Transient increases in intracellular Ca2+ activate endothelium-dependent vasodilatory pathways. This process is impaired in cerebral amyloid angiopathy, where amyloid- β(1-40) accumulates around blood vessels. In neurons, amyloid- β impairs the Ca2+-permeable N-methyl-D-aspartate receptor (NMDAR), a mediator of endothelium-dependent dilation in arteries. We hypothesized that amyloid- β(1-40) reduces NMDAR-elicited Ca2+ signals in mouse cerebral artery endothelial cells, blunting dilation. Cerebral arteries isolated from 4-5 months-old, male and female cdh5:Gcamp8 mice were used for imaging of unitary Ca2+ influx through NMDAR ( NMDAR sparklets) and intracellular Ca2+ transients. The NMDAR agonist NMDA (10 µmol/L) increased frequency of NMDAR sparklets and intracellular Ca2+ transients in endothelial cells; these effects were prevented by NMDAR antagonists D-AP5 and MK-801. Next, we tested if amyloid- β(1-40) impairs NMDAR-elicited Ca2+ transients. Cerebral arteries incubated with amyloid- β(1-40) (5 µmol/L) exhibited reduced NMDAR sparklets and intracellular Ca2+ transients. Lastly, we observed that NMDA-induced dilation of pial arteries is reduced by acute intraluminal amyloid- β(1-40), as well as in a mouse model of Alzheimer’s disease, the 5x-FAD, linked to downregulation of Grin1 mRNA compared to wild-type littermates. These data suggest that endothelial NMDAR mediate dilation via Ca2+-dependent pathways, a process disrupted by amyloid- β(1-40) and impaired in 5x-FAD mice.

Vasculopathy and nitro-oxidative stress are present in patients with Alzheimer’s disease (AD) and may contribute to disease progression and severity. Large conductance calcium activated K+ channels (BKCa) plays an important role in vasodilatory responses and maintenance of myogenic tone in resistance arteries. Opening of BKCa channels occurs upstream from localized intracellular Ca2+ release events (Ca2+ sparks), and results in K+ efflux, vascular smooth muscle cell hyperpolarization and vasorelaxation. In a pro-nitro-oxidative scenario, BKCa can be modified, resulting in decreased activity and hypercontractility, which can compromise cerebral blood flow regulation, generating an environment that may accelerate neurodegeneration. We hypothesized that reductions in BKCa-dependent vasodilation in cerebral arteries, as consequence of nitro-oxidative stress, results in neurovascular dysfunction in the 5x-FAD model of AD. Methods: Posterior communicating arteries (PComA) from 5 months-old male and female 5x-FAD and wild-type (WT) littermates were isolated and studied ex vivo using pressure myography. Smooth muscle Ca2+ transients were evaluated by spinning-disk confocal microscopy. Oxidative stress was assessed by oxidized glutathione levels in the brain using a colorimetric enzymatic assay. BKCa expression was assessed by qPCR. Nitrosylated BKCa was evaluated using Western blot. Functional hyperemia were evaluated by laser speckle contrast imaging. Data are means±SEM, 5x-FAD vs WT, analyzed by two-tailed Student’s t-test or Mann-Whitney test. Results: In females, PComA from 5x-FAD showed higher spontaneous myogenic tone than WT (Myogenic tone: 24.48 ± 3.20 vs 16.09 ± 0.93%, p<0.05, N=7). Constriction to the BKCa blocker iberiotoxin (30 nM) was smaller in 5x-FAD than WT, suggesting lower basal BKCa activity (Vasoconstriction: -4.25 ± 0.43 vs -9.22 ± 2.56%, p<0.05; N=5), which was independent of alterations in intracellular Ca2+ transients or BKCa mRNA expression. These vascular changes were associated with higher levels of oxidized glutathione in female 5x-FAD (7.83 ± 0.62 vs 5.27 ± 0.74 μM, p<0.05, N=8) and of S-nitrosylation in the BKCa α-subunit (0.68 ± 0.04 vs 0.41 ± 0.03, p<0.05, N=5). Female 5x-FAD mice showed increased expression of iNOS mRNA ([2-ΔΔCT]: 10.64 ± 5.40 vs 0.74 ± 0.19, p<0.05, N=6) and impaired functional hyperemia responses after whisker stimulation (%increase: 3.82 ± 0.64 vs. 9.91 ± 1.41%, p<0.05, N=6). No significant differences were observed between male 5x-FAD and WT for all parameters above. Conclusion: Cerebrovascular impairments were more pronounced in female 5x-FAD mice, observed as an increase in spontaneous myogenic tone, likely due to reduction in smooth muscle cell BKCa activity associated to an increase in brain nitro-oxidative stress and a blunted neurovascular coupling response. Together, they identify post-translational modifications of BKCa as a putative target to improve cerebral microvascular function in AD. National Institutes of Health (R00 HL140106, R01 AG073230) and the Alzheimer's Association (AARGD-21-850835). This is the full abstract presented at the American Physiology Summit 2023 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.

Cerebral microvascular dysfunction and nitro-oxidative stress are present in patients with Alzheimer's disease (AD) and may contribute to disease progression and severity. Large conductance Ca2+-activated K+channels (BKCa) play an essential role in vasodilatory responses and maintenance of myogenic tone in resistance arteries. BKCacan be modified in a pro-nitro-oxidative environment, resulting in decreased activity and vascular hyper-contractility, which can compromise cerebral blood flow regulation. We hypothesized that reductions in BKCafunction in cerebral arteries, as a consequence of nitro-oxidative stress, are associated with blunted neurovascular responses in the5x-FADmodel of AD. Using pressure myography, we observed that posterior communicating arteries (PComA) from 5 months-old female5x-FADmice showed higher spontaneous myogenic tone than wild-type (WT) littermates. Constriction to the BKCablocker iberiotoxin (30 ηM) was smaller in5x-FADthan WT, suggesting lower basal BKCaactivity, which was independent of alterations in intracellular Ca2+transients or BKCamRNA expression. These vascular changes were associated with higher levels of oxidative stress in female5x-FADand a higher level of S-nitrosylation in the BKCaα-subunit. In females, pre-incubation of PComA from5x-FADwith the reducing agent DTT (10 μM) rescued iberiotoxin-induced contraction. Female5x-FADmice showed increased expression of iNOS mRNA, lower resting cortical perfusion atop the frontal cortex, and impaired neurovascular coupling responses. No significant differences between male5x-FADand WT were observed for all parameters above. These data suggest that the exacerbation in BKCaS-nitrosylation contributes to cerebrovascular and neurovascular impairments in female5x-FADmice.

Introduction: Cerebral amyloid angiopathy (CAA), the accumulation of amyloid- β (1-40) (A β ) around cerebral arteries, impairs endothelial function. Endothelium-dependent dilation is a consequence of transient increases in intracellular [Ca 2+ ] in endothelial cells (EC). The Ca 2+ permeable N-methyl-D-aspartate receptor (NMDAR) mediates endothelium-dependent dilation, although if these effects are dependent on Ca 2+ influx and transients, or if they are impaired by A β , remains undetermined. Hypothesis: A β inhibits endothelial NMDAR-mediated Ca 2+ influx and transients in murine pial arteries. Methods: We performed Ca 2+ time-lapse imaging of en face pial arteries from cdh5-GCaMP8 mice to quantify EC Ca 2+ events induced by NMDAR activation. Data are means ± SEM. Results: Elemental Ca 2+ entry through NMDAR, hereon called NMDAR sparklets , was assessed in arteries incubated with EGTA-AM and cyclopiazonic acid (CPA) to inhibit intracellular Ca 2+ transients. NMDA (10 μM) induced an increase in NMDAR sparklets frequency when compared to vehicle, an effect inhibited by the NMDAR antagonist D-APV (in Hz: 0.12±0.01 vs 0.44±0.05 vs 0.21±0.02, vehicle vs NMDA vs NMDA+D-APV, p<0.05). Further, pial arteries exposed to NMDA without EGTA-AM and CPA showed an increase in the frequency of intracellular Ca 2+ transients, also blocked by D-APV (in Hz: 0.24±0.05 vs 0.53±0.10 vs 0.28±0.05, vehicle vs NMDA vs NMDA+D-APV, p<0.05). We then tested the effects of A β on Ca 2+ events in pial artery EC. We observed that 30 minutes exposure to A β (5 μM) caused a significant reduction in NMDAR sparklets (in Hz: 0.62±0.07 vs 0.22±0.03, NMDA vs NMDA + A β , p<0.05) but did not significantly alter intracellular Ca 2+ transients (in Hz: 0.62±0.37 vs 0.27±0.07, NMDA vs NMDA + A β ). Lastly, we performed pressure myography on pial arteries of wild-type and 5x-FAD mice, a model of familial Alzheimer’s disease with rapid amyloid accumulation. 5x-FAD mice displayed impaired vasodilation to NMDA (vasodilation (%): 9.86±0.64 vs 4.22±2.76, wild-type vs 5x-FAD , p<0.05). Conclusion: These preliminary data suggest that A β impairs endothelial NMDAR-associated Ca 2+ influx events in cerebral arteries, which can impair blood flow in CAA patients, thus contributing to cognitive impairment.

Alzheimer's disease (AD)‐ associated dementia, one of the most common forms of cognitive decline, shows a higher incidence in women than men. One of the underlying factors for dementia is improper regulation of cerebral blood flow, which can be a consequence of brain vascular dysfunction caused by perivascular amyloid‐β deposition, a peptide linked to the AD pathology. However, it is unknown if there are sex differences in cerebral vascular function and structure in AD that account for the sex differences with respect to AD‐associated dementia. We hypothesized that AD induces a more pronounced cerebral vascular dysfunction and remodeling in females in a mouse model of AD with pial artery amyloid‐β accumulation, the 5x‐FAD. Posterior communicating arteries (PComA) from 5x‐FAD and wild‐type (WT) mice were isolated and cannulated in a pressure myograph to analyze: spontaneous myogenic tone, autoregulation, vasoconstrictor response to endothelin‐1 (ET‐1, 30 nM) and passive vascular parameters. Data are means ± SEM. In males, spontaneous myogenic tone was significantly higher in 5x‐FAD than WT (tone (%): 20.8 ± 2.4 vs 14.2 ± 1.0, 5x‐FAD vs WT, n = 8‐7, p<0.05, Student's t‐test), without changes in autoregulation and constriction to ET‐1 between strains. PComA from 5x‐FAD males showed inward remodeling, observed as a reduction in lumen diameter across the range of intraluminal pressures (at 60 mmHg: 71.5 ± 8.0 µm vs 87.3± 8.1 µm, 5x‐FAD vs WT, n = 6‐11, p<0.05, two‐way ANOVA), without alterations in wall thickness. Distensibility was lower in male 5x‐FAD at higher pressures (% diameter increase from 5 mmHg to 120 mmHg: 63.8 ± 5.9 vs 46.9 ± 5.8, 5x‐FAD vs WT, p <0.05, two‐way ANOVA) without changes in stiffness. In females, spontaneous myogenic tone (tone (%): 20.1 ± 1.8 vs 25.5 ± 3.7, 5x‐FAD vs WT, n = 16‐8, p> 0.05), autoregulation and constriction to ET‐1 were not different between strains. Similarly, no structural remodeling was observed in the PComA of 5x‐FAD females, except for a small but significant increase in distensibility at lower pressures (at 20 mmHg: 20.2 ± 3.2 vs 16.9 ± 3.0, 5x‐FAD vs WT p<0.05), without changes in stiffness. We conclude that, contrary to the hypothesis, PComA from female 5x‐FAD do not exhibit dysfunction or remodeling at 3‐4 months of age, despite amyloid‐β deposition being evident. However, PComA from 5x‐FAD male show increased spontaneous myogenic tone, inward remodeling and a small reduction in distensibility. These results reveal that premenopausal women may be protected from AD‐associated vascular dysfunction. Further, the increase in incidence of dementia in females may occur by pathophysiological changes occurring during menopause.