Declines in vascular integrity are potential contributors to Alzheimer's disease (AD) as these result in increased blood-brain barrier permeability and, consequently, accelerate neuroinflammation and cognitive impairment. Roundabout guidance receptor 4 (Robo4) is primarily expressed in endothelial cells and stabilizes the vasculature, thereby potentially protecting the brain in AD. To study the effect of Robo4 on neuroinflammation and cognitive function in the context of AD, we compared Robo4 knockout and wild-type mice crossed with mice with and without AD mutations (APP/tau) from heterogeneous age and sex groups. We found that knockout of Robo4 led to greater astrocyte activation, as demonstrated by GFAP content, but this effect depended on the brain region studied. The knockout of Robo4 also led to greater activated microglia, as assessed by Iba1 content, but only in the presence of AD-related mutations. We found that AD mutations, but not Robo4 mutations, were associated with cognitive dysfunction, as measured by a nest-building test. Lastly, there was a non-statistically significant trend toward Robo4 deletion being associated with greater arterial stiffness. In summary, these results demonstrate that Robo4 impacts neuroinflammation and arterial stiffness; however, the impact on neuroinflammation is dependent on the presence/absence of AD-related mutations and the brain region examined.

The APOEε4 allele and oestrogen deficiency independently predispose females to an increased risk of vascular and metabolic impairments, but their cerebrovascular effects are less understood. The purpose of this study was to determine the interaction between APOE genotype and oestrogen on cerebrovascular endothelial and mitochondrial function. We studied young female homozygous APOEε3 and APOEε4 mice (n = 19-20/group; ∼6 months old) that were fed a high-fat diet and were ovariectomized (OVX), OVX and supplemented with 17β-oestradiol, or left intact. In APOEε3 mice, OVX was associated with impaired posterior cerebral artery endothelium-dependent dilatation, which was rescued by 17β-oestradiol. However, in APOEε4 mice, there was no effect of OVX or 17β-oestradiol on cerebral artery endothelial function. Carotid artery passive stiffness was greater with OVX and lower with 17β-oestradiol treatment in APOEε3 mice, but there was no impact of OVX or 17β-oestradiol in the APOEε4 mice. In cerebral arteries and arterioles, 17β-oestradiol led to higher mitochondrial complex I respiration in APOEε3 but not APOEε4 mice. These functional differences were concomitant with group differences in mitochondrial DNA copy number, antioxidant enzymes and pro-inflammatory factors. Overall these results indicate that the APOE genotype modulates the impact of OVX and oestradiol on the cerebral vasculature. We found that 17β-oestradiol enhances cerebrovascular endothelial and mitochondrial function in OVX APOEε3 mice but not in APOEε4 mice. This suggests that 17β-oestradiol supplementation may have more cerebrovascular benefits for APOEε4 non-carriers. KEY POINTS: Females have twice the risk of Alzheimer's disease than males, and the APOEε4 genetic variant has a greater risk for Alzheimer's disease than the APOEε3 variant. The risk for Alzheimer's disease increases after menopause in females, suggesting that the loss of female sex hormones may play a role. There are highly inconsistent results among past studies examining the interaction between APOE genotype and oestrogens on brain outcomes, and their impact on the vasculature has not been studied. We aimed to determine the impact of APOEε4 genotype on the cerebrovascular response to ovariectomy and oestradiol. We found that oestradiol improved cerebral artery endothelial function and mitochondrial respiration in ovariectomized APOEε3 mice following ovariectomy. In contrast APOEε4 mice were resistant to the beneficial effects of ovarian hormones on cerebrovascular and mitochondrial function. This research suggests that APOE genotype may be a consideration when weighing the risks and benefits of prescribing hormone replacement therapy to postmenopausal females.

Old age and the apolipoprotein E ε4 (APOE4) genotype are two of the greatest risk factors for late-onset Alzheimer's disease (LOAD). However, the interaction between these is poorly understood, as most preclinical studies use young mice. Therefore, we assessed the interaction between APOE genotype and age across a comprehensive set of cerebrovascular and related outcomes. We performed in vivo imaging, ex vivo cerebral artery studies, behavioral tests, and molecular analyses in male and female homozygous APOE3 and APOE4 mice at ~6 months (young) and ~24 months (old). APOE4 interacted with old age to lead to deficits in brain volume and greater microglia content. Old APOE4 mice also exhibited greater cerebral artery vasoconstriction to endothelin-1 (ET-1) than old APOE3 mice, a response concomitant with age- and genotype-related differences in the expression of ET-1 receptors and endothelin-converting enzyme. While we found several interactions between age and APOE genotype, only age impacted cognitive function, cerebral artery endothelial function, and arterial stiffness. In summary, we found that brain volume, neuroinflammation, and ET-1-related outcomes were influenced by the interaction of APOE genotype and age, while other outcomes were affected only by age. As such, an altered ET-1 response and greater neuroinflammation may contribute to the increased risk for LOAD in APOE4 carriers.

Neuroendocrine tumors (NETs) secrete vasoactive hormones that promote hemodynamic instability. This study investigated whether NETs alter vascular responsiveness using a murine model. J:Nu mice received intrasplenic injections of BON-1 NET cells (BON-1, n=21) or PBS (VEH, n=21) and monitored for 10 weeks. Liver metastases were identified by histological analysis, chromogranin A expression, and presence of ALDH1-positive cancer stem cells. Vasomotor function of isolated mesenteric arteries was assessed to acetylcholine, serotonin, vasopressin, and endothelin-1, and with nitric oxide synthase inhibition (L-NAME), 5-HT receptor blockade, or serotonin incubation. BON-1 mice exhibited impaired vasodilation to acetylcholine and serotonin compared with VEH (p<0.05), specifically in males (p<0.001). L-NAME and 5-HT1b/d receptor blockade attenuated vasodilatory responses to serotonin only in VEH mice. Serotonin incubation reduced vasopressin-mediated vasoconstriction (p<0.0001). These findings indicate that NET metastases are associated with impaired serotonin-mediated vasodilation, which is associated with reduced nitric oxide bioavailability, altered 5-HT-1b/d receptor action, and sex-dependent impairment of endothelium-dependent vasodilation. Elevated serotonin levels may further compromise vasopressin-mediated vasoconstriction. The presence of NET metastases alters vascular responsiveness, which may contribute to hemodynamic instability.

Abstract Age‐related increases in large artery stiffness contribute to cerebrovascular dysfunction and cognitive impairment. Alagebrium Chloride (ALT‐711) is a collagen crosslink breaker that reduces vascular stiffness. We hypothesized that long‐term treatment with ALT‐711 would reverse age‐related large artery stiffness, thereby preserving cerebral artery function and cognitive function. We treated old male and female C57BL/6 mice (20 months) with ALT‐711 (1 mg/kg/day) or vehicle via oral gavage for 4 months and measured arterial stiffness, cerebral artery endothelial function, cognitive function, and collagen crosslinking. Treatment with ALT‐711 did not significantly reduce stiffness in large arteries or cerebral arteries. There was no effect of ALT‐711 on cerebral artery endothelial function, cognitive function, or collagen crosslinking. However, we found that collagen crosslinking was greater in old mice than in young, untreated C57BL/6 mice. In old mice, aortic collagen crosslinking was correlated with carotid artery passive stiffness. Additionally, among old mice, the passive stiffness of the cerebral artery negatively correlated with cerebral artery endothelial function and cognitive function. Cerebral artery endothelial function was negatively correlated with Frailty Index. In sum, age‐related cerebral artery stiffness is negatively correlated with cognitive function and may be a promising therapeutic target to combat cerebrovascular dysfunction and cognitive decline with age.

Late-onset Alzheimer's disease (LOAD) is an age-related disease that is strongly associated with vascular risk factors and cerebrovascular impairments. As such, changes in the vasculature with advancing age likely contribute to LOAD, but the mechanisms underlying these contributions remain incompletely understood. With advancing age, there is dysregulation of cerebral blood flow, impairment of neurovascular coupling, and increased blood-brain barrier permeability, which may initiate or contribute to the neuropathology associated with LOAD. Changes to the vasculature outside of the brain, including increases in blood pressure and arterial stiffness, may initiate age-related cerebrovascular impairments. Age-related increases in oxidative stress and inflammatory signalling, as well as contributions to LOAD-related neuropathology, such as amyloid-β and hyperphosphorylated tau, impair cerebrovascular cells. In this review, we summarize the evidence for the role of vascular ageing in LOAD, describing age-related cerebrovascular impairments and their causes.

Apolipoprotein E4 (E4) increases the risk of Alzheimer's disease (AD) by up to 12-fold. However, understanding of the mechanisms underlying this increased risk has been limited by a lack of preclinical models that accurately reflect the effects of E4 in the presence of humanized non-mutant amyloid-β precursor protein (hAβPP). Therefore, we studied novel humanized APOE and hAβPP mice to investigate the contributions of the E4 genotype to cognitive, inflammatory, and vascular dysfunction, specifically comparing male and female E3/hAβPP and E4/hAβPP mice. E4/hAβPP mice exhibited impaired nest-building behavior and novel object recognition compared with E3/hAβPP mice. Microglial content was higher in E4/hAβPP mice, whereas astrocyte content was not different across groups. E4/hAβPP mice had greater carotid and cerebral artery stiffness, and higher collagen I content in cerebral arteries than E3/hAβPP mice. Under static pressure, cerebral artery endothelium-dependent and endothelium-independent vasodilation were similar across genotypes. However, high pulse pressure selectively impaired cerebral artery endothelial function in E4/hAβPP mice, with the greatest impairment observed in females. The E4/hAβPP mice also exhibited higher cortical expression of Nox2 and Sod1 and elevated cerebral artery Il1b expression. As such, E4/hAβPP mice exhibit convergent cognitive, inflammatory, and vascular abnormalities that recapitulate several features of AD. Elevated pulse pressure revealed an E4-dependent vulnerability of the cerebral vasculature, suggesting that vascular stress may be an important contributor to disease risk. Together, our findings support the use of the APOExhAβPP model to investigate the mechanisms by which E4 promotes vascular dysfunction, neuroinflammation, and cognitive impairment in AD.

Elastin insufficiency is associated with structural differences in the large elastic arteries and cerebral artery dysfunction. However, previous studies have not assessed potential sex differences in cerebrovascular function. We measured cerebral blood flow (CBF) using arterial spin labeling MRI at rest and in response to hypercapnia challenge (cerebrovascular responsiveness, CR) in middle-aged and old elastin haploinsufficient (Eln+/-) and wild-type (Eln+/+) mice. We also assessed neuroinflammation by microglia and astrocyte cell counts. We found that Eln+/- mice had a significantly lower resting CBF in the cerebral cortex compared with Eln+/+ mice, with similar non-significant trends in the hippocampus and thalamus. In contrast, the Eln+/- mice had an intact hypercapnic response, resulting in better CR compared with Eln+/+ in hippocampus, with a similar trend in the cerebral cortex. Sex did not impact CBF or CR. We found that Eln+/- mice had lower hippocampal volume compared with Eln+/+ mice. Glia cell counts were highly dependent on brain region, with Eln+/- mice having more microglia in the cerebral cortex, but fewer astrocytes in the hippocampus compared with Eln+/+ mice. While sex also impacted glial cell counts, we found no interactions between sex and Eln genotype. Our results demonstrate that elastin haploinsufficiency results in lower resting CBF, but greater CR.

INTRODUCTION:Cerebrovascular deficits, including cerebral amyloid angiopathy (CAA), play a key role in Alzheimer's disease (AD) pathogenesis. Here, we characterize the susceptibility of the WSB/EiJ genetic context to human AD-relevant cerebrovascular phenotypes. METHODS:CAA and parenchymal plaque analysis and in vivo neurovascular imaging were performed on WSB.APP/PS1 brains. Transcriptomics was performed on WSB.APP/PS1 and B6.APP/PS1 brains. B6 and WSB cerebrovascular reactivity was assayed ex vivo. Additional CAA and parenchymal plaque analysis was performed on WSB.APP/PS1 mice with APOE2, APOE3, or APOE4 alleles. RESULTS:WSB.APP/PS1 brains exhibited plaque deposition, CAA, transcriptomic overlap with human AD, myelin deficits, cerebrovascular/metabolic uncoupling, and altered cerebrovascular morphology. Aged WSB vasculature retained vasoreactivity but exhibited increased stiffness. Compared to WSB.APOE2/2APP/PS1, WSB.APOE4/4APP/PS1 mice had increased CAA and plaque-associated microglial area. DISCUSSION:These data illustrate the utility of the WSB genetic context to model CAA and uncover vascular contributions to AD.

Background:Postmenopausal females who carry an APOEε4 allele are at higher risk of late-onset Alzheimer's Disease compared to age-matched APOEε4 males. Estrogen deficiency predisposes females to an increased risk of vascular, cognitive, and metabolic impairments. While estrogen and APOE genotype are known to impact metabolic and mitochondrial function in the brain, their cerebrovascular effects are less understood. Thus, the purpose of this study was to determine the interaction between APOE genotype and estrogen on cerebrovascular endothelial and mitochondrial function. Methods:Young female homozygous APOEε3 and APOEε4 mice (n=19-20/group; ~6 months old) fed a high-fat diet were ovariectomized (OVX), OVX and supplemented with 17β-estradiol, or left intact. Results:In APOEε3 mice, OVX was associated with impaired posterior cerebral artery endothelium-dependent dilation, which was rescued by 17β-estradiol. However, in APOEε4 mice, there was no effect of OVX or 17β-estradiol on cerebral artery endothelial function. Carotid artery passive stiffness was greater with OVX and lower with 17β-estradiol treatment in APOEε3 mice, but there was no impact of OVX or 17β-estradiol in the APOEε4 mice. In cerebral arteries and arterioles, mitochondrial complexes I and I+II respiration were lower in APOEε4 mice compared with APOEε3 mice. 17β-estradiol led to higher mitochondrial complex I respiration in APOEε3 but not APOEε4 mice. These functional differences were concomitant with group differences in mitochondrial DNA copy number, antioxidant enzymes, and pro-inflammatory factors. In contrast to other outcomes, we found that 17β-estradiol treatment was associated with lower cerebral artery stiffness in APOEε4 but not APOEε3 mice. Conclusions:Overall, these results indicate that the APOE genotype modulates the impact of estrogen on the cerebral vasculature. We found that 17β-estradiol enhances cerebrovascular endothelial and mitochondrial function in APOEε3 mice but not in APOEε4 mice. The results suggest that 17β-estradiol supplementation has more cerebrovascular benefit for APOEε4 non-carriers. Novelty & Significance:What is known?: Females have twice the risk of Alzheimer's disease compared with males, and the APOE4 genetic variant is associated with a greater risk for Alzheimer's disease compared with the APOE3 variant. The risk for Alzheimer's disease increases after menopause in females, suggesting that the loss of female sex hormones may play a role. There are highly inconsistent results among past studies examining the interaction of APOE genotype and estrogens on cognitive function and other brain outcomes. What new information does this article contribute?: Vascular outcomes were not measured in previous studies examining the interaction between APOE genotype and estrogens. As such, we aimed to determine the impact of APOE4 genotype on the cerebrovascular response to estradiol. We found that estradiol improved cerebral artery endothelial function and mitochondrial respiration in APOE3 mice following ovariectomy. In contrast, APOE4 mice were refractory to the beneficial effects of estradiol on cerebrovascular endothelial and mitochondrial function. The broader implication of this research is that APOE genotype may be a consideration when prescribing hormone replacement therapy to menopausal females due to the impact on vascular outcomes.

Artery structural properties and Alzheimer's disease (AD) pathology are individually associated with impaired cerebrovascular function; however, the interaction of these factors is unclear. Furthermore, while elastin haploinsufficient (Eln +/- ) mice are known to have impaired cerebrovascular function, sex differences for this effect have not been previously studied. To answer these questions, we crossed middle-aged and old Eln +/- mice with 3xTg-AD mice. We measured cerebral blood flow (CBF) using arterial spin labeling MRI at rest and during hypercapnia to calculate cerebrovascular reactivity (CVR). We also assessed neuroinflammation by microglia and astrocyte cell counts. We found that Eln +/- mice had lower resting blood flow rate in the cerebral cortex compared with Eln +/+ mice, but Eln +/- mice had an intact hypercapnic response, resulting in better CVR compared with Eln +/+ in hippocampus. Sex did not impact resting blood flow or CVR. 3xTg-AD mice had a lower resting CBF than non-AD mice, and there was an interaction between Eln genotype and AD mutations on CVR, such that Eln +/- x 3xTg-AD mice had the poorest hippocampal CVR of all groups. Glia cell counts were highly dependent on brain region, with Eln +/- having more microglia but fewer astrocytes, while 3xTg-AD having higher both microglia and astrocytes. While sex also impacted glial cell counts, we found no interactions between sex and Eln genotype. Our results demonstrate that elastin haploinsufficiency and AD mutations individually result in lower resting CBF, and the combination of these leads to impaired CVR.

Stiffening of the large arteries is a hallmark feature of vascular aging and is associated with cognitive impairment and Alzheimer’s disease pathology. Increased large artery stiffness leads to higher-than-normal pulse pressure in the cerebral circulation, damaging endothelial cells. It is known that short-term exposure to stiffer large arteries causes cerebral artery endothelial dysfunction and hypoperfusion in young mice. However, the impact of long-term exposure to large artery stiffness on cerebrovascular function is unknown. As there are known sex differences in Alzheimer’s disease risk, we sought to understand the influence of sex on the impact of large artery stiffness on cerebrovascular function. We studied an established model of greater large artery stiffness, the elastin haploinsufficient (Eln±) mouse, at young (6 months) and old (24 months) ages and compared with wildtype (Eln+/+) mice. We measured endothelium-dependent dilation ex vivo in pre-constricted, pressurized posterior cerebral arteries by the maximal response to acetylcholine. Nitric oxide bioavailability was determined by the acetylcholine response with and without the presence of L-NAME, a nitric oxide synthase inhibitor. Endothelium-independent dilation was assessed by the maximal response to sodium nitroprusside. With the sexes combined, posterior cerebral artery endothelium-dependent dilation was better for young compared with old mice (p<0.001) and for Eln+/+ compared with Eln± mice (p = 0.03). In males, Eln+/+ mice had better posterior cerebral artery endothelium-dependent dilation than Eln± mice at young ages (p = 0.05), but not at old ages (p = 0.35). The opposite occurred in females, as Eln+/+ mice had better posterior cerebral artery endothelium-dependent dilation than Eln± at old ages (p = 0.04) but not at young ages (p = 0.71). Group differences in endothelium-dependent dilation were mediated by differences in nitric oxide bioavailability. There was no effect of genotype, age, or sex on endothelium-independent dilation. Our results suggest males are more vulnerable at young ages, while females are more vulnerable at old ages, to the detrimental effects of large artery stiffness on cerebrovascular function. These age-dependent differences in vulnerability could underlie sex differences in Alzheimer’s disease risk. Funded by AARG-200675709, TL1TR002371.

Age-related increases in arterial stiffness and pulse pressure are associated with cerebrovascular dysfunction; yet sex differences in these factors remain unclear. We found that cerebral artery passive stiffness was greater with age, independent of sex. With exposure to acute pulse pressure, young, compliant female cerebral arteries had a more pronounced decline in endothelial function. Thus, females may be more susceptible to cerebrovascular dysfunction due to age-related increases in pulse pressure.

Vascular contributions are now widely accepted to play a key role in many cases of dementia, including Alzheimer's disease (AD), that commonly manifest as cerebral small vessel diseases, including cerebral amyloid angiopathy (CAA). However, the mechanisms by which vascular contributions such as CAA contribute dementias such as AD are not well understood. This is due in part to the lack mouse models that develop robust CAA, hampering our ability to develop therapies that target vascular deficits. To address this, we have explored the use of distinct genetic contexts to enhance the face validity of mouse models for AD. We have previously identified the WSB/EiJ (WSB) strain as a model that shows increased susceptibility to CAA in the presence of the APP/PS1 amyloid driver, compared to the commonly used C57BL/6J (B6) strain. Here, we now perform an in-depth characterization of WSB.APP/PS1 and its WSB wild type (WT) counterpart, assessing male and female mice, at 4, 8, and 12 months of age (M). We show that WSB.APP/PS1 mice show mild CAA at 8M, with robust CAA being apparent at 14M. Transcriptional profiling showed strong correlation to AMP-AD gene expression modules highlighting the human relevance of WSB.APP/PS1 mice and predicted white matter deficits at 14M that was confirmed by immunofluorescence. PET/CT showed blood flow and metabolic deficits, and modifications in small vessel morphology in 8M WSB.APP/PS1 compared to WSB WT mice. We tested whether cerebrovascular reactivity deficits in WSB WT mice may underly the susceptibility to CAA, but interestingly, they did not show age-dependent decline in reactivity that was observed in B6 mice. Finally, using an allelic series of humanized apolipoprotein E (APOE), we show that APOE4 increased the extent of CAA in WSB.APP/PS1 mice, compared to APOE2 and APOE3, but in a sex-dependent manner. Collectively, these data show the utility of the WSB strain to uncover mechanisms of vascular contributions to Alzheimer's disease and related dementias.

Postmenopausal females who carry an APOE4 allele are at higher risk of late-onset Alzheimer’s Disease (LOAD) compared to age-matched APOE4 males. Estrogen deficiency predisposes females to an increased risk of vascular, cognitive and metabolic impairments. Estrogen and APOE genotype are known to impact metabolic and mitochondrial function in the brain, but their effects on cerebral vessels are unknown. Thus, the purpose of this study was to determine the interaction between APOE genotype and estrogen deficiency in relation to cerebrovascular mitochondrial function. Young female homozygous APOE3 and APOE4 mice (n = 6-8 per group; 6 months old) fed a high-fat diet were ovariectomized (“ovx”), ovariectomized and supplemented with 17β-estradiol (0.36 mg, 60-day release, “estradiol”), or left intact (“sham”). At 2 months after ovariectomy, a glucose tolerance test (GTT) was performed, then cerebral arteries and arterioles were dissected, incubated in saponin, then assessed for mitochondrial respiration in response to substrates probing carbohydrate metabolism (Oroboros). Data are presented as mean±SEM. There was an interaction between APOE genotype and ovx/estradiol status in relation to cerebrovascular complex I (CI)- (p = 0.04) and complex II (CII)-coupled respiration (p = 0.04). Additionally, there was a significant effect of genotype, such that vessels from APOE3 mice had greater CI-coupled respiration than vessels from APOE4 mice (p = 0.02). When examining differences between APOE3 groups, APOE3 -estradiol mice had a 53% greater CI-coupled respiration than APOE3 -sham mice (15.4±3.4 vs. 7.2±1.2 pmol/(s*mg), p = 0.03) and a 60% greater CI-coupled respiration than APOE3 -ovx mice (6.2±1.6 pmol/(s*mg), p = 0.007). The APOE3 -estradiol group also had greater CI+CII-coupled respiration compared to both APOE3 -sham (50.8±7.1 vs. 27.3±1.7 pmol/(s*mg), p = 0.006) and ovx groups (28.0±5.7 pmol/(s*mg), p = 0.009). Interestingly, CI and CI+CII coupled respiration did not differ between sham, ovx, and estradiol in APOE4 mice (p>0.05). Maximal uncoupled respiration was greater in the APOE3 mice than APOE4 mice (p = 0.01). Whole-body glucose tolerance did not differ between groups (all p>0.05). Overall, these results indicate that APOE genotype modulates the impact of estrogen on the cerebrovasculature. We found that 17β-estradiol enhances cerebrovascular mitochondrial function in APOE3 mice but not APOE4 mice. The results suggest that estradiol supplementation may have more therapeutic benefit for APOE4 non-carriers.

Elevated arterial pulse pressure (PP) is associated with cognitive decline and Alzheimer’s disease (AD). High PP damages the brain vasculature by causing endothelial cell dysfunction. Stiffer cerebral arteries have an impaired ability to dampen PP, which transmits the pulsatility further into the microvasculature, where it can damage brain tissue. At the same time, the APOE4 genotype is associated with cerebrovascular dysfunction; however, it is unknown if elevated PP amplifies this risk. Furthermore, female APOE4 carriers are at a higher risk for developing AD than males. Thus, we hypothesized that female APOE4 mice would be more vulnerable to high PP and have greater cerebral artery stiffness and cognitive impairments compared with male mice. In male and female mice homozygous for APOE4 and humanized Aβ1-42 (APOE4/hAβ, n = 26, 6 months), we assessed ex vivo endothelium-dependent vasodilation by the dose-responses to acetylcholine in isolated posterior cerebral arteries (PCAs) following exposure to static pressure (50 mmHg), low PP (50-75 mmHg), or high PP (37.5-87.5 mmHg). PCA endothelium-independent vasodilation was measured by the response to sodium nitroprusside, and stiffness was measured during the high PP condition. We assessed cognition by the Morris Water Maze. Under static pressure, PCA endothelium-dependent vasodilation was similar between male and female mice (p = 0.13). In females, PCA endothelium-dependent vasodilation was similar between static and low PP (p = 0.37), while exposure to high PP resulted in a 38% decline in endothelium-dependent vasodilation (p = 0.004 vs. static). In contrast, PCA endothelium-dependent vasodilation in male mice was similar between static and PP conditions (p>0.05). Endothelium-independent vasodilation did not differ between sexes (p>0.05). Female mice also exhibited a higher PCA β-stiffness index than male mice (p = 0.03). Additionally, during the Morris Water Maze probe trial, females crossed the platform area significantly fewer times than males (p = 0.048). Our data indicate that high PP is detrimental to cerebral artery endothelial function in female, but not male, APOE4/hAβ mice. Additionally, female APOE4/hAβ mice have stiffer cerebral arteries and poorer spatial memory than male APOE4/hAβ mice. Our findings suggest that sex may influence the interactive effect of APOE4 and PP on cerebrovascular and AD-related outcomes.

With advancing age, the cerebral vasculature becomes dysfunctional, and this dysfunction is associated with cognitive decline. However, the initiating cause of these age-related cerebrovascular impairments remains incompletely understood. A characteristic feature of the aging vasculature is the increase in stiffness of the large elastic arteries. This increase in arterial stiffness is associated with elevated pulse pressure and blood flow pulsatility in the cerebral vasculature. Evidence from both humans and rodents supports that increases in large elastic artery stiffness are associated with cerebrovascular impairments. These impacts on cerebrovascular function are wide-ranging and include reductions in global and regional cerebral blood flow, cerebral small vessel disease, endothelial cell dysfunction, and impaired perivascular clearance. Furthermore, recent findings suggest that the relationship between arterial stiffness and cerebrovascular function may be influenced by genetics, specifically APOE and NOTCH genotypes. Given the strength of the evidence that age-related increases in arterial stiffness have deleterious impacts on the brain, interventions that target arterial stiffness are needed. The purpose of this review is to summarize the evidence from human and rodent studies, supporting the role of increased arterial stiffness in age-related cerebrovascular impairments.

Two of the greatest risk factors for late-onset Alzheimer’s disease (LOAD) are age and the APOE4 genotype. The gene encoding for apolipoprotein E ( APOE) has three isoforms in humans ( E2, E3, E4). E3 has the highest prevalence, while E4 exponentially increases the risk for AD. Additionally, it is well known that the E4 genotype is accompanied by an altered cerebral blood flow. However, the mechanisms underlying these blood flow changes in E4 individuals remain unclear and may be mediated by changes to cerebral artery structure and function. We aimed to determine if APOE genotype alters the impact of age on cerebral artery endothelial function and stiffness. We studied male and female, young E3 (n=20; ~6 months), aged E3 (n=9; ~24 months young E4 (n=22; ~6 months), and aged E4 (n=18; ~24 months) mice. We assessed endothelium-dependent and -independent vasodilation and vasoconstriction in isolated, pressurized posterior cerebral arteries (PCAs). Passive stiffness was measured in isolated PCAs after incubation in a calcium-free solution. Data are mean ± SD. Both old E3 and old E4 mice had impaired PCA endothelium-dependent vasodilation to acetylcholine compared with young mice ( E3: 29±10.6% vs 46±15.2%, p=0.02; E4: 30±10.8 vs 49±14.3%, p=0.0004). Sex comparisons found that the PCA maximal dilation to acetylcholine was 21% greater in young E3 females compared with young E3 males (52±12.5% vs 41±16%, p=0.04), and 13% greater in young E4 females compared with young E4 males (52±10% vs 45±16%, p=0.07), but there were no sex differences among the old groups. Additionally, the PCA responses to acetylcholine in the presence of nitric oxide synthase inhibitor L-NAME did not differ between groups (p>0.05). Endothelium-independent dilation, measured as the PCA response to sodium nitroprusside, also did not differ between groups (p>0.05). Aged E3 mice, compared with young E3, had a lower maximal constriction to potassium chloride (20±11% vs 59±16%, p<0.0001) and endothelin-1 (23±16% vs 53±14%, p=0.0005). In contrast, young E4 and old E4 mice had a similar maximal constriction to potassium chloride (35±13% vs 43±18%, p>0.05) and endothelin-1 (48±12% vs 47±20%, p>0.05). Lastly, we found that old mice had a greater PCA β stiffness index compared with young mice for both the E3 (15.5±4.5 vs 10.6±1.4 AU, p=0.0008) and E4 (13.4±2.9 vs 10.2±2.5 AU, p>0.0001) groups. In summary, we found that age impairs cerebral artery endothelial function in both APOE3 and APOE4 mice, and young female mice have better endothelial function than their male counterparts regardless of genotype. In addition, vasoconstrictor responsiveness appears to decline in old age in APOE3 mice, whereas vasoconstrictor responsiveness is maintained in old APOE4 mice. This maintained sensitivity to vasoconstrictors in old age may explain why APOE4 individuals have reduced cerebral blood flow and greater LOAD risk. Luvaas Family Fund, Alzheimer’s Association ALZDISCOVERY-1049110. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Declines in vascular integrity are potential contributors to Alzheimer’s disease (AD) as these result in increased blood-brain barrier permeability and, as a consequence, accelerate neuroinflammation and cognitive impairment. Roundabout guidance receptor 4 (Robo4) is primarily expressed in endothelial cells and stabilizes the vasculature, and thus, has the potential to protect the brain in AD. To study the effect of Robo4 on neuroinflammation and cognitive function in the context of AD, we compared Robo4 knockout and wildtype mice crossed with mice with and without AD mutations (APP/tau). We found that the knockout of Robo4 led to greater astrocyte activation, as demonstrated by GFAP content, but this was dependent on the brain region studied. The knockout of Robo4 also led to greater activated microglia, as assessed by Iba1 content, but only in the presence of AD-related mutations. We found that AD mutations, but not Robo4, were associated with cognitive dysfunction measured by a nest-building test. In contrast, Robo4 deletion, but not AD mutations, was associated with impaired motor coordination. Lastly, Robo4 deletion was associated with greater arterial stiffness, but this trend did not reach statistical significance. In summary, these results demonstrate that Robo4 impacts neuroinflammation, motor coordination, and arterial stiffness, however, the impact on neuroinflammation is dependent on the presence/absence of AD-related mutations and the brain region examined.### Competing Interest StatementThe authors have declared no competing interest.

Age-related increases in large artery stiffness are associated with cerebrovascular dysfunction and cognitive impairment. Pyridoxamine treatment prevents large artery stiffening with advancing age, but the effects of pyridoxamine treatment on the cerebral vasculature or cognition is unknown. The purpose of this study was to investigate the effects of pyridoxamine on blood pressure, large artery stiffness, cerebral artery function, and cognitive function in old mice. Old male C57BL/6 mice consumed either pyridoxamine (2 g/L) or vehicle control in drinking water for ∼7.5 months and were compared with young male C57BL/6 mice. From pre- to post-treatment, systolic blood pressure increased in old control mice, but was maintained in pyridoxamine treated mice. Large artery stiffness decreased in pyridoxamine-treated mice but was unaffected in control mice. Pyridoxamine-treated mice had greater cerebral artery endothelium-dependent dilation compared with old control mice, and not different from young mice. Old control mice had impaired cognitive function; however, pyridoxamine only partially preserved cognitive function in old mice. In summary, pyridoxamine treatment in old mice prevented age-related increases in blood pressure, reduced large artery stiffness, preserved cerebral artery endothelial function, and partially preserved cognitive function. Taken together, these results suggest that pyridoxamine treatment may limit vascular aging.