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. Author manuscript; available in PMC: 2026 Jul 4.
Published before final editing as: J Physiol. 2026 Jan 31:10.1113/JP290174. doi: 10.1113/JP290174

Intracerebral Age-Independent Reduction of Endothelium-Dependent Hyperpolarization and Nitric Oxide Signaling in the Dp16 Mouse Model of Down Syndrome

Abigail N Russell 1, Danielle A Jeffrey 1, Gregory Seedorf 1, Hong Li 1, Kelly D Sullivan 2,3, Huntington Potter 2, Mingxia Huang 1, Fabrice Dabertrand 1,4,*
PMCID: PMC13328574  NIHMSID: NIHMS2189130  PMID: 41620391

Abstract

Background and Purpose:

Down syndrome (DS) is associated with a myriad of cardiovascular defects. Endothelial cells, which form the innermost layer of blood vessels, have been found to exhibit divergent morphology and functional impairments in the peripheral circulation of individuals with DS. DS is also a strong risk factor for Alzheimer’s disease-related neuropathology. Recent evidence linking cerebral vasculature to Alzheimer’s disease, along with established correlations between cardiovascular and cognitive functions, prompted us to investigate brain parenchymal microcirculation reactivity in a DS mouse model.

Experimental Approach:

We used brain parenchymal arterioles to investigate the impact of DS on endothelium-dependent vasodilation using the Dp16 mouse model coupled with ex vivo pressure myography and in vivo two-photon laser-scanning microscopy. Two age cohorts of mice, young and middle-aged adult, were employed to test for age-dependence with the imminent development of Alzheimer’s disease with age of individuals with DS in mind.

Key Results:

Using pressure myography combined with pharmacological inhibitors, we examined two essential vasodilatory pathways in intracerebral arterioles—endothelial nitric oxide synthase and endothelium-dependent hyperpolarization mediated by small- and intermediate-conductance potassium channels. These pathways exhibited reduced constitutive activity in the DS model in both age groups. Consistent with these impairments, we measured higher levels of pressure-induced constriction, also referred to as myogenic tone, in arterioles from the cortex of Dp16 mice seen both in vivo and ex vivo.

Conclusion and Implications:

Together, our findings demonstrate altered cerebrovascular functionality in DS and identify possible therapeutic targets for alleviating symptoms associated with DS.

Keywords: Down syndrome, Endothelial dysfunction, Parenchymal arterioles, Myogenic tone, NOS, Endothelium-dependent hyperpolarization

INTRODUCTION

Down syndrome (DS) is caused by triplication of chromosome 21 and is the most common chromosomal disorder occurring in humans, affecting approximately 1 in 700 live births in the United States(Dimopoulos et al., 2023; Hartley et al., 2015). Cardiovascular disease is a leading cause of mortality in individuals with DS, and congenital heart disease is present in up to 50% of individuals with DS(Dimopoulos et al., 2023). Multiple studies have identified associations between cardiovascular disease and increased risks of stroke, dementia and cognitive impairment(Moroni et al., 2018; Ungvari, Tarantini, Donato, Galvan, & Csiszar, 2018). Reciprocally, cardiovascular disease is a common comorbidity among patients hospitalized for cerebrovascular conditions(Nakai et al., 2022). Therefore, the stark linking of cardiovascular health with brain health in recent years necessitates further understanding of cerebrovascular dysfunction in populations known to have high rates of cardiovascular impairment, such as those with DS.

Myogenic tone is a fundamental property of resistance arteries and arterioles that confers a pre-constricted state to the vasculature(Bayliss, 1902). This pre-constricted state is both protective for the brain and necessary for proper neuronal functioning(Claassen, Thijssen, Panerai, & Faraci, 2021). Myogenic tone supports autoregulation, a regulatory system that ensures consistent cerebral blood flow over a wide range of blood pressures (BPs)(Claassen et al., 2021). The establishment of myogenic tone creates the capacity for a vascular dilation in response to a decrease in BP as well as the capacity for further constriction in response to an increase in BP(Claassen et al., 2021). The role for myogenic tone in autoregulation is to serve as an important protector of the capillary bed, which would otherwise be damaged by normal fluctuations in BP causing unrestrained fluctuations in cerebral blood flow (CBF)(Claassen et al., 2021). Myogenic tone also sets up the stage for functional hyperemia (FH), or on-demand localized blood flow in response to neural activity, possibly by creating the reserves necessary for this system(Blackwell et al., 2022; Mellander, 1989; Nippert, Biesecker, & Newman, 2018). Indisputably, proper vascular tone is then crucial for brain functioning and potential breakdowns or divergences in the processes that establish and maintain vascular tone can have large-scale effects, some of which overlap with hallmarks of DS, like intellectual disability and delayed development(Rachubinski et al., 2024; Zammit et al., 2020).

In the microvasculature, the endothelial cells (ECs) are responsible for regulating the activity of adjacent contractile myocytes, called mural cells(Sandow, Senadheera, Bertrand, Murphy, & Tare, 2012). The mural cells that surround the parenchymal arterioles are smooth muscle cells (SMCs)(Sandow et al., 2012). The SMCs possess intrinsic mechanisms for their own contraction that cause vascular constriction and are the basis for myogenic tone(Claassen et al., 2021). The ECs, on the other hand, possess mechanisms that can relax adjacent SMCs by inhibiting these intrinsic contractile processes, causing subsequent mural cell relaxation and vascular dilation(Brian, Heistad, & Faraci, 1994). Nitric oxide synthase (NOS) is an enzyme that produces nitric oxide (NO) in ECs, which can then diffuse into adjacent SMCs and induce mural cell relaxation(Brian et al., 1994). Similarly, endothelium-dependent hyperpolarization (EDH) is achieved through the activation of the small- and intermediate-conductance, Ca2+-activated, K+ (SK, IK) channels located on EC’s which cause SMCs relaxation by propagating the hyperpolarization via gap junctions(Brian et al., 1994; Feletou & Vanhoutte, 2013). These endothelium-dependent vasodilatory pathways are fundamental regulators of myogenic tone, particularly in brain parenchymal arterioles where they exhibit constitutive activity. These mechanisms are altered by several pathological conditions and with aging(Behringer, 2023). However, how they are affected in Down syndrome (DS) remains poorly understood.

There exists a plethora of mouse models of DS. In the present study, we use the Dp(16)1Yey/+ (Dp16) mouse model of DS to investigate the cerebral vasculature. The Dp16 model carries a segmental duplication of a region on murine chromosome 16 (Mmu16) which contains 113 genes orthologous to genes on the human chromosome 21 (Hsa21), making it the mouse model that contains the largest number of triplicated Hsa21 orthologs on Mmu16(Akeson et al., 2001; Li et al., 2007; Sawa et al., 2022; Tuttle et al., 2020). Encouragingly, Dp16 mice display some hallmarks of DS like intellectual disability and delayed development (Goodliffe et al., 2016; Yu et al., 2010). Here, we sought to determine whether DS alters constitutive endothelial vasodilatory influences and myogenic tone of cortical arterioles using the Dp16 model. Examining 2–3- and 9–10-month-old mice, we measured an age-independent reduction in both nitric oxide signaling and endothelium-dependent hyperpolarization. Consistent with these observations, we measured weaker arteriolar myogenic tone both ex vivo and in vivo. Importantly, we provide to our knowledge the first insights into the molecular basis of the cerebral arteriolar dysfunction associated with DS.

MATERIALS AND METHODS

Ethical Approval.

All procedures were approved (Protocol number #00817) by the University of Colorado Institutional Animal Care and Use Committee (IACUC) and were performed in accordance with National Institutes of Health guidelines for the care and use of animals in research. This study conforms to the ethical principles of The Journal.

Animals.

Male Dp(16)1Yey/+ (Dp16) mice on the C57BL/6J background were purchased from Jackson Laboratory (Stock # 013530) and crossed with female C57BL/6J (control) mice to generate the litters used for these experiments. All animals were bred at the University of Colorado Anschutz Medical Campus and maintained in a room with HEPA-filtered air and a 14:10 light:dark cycle, fed a 6% fat diet, and provided with acidified (pH 2.5–3.0) water ad libitum. Same-sex littermates were housed in the same cage after weaning. Mice were used in two separate age-dependent groups: young-adulthood (2–3 months old) and middle-adulthood (9–10 months old). All mice were euthanized by intraperitoneal injection of sodium pentobarbital (100 mg/kg) followed by decapitation.

Solutions.

A MOPS-buffered saline solution was used for all dissections. The MOPS-buffered saline was prepared in bulk, aliquoted, and stored in aliquots at −20°C until use, at which point it was thawed at 4°C. The MOPS-buffered saline had the following composition: 145 mM NaCl, 5 mM KCl, 1 mM KH2PO4, 1 mM MgSO4, 2.5 mM CaCl2, 5 mM glucose, 3 mM MOPS, 2 mM pyruvate, 10 mg/mL bovine serum albumin, pH 7.3 at 4°C. Artificial cerebral spinal fluid (aCSF) was used for perfusion during all myography (ex vivo) and acute cranial window (in vivo) experiments. Reagents perfused during these experiments were diluted in aCSF. aCSF was prepared the day of the experiment and had the following composition: 125 mM NaCl, 3 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 1 mM MgCl2, 4 mM glucose, 2 mM CaCl2, pH 7.3 at room temperature with gas aeration. The aCSF was warmed to 37°C ± 0.5°C and gassed (5% CO2, 20% O2, 75% N2) when perfusing the organ chamber. Ca2+-free aCSF was used to achieve passive diameter of arterioles in myography and perfused over the cranial window for in vivo experiments. Ca2+-free aCSF was composed of 125 mM NaCl, 3 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 1 mM MgCl2, 4 mM glucose, 5 mM EGTA, pH 7.3 at room temperature with gas aeration.

Reagents.

All drug compounds were mixed with aCSF to their respective molarities. Effects of drug exposure on arteriole diameter were recorded using the edge-detection software. Four different pharmacological agents that target different vasodilatory effectors were used in myography experiments. 100 μM L-NAME (Cat no. N5751), an NOS inhibitor, was used separately to assess one of the main vasodilatory pathways. 100 nM charybdotoxin (Cat no. C7802), an IK and large-conductance (BK) channel inhibitor, was used along with 300 nM apamin (Cat no. A95459), an SK channel inhibitor to assess EDH vasodilatory pathway. All the above chemicals and reagents were obtained from Sigma-Aldrich (USA). Paxilline (Cat no. 2006), a BK channel inhibitor, was obtained from Tocris Bioscience (USA). 1 μM paxilline was perfused prior to the charybdotoxin to narrow any effects of the charybdotoxin to only IK channel and therefore the endothelium.

Pressure Myography.

Shortly after euthanasia, the brain was removed and placed into chilled MOPS-buffered saline. The brain region containing and surrounding the middle cerebral artery from both halves of the brain was removed. These pieces were pinned to a dissecting plate and parenchymal arterioles were carefully pulled from surrounding tissue. A custom organ chamber (University of Colorado Anschutz Medical Campus IDEA Core) was then assembled by screwing in borosilicate glass micropipettes (outside diameter = 1.2 mm; inside diameter = 0.69 mm) to both ends of the chamber, assuring their tips met and aligned in the center. Miniature tubing was attached to the front-most micropipette and that micropipette was filled with aCSF via the tubing and a filtered syringe. The micropipette on the back end was then pulled back for the cannulating. An arteriole was then cannulated on and tied to the front-most glass micropipette. The micropipette in the back of the organ chamber was pulled forward and the other end of the arteriole tied off to this micropipette. The arteriole was pressurized using an arteriography system (Living Systems Instrumentation, Inc., St. Albans, VT, USA) and aCSF was perfused over the arteriole at 4 mL/min. The lumen diameter of the arteriole was continuously monitored using a CCD camera and edge-detection software (IonOptix, Westwood, MA, USA). Passive diameter was obtained in nominally Ca2+-free aCSF. Only viable arterioles, defined as those that developed pressure-induced myogenic tone at 40 mmHg, were used in the experiments. Arteriolar tone was calculated with the following equation:

[(passivediameter–activediameter)/(passivediameter)]×100.

Changes in arterial diameter were calculated as the percentage change from base-line using the following equation:

[(changeindiameter)/(initialdiameter)]×100.

Acute cranial window implantation.

Using a 150-μl retro-orbital injection, 3 mg/ml of tetramethylrhodamine (TRITC)-dextran was administered to mice under isoflurane (5% induction, 2% maintenance) anesthesia. An acute cranial window was then implanted as previously described(Dabertrand et al., 2021; Jeffrey, Fontaine, & Dabertrand, 2022; Longden et al., 2017). Following TRITC-dextran injection, the skull was exposed, a 8 mm in diameter window was drilled above the somatosensory cortex and the dura mater delicately removed. A custom stainless steel head place with a corresponding 10-mm hole was then placed over the exposed brain and secured to the skull using a mixture of superglue and dental cement. Throughout the surgery isoflurane anesthesia was weaned off while α-chloralose (50 mg/kg) and urethane (750 mg/kg) were administered via intraperitoneal injection. Mice were monitored throughout the surgery with a rectal thermometer and kept at 37°C with a feedback-controlled electric heating pad.

Fluorescent image acquisition and analysis.

In vivo cranial window two-photon laser scanning microscopy (TPLSM) was performed using a Bruker Ultima Investigator multiphoton microscope with Prairie View software and a Spectra-Physics Mai Tai® eHP DeepSeeTM laser at 810 nm as previously described(Jeffrey et al., 2022). The laser was tuned to 810 nm excitation when imaging TRITC-dextran. Collection of red emission was achieved with 595/50 nm bandpass filters, detected via GaAsP photomultiplier tubes. To image the vasculature, a Nikon 16x (0.80 NA, CFI75 LWD) water-immersion objective was used. Z-stacks were collected at 1.0 μm z increments at 1024 × 1024 pixel resolution using a 3.6 μs/pixel dwell time to capture the vascular network. Parenchymal arterioles were identified by the direction of red blood cell flow into the brain. Two z-stacks with the same X/Y/Z coordinates were captured for each mouse, one in an active condition, normal aCSF applied, and one in a passive condition, 0 Ca2+ aCSF applied, treatments to compare vessel diameter changes. Oxygenated and warmed (32–34°C) aCSF and 0 Ca2+ aCSF were manually applied over the cranial window and refreshed every 1 minute for a total of 15 minutes. Fig. 6 shows our Imaris software quantitative workflow for z-stack network recreation which has been previously validated against full-width at half maximum validation(Ferris et al., 2025). Image files were converted into Imaris and then a gaussian filter was applied to all images to reduced noise in the samples. Then surfaces were created in Imaris using TRITC-dextran as in the input. Imaris’ machine learning software was utilized to define lumen borders as foreground and all other space as background. A training dataset was created using many training points throughout the entire z-stack and then a classifier was created that was used to train all images to render surfaces on the vasculature. Once surfaces were created then the channel was masked as an input for the filament creation in Imaris. The Automated Autopath Algorithm was used in Imaris to produce filaments with loops connected by seed points. Seed points were set with the same range, 3 μm to 27 μm, to represent the range of the desired vasculature. Segments were created with these trained seed points, followed by filaments. Segments under 4 μm were filtered out. Post network creation in Imaris, the average segment diameter was obtained from 3 to 5 segments of parenchymal arterioles per mouse in both active (aCSF) and passive (0 Ca2+ aCSF). Myogenic tone was calculated using the following equation:

[(passivediameter–activediameter)/(passivediameter)]x100.

Figure 6. Imaris filament creation workflow.

Figure 6.

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Visualization of the step-wise analysis process using Imaris unbiased 3D reconstruction and quantification of microvascular diameter. (A) Raw z-stacks are acquired with two-photon laser scanning microscopy, the vasculature is visualized with TRITC-dextran, a fluorescent probe that labels the vascular lumen. (B) Machine learning is then trained in the Imaris program creating a surface representative of the vascular network. (C) Surfaces are masked and filaments are rendered using frustums. Filaments are then labelled with a heatmap, with warm colors representing larger diameters and cooler colors representative of smaller diameters. (D) Single parenchymal arterioles are then selected, and the software generates exact values for mean diameter of each selected segment of parenchymal arteriole which are then used for calculations of myogenic tone.

Vascular density was calculated by considering the total volume the vessels (using TRITC-dextran) occupied compared to the entire volume of the capture. Filaments were created and then segment volume was calculated using SV=πr2h where r = radius and h = height. Microvascular area fraction was then calculated by taking the SV divided by the total volume capture area (detailed in Fig. 7E–F).

Figure 7. In vivo interrogation reveals higher level of myogenic tone in Dp16 cortical arterioles but consistent vascular percent fraction.

Figure 7.

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(A) Cartoon demonstrating experimental technique used for 2-photon imaging. (B) Representative z-stacks of vascular network visualized by 2-photon microscopy from both control and Dp16 animals in both active (treated with aCSF) and passive (treated 0 Ca2+ aCSF) conditions. Raw images are shown grayscale and Imaris network reconstruction represented by selected arterioles in blue and orange. Pop-out zoomed in areas indicate lumen diameter generated in Imaris with heatmap, corresponding diameters are also noted as well as calculated tone for both Control and Dp16 mice. (C) Summary data showing average arteriole passive diameter. Unpaired t-test revealed no statistically significantly difference between the groups, control n = 10 PAs from 3 mice, Dp16 n = 14 PAs from 3 mice. (D) Summary data indicating higher levels of myogenic tone in Dp16 arterioles compared to littermate controls. Unpaired t-test revealed p = 0.0093, control n = 10 PAs from 3 mice, Dp16 n = 14 PAs from 3 mice, ns = not significant. (E) Microvascular area fraction calculation using Imaris representative image of filament creation using TRITC-dextran as lumen diameter indicator. Total segment volume (SV) over total slice volume (shown in yellow) was calculated to generate the vascular area fraction. (F) Vascular area fraction summary data, n = 3 mice for control and Dp16 groups, data non-significant per unpaired t-test, p = 0.7916, ns = not significant.

Statistical analysis.

Data are presented as mean ± standard error of the mean. When applicable, technical replicates within an experiment are shown as replicate data points. All data were checked for normality using the Shapiro-Wilks test, skewness and frequency histograms were observed, and if normally distributed a paired t-test, (all dependent data or microvasculature from the same mouse) or unpaired (all independent data or microvasculature from different mice) was used (Fig. 1, 2, 4, and 7). Data with multiple comparisons were analyzed by a two-way analysis of variance (ANOVA) (Fig. 3). Šídák’s multiple comparisons test was used post hoc if the ANOVA was significant (Fig. 3). All tests were performed using GraphPad Prism 10 software (GraphPad Software, Boston, MA, USA). Significance was assigned as α < 0.05 and error bars denote ± S.D (standard deviation). All statistical methods are indicated in figure legends with corresponding independent numbers.

Figure 1. Constriction in response to NOS inhbitor is blunted in arterioles from Dp16 mice compared to those of littermate controls.

Figure 1.

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(A) Images of ex vivo pressure myography workflow. (B) Representative image of edge-detection software applied to cannulated arteriole. (C-D) Representative trace of arteriole diameter over time in 2–3-month-old (C) littermate control and (D) Dp16 mice. In all representative traces, 100 μM L-NAME, a NOS inhibitor, was perfused over the arteriole causing vasoconstriction. (E) Summary data revealing that the constriction of arterioles from young-adulthood Dp16 mice was statistically significantly less than that of control arterioles in response to L-NAME. Significance per unpaired t-test, control n = 5 mice, Dp16 n = 5 mice. (F-G) Representative trace of arteriole diameter over time in 9–10-month-old (F) littermate control and (G) Dp16 mice. (H) Summary data revealing that the constriction of arterioles from middle-adulthood Dp16 mice was statistically significantly less than that of control arterioles in response to L-NAME. Unpaired t-test reveals significance, control n = 5 mice, Dp16 n = 8 mice.

Figure 2. Constriction in response to SK and IK channel inhbitors is blunted in arterioles from Dp16 mice compared to those of littermate controls.

Figure 2.

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(A-B) Representative trace of arteriole diameter over time, bath-application of paxilline is indicated for both control and Dp16 mice. (C) Summary data showing no statistical difference (p = 0.06134) between control and Dp16 mice per unpaired t-test, n = 5 and 7 mice per group, ns = not significant. (D-E) Representative trace of arteriole diameter over time in 2–3-month-old (D) littermate control and (E) Dp16 mice. In all representative traces, 100 nM charybdotoxin and 300 nM apamin, IK and SK channel blockers, respectively, were perfused over the arteriole in the presence of 1 μM paxilline, a BK channel blocker used to narrow the effects of the non-specific charybdotoxin. (F) Summary data revealing that the constriction of arterioles from young-adulthood Dp16 mice was statistically significantly less than that of control arterioles in response to apamin and charybdotoxin. Unpaired t-test reveals significance, control n = 7 mice, Dp16 n = 6 mice. (G-H) Representative trace of arteriole diameter over time in 9–10-month-old (G) littermate control and (H) Dp16 mice. (I) Summary data revealing that the constriction of arterioles from middle-adulthood Dp16 mice was statistically significantly less than that of control arterioles in response to apamin and charybdotoxin. Unpaired t-test reveals significance, control n = 7 mice, Dp16 n = 6 mice.

Figure 4. High K+–induced constriction is similar in control and Dp16 mice, whereas correction for myogenic tone alters L-NAME–induced constriction without affecting impaired endothelium-dependent hyperpolarization.

Figure 4.

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(A-B) Representative trace of arteriole diameter over time and the addition of 60 mM K+ is indicated for both control and Dp16 mice. (C) Summary data showing no statistical difference between control and Dp16 mice per unpaired t-test, n = 5 and 4 mice. (D-E) Representative trace of arteriole diameter over time and the addition of L-NAME is indicated for both control and Dp16 mice and corresponding luminal pressure. (F) Summary data showing no statistical difference between control and Dp16 mice per unpaired t-test, n = 5 mice per group. (G-H) Representative trace of arteriole diameter over time and the addition of apamin and TRAM 34. (I) Summary data showing statistical significance per unpaired t-test, n = 5 mice per group.

Figure 3. Ex vivo interrogation reveals higher level of myogenic tone in the Dp16 cortical arterioles.

Figure 3.

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(A-B) Representative trace of real-time diameter fluctuations over a range of physiological pressures in myography experiments from (A) littermate control and (B) Dp16 mice. Pressure myography experiments track lumen diameter fluctuations over time (active condition) and are plotted below traces in 0 Ca2+ aCSF which elicits the maximal dilation of an arteriole (passive condition). (C) Summary data of passive diameters of littermate control and Dp16 arterioles. Two-way repeated measures ANOVA reveals no statistical significance, p > 0.1 for each pressure level indicating no difference in maximal diameters between the groups. (D) Summary data of measured myogenic tone over a set of experiments. Two-way repeated measures ANOVA followed by Šídák’s post hoc test indicates statistical significance for physiologically relevant pressures 40 mmHg and above, n = 5 mice for each group.

RESULTS

Brain parenchymal arterioles (PAs) from young and middle-aged Dp16 mice exhibit reduced sensitivity to NOS inhibition compared to those from littermate controls.

We first investigated the cerebral vasculature of the Dp16 mice, and their littermate controls, through a pharmacological approach in physiological assays. Our focus was on evaluating endothelium-dependent vasodilatory mechanisms across two age groups: 2–3-month-old mice (young adulthood), approximating a 20-year-old human, prior to expected vascular function decline in DS(Ungvari et al., 2018), and 9–10-month-old mice (middle-adulthood), representing the human equivalent of 40–50 years of age, an age associated with vascular senescence, even in healthy individuals and especially in individuals with DS(Hartley et al., 2015; Ungvari et al., 2018).

In the cerebral vasculature, nitric oxide is constitutively produced by NOS, causing mural cell relaxation, and therefore acting as a brake on pressure-induced constriction(Faraci & Brian, 1994). To evaluate its activity in PAs from control and Dp16 mice, we isolated and pressurized cortical arterioles (20 to 30 μm of diameter), and bath applied 100 μM L-NAME, a NOS inhibitor (Fig. 1A). We then measured the arteriolar diameter’s percent change from baseline in response (Fig. 1B). Arterioles from both Dp16 and control mice in both age cohorts constricted in response to L-NAME, indicating constitutive NOS activity in all groups (Fig. 1C–F). However, the average constriction observed in Dp16 arterioles was less than that observed in the control arterioles across both age cohorts (Fig. 1C–F). In the young-adulthood cohort, the mean constriction for Dp16 arterioles was 2.8-fold less compared to control PAs (Fig. 1C and D). Per an unpaired t-test this difference was statistically significant (Fig. 1E). Notably, this significant difference was sustained in the middle-adulthood cohort, wherein average vasoconstriction of Dp16 arterioles remained 2.2-fold less compared to littermate controls. (Fig. 1G–H). These observations are consistent with lower levels of constitutive activity of NOS in the Dp16 mouse model.

Cortical arterioles from young- and middle-adulthood Dp16 mice are less sensitive to SK and IK channels inhibitors than littermate controls.

Because of its constitutive activity, endothelium-derived hyperpolarization (EDH) via SK and IK channel activation, is particularly preponderant in PAs compared to the peripheral circulation (Brian et al., 1994; Feletou & Vanhoutte, 2013) and even pial arteries(Cipolla, Godfrey, & Wiegman, 2009; Dabertrand et al., 2015; Hannah, Dunn, Bonev, & Nelson, 2011). We perfused 300 nM apamin to block SK channels and 100 nM charybdotoxin to block IK channels. 1 μM paxilline, a BK channel-blocker, was perfused prior to and during the perfusion of charybdotoxin to narrow any effects of charybdotoxin to IK channel as charybdotoxin also inhibits BK channels. Consistent with previous reports on parenchymal arterioles(Dabertrand et al., 2015; Hannah et al., 2011), paxilline alone had minimal effects on baseline diameters in both control and Dp16 groups across age cohorts (Fig. 2A–C). Pressure myography arteriole preparations from both Dp16 and littermate control mice across both age groups constricted in response to the combination of inhibitors (Fig. 2D–I). However, the average constriction observed in control mice was again significantly greater than that of the Dp16 mice in both the young- and middle-adulthood groups (Fig. 2F and I). In the young-adulthood and the middle-adulthood cohorts, the constrictions induced by the inhibitors cocktail was 2 and 2.2-fold less in Dp16 mice compared to littermate controls, respectively. As with NOS, these measurements indicate lower levels of basal activity of EDH via the SK and IK channels in the Dp16 mice model of DS.

Endothelial dysfunction in Dp16 PAs is similar between young and middle-aged adults.

Having identified dysfunction of two vasodilatory mechanisms rooted in the cerebral endothelium of Dp16 mice to be similar across the young adulthood and middle adulthood groups, we conducted statistical analysis between age cohorts. Unpaired t-tests, matched for treatment and genetic group, indicated no difference across the age cohorts (Table 1). For L-NAME treatment, p values per unpaired t-test were 0.3959 and 0.5633 for WT and Dp16, respectively. For apamin and charybdotoxin treatment, p values, again per unpaired t-test, were 0.9923 and 0.6901 for WT and Dp16, respectively. With established non-age-dependence for these findings, our experiments moving forward focused on a range in age cohort, grouping young- and middle-adulthood together.

Table 1. Statistical testing reveals no difference in percent change from baseline between young- and middle-adulthood Dp16 mice in their endothelial cell dysfunction.

L-NAME-induced (NOS inhibitor) vasodilation results in arteriolar constriction of similar levels in 2–3-month-old and 9–10-month-old Dp16 mice. Using apamin and charybdotoxin, to block the SK and IK channels respectively, and the downstream signaling that leads to vasodilation, causes arteriolar constriction of similar levels in 2–3-month-old and 9–10-month-old Dp16 mice. The littermate controls tested similarly displayed no significant differences between the age groups in response to either treatment.

L-NAME Treatment Apamin & Charybdotoxin Treatment
Control Dp16 Control Dp16
2–3-MO 9–10-MO 2–3-MO 9–10-MO 2–3-MO 9–10-MO 2–3-MO 9–10-MO
Mean 32.37 27.76 10.54 12.18 21.61 21.56 10.76 9.06
S.E.M. 3.307 3.695 1.180 2.013 3.281 3.292 3.393 2.360
Unpaired t-test 0.3959 0.5633 0.9923 0.6901
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Dp16 parenchymal arterioles display higher levels of myogenic tone compared to controls from littermates in the physiological range of pressures.

The observed divergence in endothelium-dependent vasodilatory pathways in Dp16 mice prompted us to test whether myogenic responses of PAs were subsequently affected. To address this question, we again utilized myography preparations of isolated and pressurized PAs, this time manipulating the luminal pressure and observing subsequent diameter changes (Fig. 3A and B). Experiencing 20 mmHg of luminal pressure, cortical arterioles from the Dp16 and controls constricted to a similar extent ~7%, p = 0.8791 per Two-way repeated measures ANOVA followed by Šídák’s post hoc test (Fig. 3D). However, increasing luminal pressure to 40 mmHg, 60 mmHg, and 80 mmHg resulted in cortical arterioles from the Dp16 mice to constrict more than littermate controls (Fig. 3D). Specifically, at 40 mmHg, the physiologically relevant pressure, Dp16 cortical arterioles constricted over 2.2-fold more than control PAs (Fig. 3D). The difference in constriction was statistically significant at 40 mmHg, 60 mmHg and 80 mmHg (p = 0.0275, 0.0192 and 0.0117, respectively per Two-way repeated measures ANOVA followed by Šídák’s post hoc test), while average passive diameters were virtually identical between the two groups at all pressures (Fig. 3C and D).

We next assessed smooth muscle contractility using high K+ concentrations and found that 60 mM K+ elicited comparable vasoconstriction in control and Dp16 mice (Fig. 4A–C). To dissociate the influence of baseline tone from endothelial function, we compared NOS- and EDH-inhibition responses in control arterioles pressurized at 40 mmHg (13.76 ± 1.6% tone) with Dp16 arterioles pressurized at 20 mmHg (14.07 ± 0.5% tone), thereby matching tone between groups. This approach isolates endothelial contributions from differences in myogenic tone. Although L-NAME responses tended to be smaller in Dp16 arterioles, the difference did not reach statistical significance (Fig. 4D–F). In contrast, the combination of apamin and TRAM-34, an IK channel inhibitor distinct from charybdotoxin, produced a markedly reduced constriction in Dp16 arterioles compared with controls (Fig. 4G–I). These findings are consistent with diminished constitutive activity of endothelial vasodilatory mechanisms in the Dp16 model and suggest that parenchymal arterioles in Dp16 mice may exist in a more pre-constricted state than those of control mice.

In vivo network interrogation reveals heightened myogenic tone in cortical arterioles of Dp16 mice compared to littermate controls.

In a final set of experiments, we investigated whether the observed differences in myogenic tone identified ex vivo were also detectable in vivo. Following acute cranial window surgery, we acquired cortical z-stacks using two-photon laser-scanning microscopy in anesthetized mice (Fig. 5) in the presence or absence of extracellular Ca2+ in the aCSF, as previously established(Ferris et al., 2025; Jeffrey et al., 2025). Filament were then created using Imaris software (Fig. 6). We determined no statistical difference (p = 0.3352) between luminal diameter of Dp16 and control arterioles via a permutations test with an average of 11.66 ± 0.98 μm for the Dp16 and 10.31 ± 0.90 μm for the littermate control PAs (Fig. 7A–C). Using arteriolar diameter values from both the active and passive conditions, we then calculated levels of arteriolar tone. It was found that levels of constriction in Dp16 arterioles were higher than that of control arterioles, 35.77 ± 2.12% versus 25.73 ± 2.93%, respectively. Using an unpaired t-test, this difference was found to be statistically significant (p = 0.0093) (Fig. 7D). We also found no differences in microvascular area fraction between groups (Fig. 7E–F). Collectively, these data suggest that Dp16 arterioles indeed display higher levels of arteriolar tone comparable to what is observed ex vivo. These findings support the physiological relevance of our ex vivo observations while acknowledging that, in vivo, pressure-induced constriction is shaped by inputs from additional cell types and regulatory factors, and therefore reflects integrated vascular regulation rather than myogenic tone alone.

Figure 5. Two-photon raw images in active and passive conditions that allow for in vivo elucidation of myogenic tone.

Figure 5.

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(A-B) Raw representative z-stacks of vascular network visualized by two-photon microscopy following intravenous injection of TRITC-dextran from (A) littermate controls and (B) Dp16 animals in both active (treated with aCSF) and passive (treated 0 Ca2+ aCSF) conditions. Enlarged sections highlighting a parenchymal arteriole in yellow boxes demonstrate diameter increase after treatment with 0 Ca2+ aCSF (passive condition).

DISCUSSION

This study is the first to investigate endothelial function and myogenic responses of intracerebral arterioles, PAs, in a mouse model of Down syndrome. Using Dp16 mice, we found evidence of profound endothelial dysfunction, reflected by blunted responses to the inhibition of two distinct, constitutively active, endothelium-dependent vasodilatory mechanisms. Specifically, basal activity of both NOS and EDH, mediated by SK and IK channels, was reduced compared to littermate controls. Consistent with these findings, PAs from Dp16 cortices exhibited elevated myogenic tone both ex vivo and in vivo.

Our findings are congruous with previous reports of altered endothelial integrity in the peripheral circulation of individuals with Down syndrome. For instance, a study of the pulmonary vasculature reported divergent endothelial function in patients with DS(Cappelli-Bigazzi et al., 2004). Additionally, Costa et al. (Costa et al., 2010)identified a significant reduction in the number of endothelial progenitor cells, accompanied by increased cell size and detrimental morphological changes in individuals with DS. Interferon (IFN) signaling is overactive in DS because the genes encoding four IFN receptors, IFNAR1, IFNAR2, IFNGR2, and IL10RB, are located on chromosome 21. These genes are also triplicated in the Dp16 mouse model, which is a major feature of this model leading to heightened sensitivity to innate immune activation(Notarangelo & Bosticardo, 2022; Powers et al., 2019; Sullivan et al., 2016; Tuttle et al., 2020; Waugh et al., 2023). This hyperactive interferon signaling may contribute to the endothelial damage observed in Down syndrome, as elevated expression and levels of type I interferons (IFN-Is) have been associated with endothelial dysfunction(Galbraith et al., 2022; Jones Buie & Oates, 2014). Although the precise mechanisms linking hyperactive IFN signaling to endothelial dysfunction in DS remain to be fully elucidated, this body of evidence provided the rationale for our investigation of endothelium-dependent vasodilation in PAs in the Dp16 mouse model.

In addition to the high risk for cardiac defects, persons with DS are at increased risk of Alzheimer’s disease (AD). Due to a large increase in the life expectancy of people with DS, the prevalence of AD has become a much more significant issue in recent years. Nearly all adults with DS will develop the neuropathology consistent with AD by their 40’s, and by age 60, the current average life expectancy for people with DS, around 70% will develop dementia (Hartley et al., 2015) Thalman et al. found evidence that vascular perfusion is compromised in older adults with DS relative to younger individuals and is further exacerbated in those with dementia(Thalman et al., 2020). Similarly, Melamed at al. found reduced cerebral blood flow in 13/14 subjects with DS as compared to age-matched controls(Melamed, Mildworf, Sharav, Belenky, & Wertman, 1987). At the level of the microcirculation, DS has been associated with reduced endothelial integrity and decreased microvascular density, paralleling observations in patients with Alzheimer’s disease(Drachman, Smith, Alkamachi, & Kane, 2017). The Dp16 mouse model is characterized by developmental delays and cognitive deficits. Waugh et al. evaluated these impairments using contextual fear conditioning and the Morris water maze, reporting significantly reduced freezing behavior and impaired spatial learning(Waugh et al., 2023). However, the vascular contribution to dementia in people with DS remains relatively unexplored and certainly inconclusive mechanistically. Our findings shed some light on mechanisms that could be causative of altered blood flow in DS and provide potential insights for AD in DS.

Our finding of higher levels of myogenic tone in the cortex of Dp16 mice certainly could be linked with AD progression in DS as well as the general cognitive impairment seen in DS. In VCID, the emerging consensus is that endothelial dysfunction impairs myogenic tone, and therefore autoregulation and neurovascular coupling, ultimately leading to reduced blood flow and increased BBB permeability(Bosetti et al., 2016). Additionally, others have reported decreased microvascular density in Down syndrome with AD-type pathology(Drachman et al., 2017), however we found our mice to have similar microvascular area fractions, perhaps due to the mouse age range we profiled. Knowing that cerebral amyloid angiopathy (CAA), a type of VCID is present in 86% of AD cases, these findings point to parallels between VCID and aging with DS and further investigation are needed to confirm whether VCID can truly serve as model for cerebral progression in DS or not(Bergeron, Ranalli, & Miceli, 1987). As to effect of higher cortical tone on the general cognitive impairment in DS, several studies, like the above, have linked higher levels of myogenic tone to reduced CBF(Bosetti et al., 2016; Zammit et al., 2020). Additional studies have demonstrated a correlation between CBF and cognition with Ogoh et al. observing reduced perfusion to the hippocampus to result in cognitive impairment and Zou et al. uncovering that revascularization and subsequent increased CBF improved cognition in moyamoya disease patients(Ogoh, 2017; Zou et al., 2023).

Dysfunction of the small- and intermediate-conductance calcium-gated K+ channels is not unique to DS. Dysfunction of these endothelial K+ channels and subsequent reduced endothelium-derived hyperpolarization have been found not only in several cardiovascular pathologies like diabetes, atherosclerosis and post-angioplastic neointima formation but also in inflammatory disease, cancer and organ fibrosis(Kohler, Olivan-Viguera, & Wulff, 2016). Observations of SK and IK channel dysfunction in inflammatory disease aligns with our findings as DS can be considered an inflammatory disease. Together, these evidence strengthen the link between SK and IK channel dysfunction, enhanced interferon signaling, and chronic inflammation in the context of DS.

Observed NOS dysfunction could also be related to hyperactive IFN signaling in DS and the Dp16 mouse model. A study in systemic lupus erythematosus, a chronic autoimmune disorder that is a known risk factor for endothelial function, links IFN signaling with NOS dysfunction. Their study showed the negative impact of IFN-alpha on NOS3 mRNA expression, NOS protein expression and insulin-mediated NO production in endothelial cells(Buie, Renaud, Muise-Helmericks, & Oates, 2017). In this study, the authors postulate that the connection could be via depletion of NOS cofactors, altered expression of transcription factors essential for NOS promotor activation or changes in NOS phosphorylation through downregulation of Vascular Endothelial Growth Factor (VEGF) and loss of kinase activation(Buie et al., 2017). Again, with our groups decision to use the Dp16 mouse model of DS, which focuses on DS as an interferonopathy(Galbraith et al., 2022; Waugh et al., 2023), this evidence provides possible explanations for our observations.

Recent advances in therapeutic strategies targeting interferon signaling have led to the development and clinical testing of Janus kinase (JAK) inhibitors in Down syndrome and related neurological conditions. A completed clinical trial (NCT04246372) evaluated the safety and efficacy of baricitinib, a JAK1/2 inhibitor, in individuals with DS, with promising results suggesting modulation of interferon-driven inflammation(Rachubinski et al., 2024). Additionally, an ongoing trial (NCT05662228) is investigating JAK inhibition in a neurological context, further supporting the therapeutic potential of targeting this pathway. The NO donor nitroglycerin, which has established clinical use in conditions such as cerebral ischemia and angina, could present therapeutic benefit in the context of reduced NOS activity observed in Dp16 mice by promoting vasodilation(Cappelli-Bigazzi et al., 2004; Fenton, Wellington, & Easthope, 2006; Kim, Adnan, & Schaller, 2025; Kirwan, Duffy, & French, 2024). However, individuals with DS often exhibit baseline hypotension, raising the possibility that exogenous NO donors could exacerbate hemodynamic instability rather than provide benefit. This concern is further complicated by nitroglycerin’s limited clinical applicability, given its short half-life (2–3 minutes) and significant adverse effects(Kang et al., 2022; Kim et al., 2025). Therefore, tightly regulated and individualized dosing strategies would be required for any potential therapeutic use. Nevertheless, these clinical efforts and considerations underscore the translational relevance of our findings and highlight the importance of endothelial function as a downstream target of interferon signaling in DS.

In conclusion, our group uncovered two dysfunctional vasodilatory mechanisms rooted in the cerebral endothelial cells of Dp16 mice intracerebral arterioles. Further, higher levels of myogenic tone were observed. The implications of this divergent functionality in the broader scope of DS remain to be elucidated but the literature points to several ways in which it could be contributing to the hallmark cognitive impairment in DS as well as the AD neuropathology characteristic of DS. Having focused mechanistically on the cerebral arterioles, this work uncovers potential therapeutic targets, that is the SK and IK channels and NOS, that could rescue the observed higher levels of myogenic tone and potentially alleviate cognitive and dementia symptoms in persons with DS.

What is already known

  • Down syndrome is associated with cardiovascular diseases.

What this study adds

  • EDH and NOS activity are reduced in Dp16 brain parenchymal arterioles.

  • Parenchymal arterioles myogenic tone is elevated in Dp16 mice both ex vivo and in vivo.

Clinical significance

  • Endothelial dysfunction is a therapeutic target in Down syndrome.

  • Restoration of cerebral blood flow through vascular modulation may be beneficial.

ACKNOWLEDGEMENTS

We would like to thank the Department of Anesthesiology for the use of the two-photon laser scanning microscope and Imaris software.

FUNDING INFORMATION

This study was supported by the Department of Anesthesiology at Anschutz Medical Campus, 2 research grants from the Ludeman Family Center for Women’s Health Research located at the University of Colorado Anschutz Medical Campus (2019 and 2024) to FD; 2 research grants from the University of Pennsylvania Orphan Disease Center in partnership with the cureCADASIL (2019 and 2022) to FD, a research grant from the Leducq Foundation for Cardiovascular Research (Leducq Transatlantic Network of Excellence 22CVD01 BRENDA), the National Institute of General Medical Sciences 3R01GM123746-02S1 to S.M. Todorovic and V. Jevtovic-Todorovic, the National Institute of Neurological Disorders and Stroke RF1NS128739 to KDS, HP and MH, RF1NS129022 and RF1NS140137 to FD, and the National Heart, Lung, and Blood Institute R01HL136636 to FD, 5T32GM007635 and F31HL170645 to DAJ.

Abbreviations:

AD

Alzheimer’s Disease

aCSF

Artificial Cerebrospinal Fluid

BBB

Blood-Brain Barrier

BK

Big-Conductance Calcium-Activated Potassium Channel

BP

Blood Pressure

CAA

Cerebral Amyloid Angiopathy

CBF

Cerebral Blood Flow

Dp16

Dp(16)1Yey/+ Mouse Model

DS

Down Syndrome

EC

Endothelial Cell

EDH

Endothelium-Dependent Hyperpolarization

NOS

Endothelial Nitric Oxide Synthase

FH

Functional Hyperemia

IFN

Interferon

IK

Intermediate-Conductance Calcium-Activated Potassium Channel

JAK

Janus Kinase

L-NAME

Nω-Nitro-L-arginine methyl ester

MOPS

3-(N-morpholino)propanesulfonic acid

MRI

Magnetic Resonance Imaging

Mmu16

Mouse Chromosome 16

NO

Nitric Oxide

NOS

Nitric Oxide Synthase

PA

Parenchymal Arteriole

SK

Small-Conductance Calcium-Activated Potassium Channel

SMC

Smooth Muscle Cell

TPLSM

Two-Photon Laser Scanning Microscopy

TRITC

Tetramethylrhodamine

VCID

Vascular Contributions to Cognitive Impairment and Dementia

VEGF

Vascular Endothelial Growth Factor

WT

Wild Type

Footnotes

COMPETING INTERESTS

The authors declare no competing interests.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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