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.

Collecting cervical lymphatic vessels pump lymph and macromolecules drained by brain meningeal lymphatics to the cervical lymph nodes. Collecting lymphatics vessels rely on spontaneous contractions to propel lymph against a pressure gradient to re-enter the venous circulation. The calcium permeable transient receptor potential vanilloid 4 channel (TRPV4) has been shown to mediate intraluminal pressure-induced vasodilatory responses in small arteries. However, whether TRPV4 participates in pressure-dependent contractile responses in lymphatic vessels remains unclear. We hypothesized that increases in intraluminal pressure leads to TRPV4-dependent increases in lymphatic pump function. TRPV4 expression was assessed by PCR assay in isolated superficial cervical lymphatic vessels (sCLV). Using pressure myography, contractile function of isolated sCLV was assessed in the presence of TRPV4 antagonist (1μM HC-067047) or vehicle (DMSO). To quantify lymphatic pump function (FPF), the product of contractile frequency (FREQ) and ejection fraction (EF) was analyzed using repeated end-diastolic and end-systolic diameter measurements. We detected TRPV4 mRNA expression following PCR amplification from isolated sCLV tissue of young C57Bl/6J mice (4-6 months). In pressurized sCLV, TRPV4 activation with GSK 1016790A (10 nM) abolished spontaneous rhythm contractions by causing a sustained constriction, consequently leading to a significant decrease in lymphatic pump function (FPF: 4.67±0.58 vs 1.17±0.86, Vehicle vs GSK 1016790A, n=8, p<0.05) at baseline intraluminal pressure (3 cm H2O). TRPV4 antagonism (HC-067047, 1 μM) did not affect basal sCLV contractility (FPF: 5.15±0.6 vs 3.44±0.43, Vehicle vs HC 067047, n=5, p>0.05). Further, when HC-067047 was applied to the bath and intraluminal pressure was maintained at 2 cm H2O, no changes were observed in FREQ (9.3±1.4 vs 11.7±1.3 Hz, Vehicle vs. HC-067047, n=7 / 9, p>0.05), EF (0.253±0.040 vs 0.268±0.037, Vehicle vs. HC-067047, n=7 / 9, p>0.05), or contractile amplitude (22.54±6.769 vs 26.76±3.028 μm, Vehicle vs HC-067047, n=7 / 9, p>0.05). Similarly, TRPV4 blockade did not alter FREQ (12.4±1.3 vs 14.3±1.4 Hz, Vehicle vs. HC-067047, n=7 / 9, p>0.05), EF (0.090±0.024 vs 0.092±0.019, Vehicle vs HC-067047) or contractile amplitude (7.98±2.814 vehicle vs 9.27±1.88 μm, Vehicle vs HC-067047) at the higher intraluminal pressure of 7 cm H2O. Collectively, these data suggest that TRPV4 channels are not involved in pressure-dependent contractile responses of sCLV in mice, despite inducing a robust constriction upon pharmacological activation. National Institutes of Health (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.