mice may be indicative general growth factor suppression or a form of accelerated aging/senescence.
The effects of
APOE in AD are multifactorial, complex at the structural and mechanistic level and an area of intense research (reviewed extensively in [
23,
34,
37,
43,
55,
59]). On a simplified level apoE4 can effect CV length through modulating Aβ levels and via signaling to cells at the blood-brain barrier (BBB) to alter Aβ-dependent and independent effects. As discussed, there are higher Aβ levels in
APOE4 AD patients and E4FADF mice in this study. Aβ can cause vessel disruption by directly signaling to brain endothelial cells and indirectly via neuronal dysfunction and activation of astrocytes, microglia and pericytes. ApoE also signals to different cells types in the brain to disrupt vessel length. In astrocytes and microglia apoE4 is associated with a detrimental stress-induced (including Aβ) neuroinflammatory response [
49]; mediators described as inflammatory can affect brain endothelial function (e.g. cytokines, matrix metalloproteases). In pericytes apoE4 is less effective at suppressing motility [
15] and preventing MMP9 production [
4,
22], both of which disrupt total vessel length. ApoE4 also directly disrupts brain endothelial cell tight junctions and effects peripheral lipid metabolism to cause brain endothelial dysfunction.
Overall, the female sex-induced, APOE4 modulated, Aβ accelerated changes in homeostatic signaling at the CV may ultimately converge to predispose and/or amplify stress-induced brain endothelial damage. Our ongoing studies are focused on delineating the role of APOE, aging, sex and peripheral risk factors on CV length.
EGF prevents cognitive and CV dysfunction in E4FADF mice
An important finding here is that EGF prevented CV dysfunction in E4FADF mice. These data are in agreement with the lower EGF plasma levels observed in humans and the beneficial effects of EGF in models of stroke and traumatic brain injury. In contrast improved cognition after blocking the tyrosine kinase activity of the EGF receptor (EGFR) with gefitinib has been reported in drosophilia and AD-Tg mice [
57]. Aside methodological differences that commonly impact data (e.g. mouse model, time of treatment), the specificity of gefitinib for the EGFR has been drawn into question. Recently, gefitinib was demonstrated to antagonize a number of G-protein coupled receptors including adrenoreceptors, chemokine receptors, histamine receptors and other neuronal receptors [
60]. Gefitinib is reported as a brain penetrant and therefore its beneficial effects in AD-Tg mice could be mediated by non-EGFR targets that play a role in AD-like pathology.
As EGF induced a pronounced effect on preventing cognitive decline in E4FADF mice, it is important to consider potential mechanisms of action. Our data support that the primary mode of action is not on cells within the brain, since brain EGF levels were not altered after EGF treatment. One caveat is that the technique used to measured EGF (ELISA of whole cortex or hippocampus) may have failed to detect regional high concentrations of EGF. Further, EGF did not modulate Aβ levels unlike VEGF in AD-Tg mice [
58]. Although VEGF lowers Aβ levels in slice cultures [
11] a role for EGF in this process has not been assessed. As EGF did not change Aβ levels, one hypothesis is that EGF acts directly on brain endothelial cells to prevent disrupted signaling induced by the interactive effects of female sex,
APOE4 and Aβ in E4FADF mice. This is consistent with our in vitro data [
31], the beneficial effect of EGF in wound healing, the promotion of angiogenesis in cancers that produce high EGF levels or that contain a mutation in erbb2 (a dimerization receptor partner of the EGFR), the protective effect of EGF-induced angiogenesis in stroke models and the general protective function of EGF in epithelial tissue (reviewed in [
16]). Further, during the course of treatment EGF may have prevented a loss of key proteins involved in the homeostatic functions of brain endothelial cells, prior to vessel degeneration. For example key proteins involved in the nutrient transport/signaling molecule supply, waste product removal and efflux/metabolic/structural barrier functions of brain endothelial cells. For microbleeds a key question remains on whether EGF prevented global leakiness deficits in capillaries (e.g. via preventing tight junction changes or basement membrane degradation) and/or dysfunction at the post capillary venule level (the site of cellular trafficking).
An alternative hypothesis is that EGF activated receptors outside of brain endothelial cells in the CNS and/or the periphery to cause functional effects that prevent changes in CV coverage. For example, subtle increases in brain EGF levels may induce a beneficial response in pericytes, astrocytes, microglia or neurons to modulate levels of soluble molecules that maintain CV length. In the periphery EGF can signal in cells of virtually every organ in the body [
15] and is associated with changes in metabolism and insulin signaling, both of which may protect the CV. An indicator of the peripheral effects is the lower body weight in EGF treated mice. EGF may also prevent cognitive decline independent of any CV changes through the same CNS and peripheral processes described above, in addition to promoting neurogenesis. Our data provide the basis for further studies dissecting if brain endothelial cells are the primary target of EGF.
CV length and AGFs as potential therapeutic targets in AD
What do the data mean in the context of CV length in AD and therapeutic options for AD patients? There is no clear in vitro or in vivo consensus on whether there is higher or lower CV in AD. One proposal is that lower CV density contributes to AD progression by disrupting cerebral blood flow and the complex transport and metabolic systems of the CV [
18,
25,
29,
39]. The counter argument is that AD-pathways induce angiogenesis, causing hypersprouting and tight junction disruption, which increase CV permeability [
7,
9,
13]. Traditional angiogenic signaling, typically observed over the short-term is likely not applicable to a chronic neurodegenerative condition such as AD. Pathological angiogenesis, described as the angiogenesis hypothesis [
3] may be more applicable for AD: AD-processes not only cause direct vessel degeneration but also induce signaling cascades consistent with increased angiogenesis, however there is a failure to complete maturation and vessel formation. AD-risk factors both in the CNS and periphery could predispose and amplify the disrupted angiogenic signaling in brain endothelial cells.
There is a drive to develop novel strategies for AD as an alternative or complimentary approach for Aβ or tau-based therapeutics. AGF-like drugs may meet this criteria, yet are frequently overlooked in drug development programs. Indeed, an alternative focus is proposed of using therapeutics to block angiogenesis-like processes [
54]. Our data with EGF and published data with other factors that increase total vessel coverage support an alternative approach [
3,
26,
27,
32], which is to stabilize brain endothelial cells and maintain or increase vessel length. Further research is required to fully dissect the use of AGF-based treatments for AD. Key research areas include the potential for AGFs to reverse AD-like pathology in vivo, distinguishing CV versus peripheral effects and dissecting whether EGF-like molecules act on cells within the brain (i.e., would a brain penetrant AGF-like drug or treatment protocol be more efficacious than non-CNS penetrant). Further research is needed to address the concerns that agonists for AGF receptors could increase cancer risk. Cancer cells themselves are often the origin of high AGF levels; therefore finding a dosing strategy that negates this concern could be important in the elderly AD population.