Impact of aging on vascular ion channels: perspectives and knowledge gaps across major organ systems.

Individuals aged ≥65 yr will comprise ∼20% of the global population by 2030. Cardiovascular disease remains the leading cause of death in the world with age-related endothelial "dysfunction" as a key risk factor. As an organ in and of itself, vascular endothelium courses throughout the mammalian body to coordinate blood flow to all other organs and tissues (e.g., brain, heart, lung, skeletal muscle, gut, kidney, skin) in accord with metabolic demand. In turn, emerging evidence demonstrates that vascular aging and its comorbidities (e.g., neurodegeneration, diabetes, hypertension, kidney disease, heart failure, and cancer) are "channelopathies" in large part. With an emphasis on distinct functional traits and common arrangements across major organs systems, the present literature review encompasses regulation of vascular ion channels that underlie blood flow control throughout the body. The regulation of myoendothelial coupling and local versus conducted signaling are discussed with new perspectives for aging and the development of chronic diseases. Although equipped with an awareness of knowledge gaps in the vascular aging field, a section has been included to encompass general feasibility, role of biological sex, and additional conceptual and experimental considerations (e.g., cell regression and proliferation, gene profile analyses). The ultimate goal is for the reader to see and understand major points of deterioration in vascular function while gaining the ability to think of potential mechanistic and therapeutic strategies to sustain organ perfusion and whole body health with aging.

Endothelial KIR2 channel dysfunction in aged cerebral parenchymal arterioles.

Aging is associated with cognitive decline via incompletely understood mechanisms. Cerebral microvascular dysfunction occurs in aging, particularly impaired endothelium-mediated dilation. Parenchymal arterioles are bottlenecks of the cerebral microcirculation, and dysfunction causes a mismatch in nutrient demand and delivery, leaving neurons at risk. Extracellular nucleotides elicit parenchymal arteriole dilation by activating endothelial purinergic receptors (P2Y), leading to opening of K+ channels, including inwardly-rectifying K+ channels (KIR2). These channels amplify hyperpolarizing signals, resulting in dilation. However, it remains unknown if endothelial P2Y and KIR2 signaling are altered in brain parenchymal arterioles during aging. We hypothesized that aging impairs endothelial P2Y and KIR2 function in parenchymal arterioles. We observed reduced dilation to the purinergic agonist 2-methyl-S-ADP (1 µM) in arterioles from Aged (>24-month-old) mice when compared to Young (4-6 months of age) despite similar hyperpolarization in endothelial cells tubes. No differences were observed in vasodilation or endothelial cell hyperpolarization to activation of small- and intermediate-conductance Ca2+-activated K+ channels (KCa2.3 / KCa3.1) by NS309. Hyperpolarization to 15 mM [K+]E was smaller in Aged than Young mice, despite a paradoxical increased dilation in Aged arterioles to 15 mM [K+]E that was unchanged by endothelium removal. KIR2 Inhibition attenuated vasodilatory responses to 15 mM [K+]E and 1 µM 2-me-S-ADP in both Young and Aged arterioles. Further, we observed a significant increase in myogenic tone in Aged parenchymal arterioles, which was not enhanced by endothelium removal. We conclude that aging impairs endothelial KIR2 channel function in the cerebral microcirculation with possible compensation by smooth muscle cells.

KIR channel regulation of electrical conduction along cerebrovascular endothelium: Enhanced modulation during Alzheimer's disease.

OBJECTIVE: Endothelial cell (EC) coupling occurs through gap junctions and underlies cerebral blood flow regulation governed by inward-rectifying K+ (KIR ) channels. This study addressed effects of KIR channel activity on EC coupling before and during Alzheimer's disease (AD). METHODS: Intact EC tubes (width: ~90-100 µm; length: ~0.5 mm) were freshly isolated from posterior cerebral arteries of young Pre-AD (1-3 months) and aged AD (13-18 months) male and female 3xTg-AD mice. Dual intracellular microelectrodes applied simultaneous current injections (±0.5-3 nA) and membrane potential (Vm ) recordings in ECs at distance ~400 µm. Elevated extracellular potassium ([K+ ]E ; 8-15 mmol/L; reference, 5 mmol/L) activated KIR channels. RESULTS: Conducted Vm (∆Vm ) responses ranged from ~-30 to 30 mV in response to -3 to +3 nA (linear regression, R2 ≥ .99) while lacking rectification for charge polarity or axial direction of spread. Conduction slope decreased ~10%-20% during 15 mmol/L [K+ ]E in Pre-AD males and AD females. 15 mmol/L [K+ ]E decreased conduction by ~10%-20% at lower ∆Vm thresholds in AD animals (~±20 mV) versus Pre-AD (~±25 mV). AD increased conducted hyperpolarization by ~10%-15% during 8-12 mmol/L [K+ ]E . CONCLUSIONS: Brain endothelial KIR channel activity modulates bidirectional spread of vasoreactive signals with enhanced regulation of EC coupling during AD pathology.

Functional Interaction among KCa and TRP Channels for Cardiovascular Physiology: Modern Perspectives on Aging and Chronic Disease.

Effective delivery of oxygen and essential nutrients to vital organs and tissues throughout the body requires adequate blood flow supplied through resistance vessels. The intimate relationship between intracellular calcium ([Ca2+]i) and regulation of membrane potential (Vm) is indispensable for maintaining blood flow regulation. In particular, Ca2+-activated K⁺ (KCa) channels were ascertained as transducers of elevated [Ca2+]i signals into hyperpolarization of Vm as a pathway for decreasing vascular resistance, thereby enhancing blood flow. Recent evidence also supports the reverse role for KCa channels, in which they facilitate Ca2+ influx into the cell interior through open non-selective cation (e.g., transient receptor potential; TRP) channels in accord with robust electrical (hyperpolarization) and concentration (~20,000-fold) transmembrane gradients for Ca2+. Such an arrangement supports a feed-forward activation of Vm hyperpolarization while potentially boosting production of nitric oxide. Furthermore, in vascular types expressing TRP channels but deficient in functional KCa channels (e.g., collecting lymphatic endothelium), there are profound alterations such as downstream depolarizing ionic fluxes and the absence of dynamic hyperpolarizing events. Altogether, this review is a refined set of evidence-based perspectives focused on the role of the endothelial KCa and TRP channels throughout multiple experimental animal models and vascular types. We discuss the diverse interactions among KCa and TRP channels to integrate Ca2+, oxidative, and electrical signaling in the context of cardiovascular physiology and pathology. Building from a foundation of cellular biophysical data throughout a wide and diverse compilation of significant discoveries, a translational narrative is provided for readers toward the treatment and prevention of chronic, age-related cardiovascular disease.

Biophysical properties of microvascular endothelium: Requirements for initiating and conducting electrical signals.

OBJECTIVE: Electrical signaling along the endothelium underlies spreading vasodilation and blood flow control. We use mathematical modeling to determine the electrical properties of the endothelium and gain insight into the biophysical determinants of electrical conduction. METHODS: Electrical conduction data along endothelial tubes (40 µm wide, 2.5 mm long) isolated from mouse skeletal muscle resistance arteries were analyzed using cable equations and a multicellular computational model. RESULTS: Responses to intracellular current injection attenuate with an axial length constant (λ) of 1.2-1.4 mm. Data were fitted to estimate the axial (ra ; 10.7 MΩ/mm) and membrane (rm ; 14.5 MΩ∙mm) resistivities, EC membrane resistance (Rm ; 12 GΩ), and EC-EC coupling resistance (Rgj ; 4.5 MΩ) and predict that stimulation of ≥30 neighboring ECs is required to elicit 1 mV of hyperpolarization at distance = 2.5 mm. Opening Ca2+ -activated K+ channels (KCa ) along the endothelium reduced λ by up to 55%. CONCLUSIONS: High Rm makes the endothelium sensitive to electrical stimuli and able to conduct these signals effectively. Whereas the activation of a group of ECs is required to initiate physiologically relevant hyperpolarization, this requirement is increased by myoendothelial coupling and KCa activation along the endothelium inhibits conduction by dissipating electrical signals.

Calcium and electrical dynamics in lymphatic endothelium.

KEY POINTS: Endothelial cell function in resistance arteries integrates Ca2+ signalling with hyperpolarization to promote relaxation of smooth muscle cells and increase tissue blood flow. Whether complementary signalling occurs in lymphatic endothelium is unknown. Intracellular calcium and membrane potential were evaluated in endothelial cell tubes freshly isolated from mouse collecting lymphatic vessels of the popliteal fossa. Resting membrane potential measured using intracellular microelectrodes averaged ∼-70 mV. Stimulation of lymphatic endothelium by acetylcholine or a TRPV4 channel agonist increased intracellular Ca2+ with robust depolarization. Findings from Trpv4-/- mice and with computational modelling suggest that the initial mobilization of intracellular Ca2+ leads to influx of Ca2+ and Na+ through TRPV4 channels to evoke depolarization. Lymphatic endothelial cells lack the Ca2+ -activated K+ channels present in arterial endothelium to generate endothelium-derived hyperpolarization. Absence of this signalling pathway with effective depolarization may promote rapid conduction of contraction along lymphatic muscle during lymph propulsion. ABSTRACT: Subsequent to a rise in intracellular Ca2+ ([Ca2+ ]i ), hyperpolarization of the endothelium coordinates vascular smooth muscle relaxation along resistance arteries during blood flow control. In the lymphatic vasculature, collecting vessels generate rapid contractions coordinated along lymphangions to propel lymph, but the underlying signalling pathways are unknown. We tested the hypothesis that lymphatic endothelial cells (LECs) exhibit Ca2+ and electrical signalling properties that facilitate lymph propulsion. To study electrical and intracellular Ca2+ signalling dynamics in lymphatic endothelium, we excised collecting lymphatic vessels from the popliteal fossa of mice and removed their muscle cells to isolate intact LEC tubes (LECTs). Intracellular recording revealed a resting membrane potential of ∼-70 mV. Acetylcholine (ACh) increased [Ca2+ ]i with a time course similar to that observed in endothelium of resistance arteries (i.e. rapid initial peak with a sustained 'plateau'). In striking contrast to the endothelium-derived hyperpolarization (EDH) characteristic of arteries, LECs depolarized (>15 mV) to either ACh or TRPV4 channel activation. This depolarization was facilitated by the absence of Ca2+ -activated K+ (KCa ) channels as confirmed with PCR, persisted in the absence of extracellular Ca2+ , was abolished by LaCl3 and was attenuated ∼70% in LECTs from Trpv4-/- mice. Computational modelling of ion fluxes in LECs indicated that omitting K+ channels supports our experimental results. These findings reveal novel signalling events in LECs, which are devoid of the KCa activity abundant in arterial endothelium. Absence of EDH with effective depolarization of LECs may promote the rapid conduction of contraction waves along lymphatic muscle during lymph propulsion.

Calcium and electrical signaling in arterial endothelial tubes: New insights into cellular physiology and cardiovascular function.

The integral role of the endothelium during the coordination of blood flow throughout vascular resistance networks has been recognized for several decades now. Early examination of the distinct anatomy and physiology of the endothelium as a signaling conduit along the vascular wall has prompted development and application of an intact endothelial "tube" study model isolated from rodent skeletal muscle resistance arteries. Vasodilatory signals such as increased endothelial cell (EC) Ca2+ ([Ca2+ ]i ) and hyperpolarization take place in single ECs while shared between electrically coupled ECs through gap junctions up to distances of millimeters (≥2 mm). The small- and intermediate-conductance Ca2+ activated K+ (SKCa /IKCa or KCa 2.3/KCa 3.1) channels function at the interface of Ca2+ signaling and hyperpolarization; a bidirectional relationship whereby increases in [Ca2+ ]i activate SKCa /IKCa channels to produce hyperpolarization and vice versa. Further, the spatial domain of hyperpolarization among electrically coupled ECs can be finely tuned via incremental modulation of SKCa /IKCa channels to balance the strength of local and conducted electrical signals underlying vasomotor activity. Multifunctional properties of the voltage-insensitive SKCa /IKCa channels of resistance artery endothelium may be employed for therapy during the aging process and development of vascular disease.

Membrane potential governs calcium influx into microvascular endothelium: integral role for muscarinic receptor activation.

In resistance arteries, coupling a rise of intracellular calcium concentration ([Ca(2+)]i) to endothelial cell hyperpolarization underlies smooth muscle cell relaxation and vasodilatation, thereby increasing tissue blood flow and oxygen delivery. A controversy persists as to whether changes in membrane potential (V(m)) alter endothelial cell [Ca(2+)]i. We tested the hypothesis that V(m) governs [Ca(2+)]i in endothelium of resistance arteries by performing Fura-2 photometry while recording and controlling V(m) of intact endothelial tubes freshly isolated from superior epigastric arteries of C57BL/6 mice. Under resting conditions, [Ca(2+)]i did not change when V(m) shifted from baseline (∼-40 mV) via exposure to 10 µM NS309 (hyperpolarization to ∼-80 mV), via equilibration with 145 mm [K(+)]o (depolarization to ∼-5 mV), or during intracellular current injection (±0.5 to 5 nA, 20 s pulses) while V(m) changed linearly between ∼-80 mV and +10 mV. In contrast, during the plateau (i.e. Ca(2+) influx) phase of the [Ca(2+)]i response to approximately half-maximal stimulation with 100 nm ACh (∼EC50), [Ca(2+)]i increased as V(m) hyperpolarized below -40 mV and decreased as V(m) depolarized above -40 mV. The magnitude of [Ca(2+)]i reduction during depolarizing current injections correlated with the amplitude of the plateau [Ca(2+)]i response to ACh. The effect of hyperpolarization on [Ca(2+)]i was abolished following removal of extracellular Ca(2+), was enhanced subtly by raising extracellular [Ca(2+)] from 2 mm to 10 mm and was reduced by half in endothelium of TRPV4(-/-) mice. Thus, during submaximal activation of muscarinic receptors, V(m) can modulate Ca(2+) entry through the plasma membrane in accord with the electrochemical driving force.

Advanced age protects microvascular endothelium from aberrant Ca(2+) influx and cell death induced by hydrogen peroxide.

KEY POINTS: Calcium signalling in endothelial cells of resistance arteries is integral to blood flow regulation. Oxidative stress and endothelial dysfunction can prevail during advanced age and we questioned how calcium signalling may be affected. Intact endothelium was freshly isolated from superior epigastric arteries of Young (∼4 months) and Old (∼24 months) male C57BL/6 mice. Under resting conditions, with no difference in intracellular calcium levels, hydrogen peroxide (H2 O2 ) availability was ∼1/3 greater in endothelium of Old mice while vascular catalase activity was reduced by nearly half. Compared to Old, imposing oxidative stress (200 µm H2 O2 ) for 20 min increased intracellular calcium to 4-fold greater levels in endothelium of Young in conjunction with twice the calcium influx. Prolonged (60 min) exposure to H2 O2 induced 7-fold greater cell death in endothelium of Young. Microvascular adaptation to advanced age may protect endothelial cells during elevated oxidative stress to preserve functional viability of the intima. ABSTRACT: Endothelial cell Ca(2+) signalling is integral to blood flow control in the resistance vasculature yet little is known of how its regulation may be affected by advancing age. We tested the hypothesis that advanced age protects microvascular endothelium by attenuating aberrant Ca(2+) signalling during oxidative stress. Intact endothelial tubes (width, ∼60 µm; length, ∼1000 µm) were isolated from superior epigastric arteries of Young (3-4 months) and Old (24-26 months) male C57BL/6 mice and loaded with Fura-2 dye to monitor [Ca(2+) ]i . At rest there was no difference in [Ca(2+) ]i between age groups. Compared to Young, the [Ca(2+) ]i response to maximal stimulation with acetylcholine (3 µm, 2 min) was ∼25% greater in Old, confirming signalling integrity with advanced age. Basal H2 O2 availability was ∼33% greater in Old while vascular catalase activity was reduced by half. Transient exposure to elevated H2 O2 (200 µm, 20 min) progressively increased [Ca(2+) ]i to ∼4-fold greater levels in endothelium of Young versus Old. With no difference between age groups at rest, Mn(2+) quench of Fura-2 fluorescence revealed 2-fold greater Ca(2+) influx in Young during elevated H2 O2 ; this effect was attenuated by ∼75% using ruthenium red (5 µm) as a broad-spectrum inhibitor of transient receptor potential channels. Prolonged exposure to H2 O2 (200 µm, 60 min) induced ∼7-fold greater cell death in endothelium of Young versus Old. Thus, microvascular endothelium can adapt to advanced age by reducing Ca(2+) influx during elevated oxidative stress. Protection from cell death during oxidative stress will sustain endothelial integrity during ageing.

Tuning electrical conduction along endothelial tubes of resistance arteries through Ca(2+)-activated K(+) channels.

RATIONALE: Electrical conduction through gap junction channels between endothelial cells of resistance vessels is integral to blood flow control. Small and intermediate-conductance Ca(2+)-activated K(+) channels (SK(Ca)/IK(Ca)) initiate electrical signals in endothelial cells, but it is unknown whether SK(Ca)/IK(Ca) activation alters signal transmission along the endothelium. OBJECTIVE: We tested the hypothesis that SK(Ca)/IK(Ca) activity regulates electrical conduction along the endothelium of resistance vessels. METHODS AND RESULTS: Freshly isolated endothelial cell tubes (60 µm wide; 1-3 mm long; cell length, ≈35 µm) from mouse skeletal muscle feed (superior epigastric) arteries were studied using dual intracellular microelectrodes. Current was injected (±0.1-3 nA) at site 1 while recording membrane potential (V(m)) at site 2 (separation distance=50-2000 µm). SK(Ca)/IK(Ca) activation (NS309, 1 µmol/L) reduced the change in V(m) along endothelial cell tubes by ≥50% and shortened the electrical length constant (λ) from 1380 to 850 µm (P<0.05) while intercellular dye transfer (propidium iodide) was maintained. Activating SK(Ca)/IK(Ca) with acetylcholine or SKA-31 also reduced electrical conduction. These effects of SK(Ca)/IK(Ca) activation persisted when hyperpolarization (>30 mV) was prevented with 60 mmol/L [K(+)](o). Conversely, blocking SK(Ca)/IK(Ca) (apamin+charybdotoxin) depolarized cells by ≈10 mV and enhanced electrical conduction (ie, changes in V(m)) by ≈30% (P<0.05). CONCLUSIONS: These findings illustrate a novel role for SK(Ca)/IK(Ca) activity in tuning electrical conduction along the endothelium of resistance vessels by governing signal dissipation through changes in membrane resistance. Voltage-insensitive ion channels can thereby tune intercellular electrical signaling independent from gap junction channels.

Aging impairs electrical conduction along endothelium of resistance arteries through enhanced Ca2+-activated K+ channel activation.

OBJECTIVE: Intercellular conduction of electrical signals underlies spreading vasodilation of resistance arteries. Small- and intermediate-conductance Ca(2+)-activated K(+) channels of endothelial cells serve a dual function by initiating hyperpolarization and modulating electrical conduction. We tested the hypothesis that regulation of electrical signaling by small- and intermediate-conductance Ca(2+)-activated K(+) channels is altered with advancing age. APPROACH AND RESULTS: Intact endothelial tubes (60 µm wide; 1-3 mm long) were freshly isolated from male C57BL/6 mouse (Young: 4-6 months; Intermediate: 12-14 months; Old: 24-26 months) superior epigastric arteries. Using dual intracellular microelectrodes, current was injected (± 0.1-3 nA) at site 1 while recording membrane potential (Vm) at site 2 (separation distance: 50-2000 µm). Across age groups, greatest differences were observed between Young and Old. Resting Vm in Old (-38 ± 1 mV) was more negative (P<0.05) than Young (-30 ± 1 mV). Maximal hyperpolarization to both direct (NS309) and indirect (acetylcholine) activation of small- and intermediate-conductance Ca(2+)-activated K(+) channels was sustained (ΔVm ≈-40 mV) with age. The length constant (λ) for electrical conduction was reduced (P<0.05) from 1630 ± 80 µm (Young) to 1320 ± 80 µm (Old). Inhibiting small- and intermediate-conductance Ca(2+)-activated K(+) channels with apamin+charybdotoxin or scavenging hydrogen peroxide (H2O2) with catalase improved electrical conduction (P<0.05) in Old. Exogenous H2O2 (200 µmol/L) in Young evoked hyperpolarization and impaired electrical conduction; these effects were blocked by apamin+charybdotoxin. CONCLUSIONS: Enhanced current loss through Ca2+-activated K+ channel activation impairs electrical conduction along the endothelium of resistance arteries with aging. Attenuating the spatial domain of electrical signaling will restrict the spread of vasodilation and thereby contribute to blood flow limitations associated with advanced age.

Cerebrovascular miRNAs Track Early Development of Alzheimer's Disease and Target Molecular Markers of Angiogenesis and Blood Flow Regulation.

BACKGROUND: Alzheimer's disease (AD) is associated with impaired cerebral circulation which underscores diminished delivery of blood oxygen and nutrients to and throughout the brain. In the 3xTg-AD mouse model, we have recently found that > 10 cerebrovascular miRNAs pertaining to vascular permeability, angiogenesis, and inflammation (e.g., let-7d, miR-99a, miR-132, miR-133a, miR-151-5p, and miR-181a) track early development of AD. Further, endothelial-specific miRNAs (miR-126-3p, miR-23a/b, miR-27a) alter with onset of overall AD pathology relative to stability of smooth muscle/pericyte-specific miRNAs (miR-143, miR-145). OBJECTIVE: We tested the hypothesis that cerebrovascular miRNAs indicating AD pathology share mRNA targets that regulate key endothelial cell functions such as angiogenesis, vascular permeability, and blood flow regulation. METHODS: As detected by NanoString nCounter miRNA Expression panel for 3xTg-AD mice, 61 cerebrovascular miRNAs and respective mRNA targets were examined using Ingenuity Pathway Analysis for canonical Cardiovascular (Cardio) and Nervous System (Neuro) Signaling. RESULTS: The number of targets regulated per miRNA were 21±2 and 33±3 for the Cardio and Neuro pathways respectively, whereby 14±2 targets overlap among pathways. Endothelial miRNAs primarily target members of the PDE, PDGF, SMAD, and VEGF families. Individual candidates regulated by≥4 miRNAs that best mark AD pathology presence in 3xTg-AD mice include CFL2, GRIN2B, PDGFB, SLC6A1, SMAD3, SYT3, and TNFRSF11B. CONCLUSION: miRNAs selective for regulation of endothelial function and respective downstream mRNA targets support a molecular basis for dysregulated cerebral blood flow regulation coupled with enhanced cell growth, proliferation, and inflammation.

Spreading the signal for vasodilatation: implications for skeletal muscle blood flow control and the effects of ageing.

Blood flow control requires coordinated contraction and relaxation of smooth muscle cells (SMCs) along and among the arterioles and feed arteries that comprise vascular resistance networks. Whereas smooth muscle contraction of resistance vessels is enhanced by noradrenaline release along perivascular sympathetic nerves, the endothelium is integral to coordinating smooth muscle relaxation. Beyond producing nitric oxide in response to agonists and shear stress, endothelial cells (ECs) provide an effective conduit for conducting hyperpolarization along vessel branches and into surrounding SMCs through myoendothelial coupling. In turn, bidirectional signalling from SMCs into ECs enables the endothelium to moderate adrenergic vasoconstriction in response to sympathetic nerve activity. This review focuses on the endothelium as the cellular pathway that coordinates spreading vasodilatation. We discuss the nature and regulation of cell-to-cell coupling through gap junctions, bidirectional signalling between ECs and SMCs, and how oxidative stress during ageing may influence respective signalling pathways. Our recent findings illustrate the role of small (SK(Ca)) and intermediate (IK(Ca)) Ca(2+) activated K(+) channels as modulators of electrical conduction along the endothelium. Gaps in current understanding indicate the need to determine mechanisms that regulate intracellular Ca(2+) homeostasis and ion channel activation in the resistance vasculature with advancing age.

Coordination of intercellular Ca(2+) signaling in endothelial cell tubes of mouse resistance arteries.

OBJECTIVE: To test the hypothesis that Ca(2+) responses to GPCR activation are coordinated between neighboring ECs of resistance arteries. METHODS: EC tubes were freshly isolated from superior epigastric arteries of C57BL/6 mice. Intercellular coupling was tested using microinjection of propidium iodide. Following loading with fluo-4 dye, intracellular Ca(2+) responses to ACh were imaged with confocal microscopy. RESULTS: Cell-to-cell transfer of propidium iodide confirmed functional GJCs. A 1 µm ACh stimulus evoked Ca(2+) responses (9.8 ± 0.8/min, F/F(0) = 3.11 ± 0.2) which pseudo-line-scan analysis revealed as composed of Ca(2+) waves and spatially restricted Ca(2+) release events. A 100 nm ACh stimulus induced Ca(2+) responses of lower frequency (4.5 ± 0.7/min) and amplitude (F/F(0) = 1.95 ± 0.11) composed primarily of spatially restricted events. The time interval between Ca(2+) waves in adjacent cells (0.79 ± 0.12 s) was shorter (p < 0.05) than that between nonadjacent cells (1.56 ± 0.25 s). Spatially restricted Ca(2+) release events had similar frequencies and latencies between adjacent and nonadjacent cells. Inhibiting intracellular Ca(2+) release with 2-APB, Xestospongin C or thapsigargin eliminated Ca(2+) responses. CONCLUSIONS: With moderate GPCR stimulation, localized Ca(2+) release events predominate among cells. Greater GPCR stimulation evokes coordinated intercellular Ca(2+) waves via the ER. Calcium signaling during GPCR activation is complex among cells, varying with stimulus intensity and proximity to actively signaling cells.

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma.

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow increases with decreasing diameter from arteries to arterioles that ultimately feed into capillaries. Due to their smaller size and location in the parenchyma, arterioles have been relatively understudied and with less reproducibility in findings than surface pial arteries. Regardless, arteriolar endothelial cell structure and function-integral to the physiology and etiology of chronic degenerative diseases-requires extensive investigation. In particular, emerging evidence demonstrates that compromised endothelial function precedes and exacerbates cognitive impairment and dementia. In the parenchymal microcirculation, endothelial K+ channel function is the most robust stimulus to finely control the spread of vasodilation to promote increases in blood flow to areas of neuronal activity. This paper illustrates a refined method for freshly isolating intact and electrically coupled endothelial "tubes" (diameter, ~25 µm) from mouse brain parenchymal arterioles. Arteriolar endothelial tubes are secured during physiological conditions (37 °C, pH 7.4) to resolve experimental variables that encompass K+ channel function and their regulation, including intracellular Ca2+ dynamics, changes in membrane potential, and membrane lipid regulation. A distinct technical advantage versus arterial endothelium is the enhanced morphological resolution of cell and organelle (e.g., mitochondria) dimensions, which expands the usefulness of this technique. Healthy cerebral perfusion throughout life entails robust endothelial function in parenchymal arterioles, directly linking blood flow to the fueling of neuronal and glial activity throughout precise anatomical regions of the brain. Thus, it is expected that this method will significantly advance the general knowledge of vascular physiology and neuroscience concerning the healthy and diseased brain.

Cerebrovascular microRNA Expression Profile During Early Development of Alzheimer's Disease in a Mouse Model.

BACKGROUND: Emerging evidence demonstrates association of Alzheimer's disease (AD) with impaired delivery of blood oxygen and nutrients to and throughout the brain. The cerebral circulation plays multiple roles underscoring optimal brain perfusion and cognition entailing moment-to-moment blood flow control, vascular permeability, and angiogenesis. With currently no effective treatment to prevent or delay the progression of AD, cerebrovascular microRNA (miRNA) markers corresponding to post-transcriptional regulation may distinguish phases of AD. OBJECTIVE: We tested the hypothesis that cerebrovascular miRNA expression profiles indicate developmental stages of AD pathology. METHODS: Total RNA was isolated from total brain vessel segments of male and female 3xTg-AD mice [young, 1-2 mo; cognitive impairment (CI), 4-5 mo; extracellular amyloid-ß plaques (Aß), 6-8 mo; plaques+neurofibrillary tangles (AßT), 12-15 mo]. NanoString technology nCounter miRNA Expression panel for mouse was used to screen for 599 miRNAs. RESULTS: Significant (p < 0.05) downregulation of various miRNAs indicated transitions from young to CI (e.g., let-7g & miR-1944, males; miR-133a & miR-2140, females) and CI to Aß (e.g., miR-99a, males) but not from Aß to AßT. In addition, altered expression of select miRNAs from overall Pre-AD (young + CI) versus AD (Aß+ AßT) were detected in both males (let-7d, let-7i, miR-23a, miR-34b-3p, miR-99a, miR-126-3p, miR-132, miR-150, miR-151-5p, miR-181a) and females (miR-150, miR-539). Altogether, at least 20 cerebrovascular miRNAs effectively delineate AD versus Pre-AD pathology. CONCLUSION: Using the 3xTg-AD mouse model, these data demonstrate that cerebrovascular miRNAs pertaining to endothelial function, vascular permeability, angiogenesis, inflammation, and Aß/tau metabolism can track early development of AD.

Progressive Vascular Abnormalities in the Aging 3xTg-AD Mouse Model of Alzheimer's Disease.

Vascular dysfunction and structural abnormalities in Alzheimer's disease (AD) are known to contribute to the progression of the pathology, and studies have tended to ignore the role of the vasculature in AD progression. We utilized the 3xTg-AD mouse model of AD to examine individual cerebral vessels and the cortical vascular network across the lifespan. Our vessel painting approach was used to label the entire cortical vasculature, followed by epifluorescence microscopy. The middle cerebral artery (MCA) tree was assessed with confocal microscopy, and a new method was developed to assess branching patterns as a measure of aging-related changes. We found that vascular remodeling was profoundly altered at 4-6 months of age, when the 3xTg-AD mouse is known to transition to cognitive impairment and Aß deposition in both sexes. Analysis of vascular features (density, junctions, length) of the MCA territory highlighted sex-dependent differences across the 3xTg-AD mouse lifespan, with no alterations in branching patterns. Our current cerebrovascular angioarchitectural analyses demonstrate progressive alterations in individual cortical vessels, as well as in the vascular network of the cortex. These new findings advance our understanding of brain anatomy and physiology in the 3xTg-AD mouse, while potentially identifying unique diagnostic signatures of AD progression.

Methyl-Beta-Cyclodextrin Restores KIR Channel Function in Brain Endothelium of Female Alzheimer's Disease Mice.

BACKGROUND: As the sixth-leading cause of death in the United States, Alzheimer's disease (AD) entails deteriorating endothelial control of blood flow throughout the brain. In particular, reduced inward-rectifying K+ (KIR) channel function in animal models of aging and AD compromises endothelial function and optimal perfusion of brain parenchyma. Deficient endothelial KIR channels may result from aberrant interaction with plasma membrane cholesterol as a primary regulator of membrane fluidity and ion channels. OBJECTIVE: We tested the hypothesis that mild methyl-ß-cyclodextrin (MßCD) treatment to reduce membrane cholesterol may restore endothelial KIR channel function in brain endothelium of old AD mice. METHODS: Membrane potential was continuously measured in isolated endothelial tubes from posterior cerebral arteries of young (1 to 3 months) and old (16 to 19 months) female 3xTg-AD mice before and after mild treatment with the cholesterol-removing agent MßCD (1 mmol/L). Elevated extracellular potassium ([K+]E; 15 mmol/L) and NS309 (1µmol/L) activated KIR and Ca2+-activated K+ (SKCa/IKCa) channels respectively before and after MßCD treatment. RESULTS: SKCa/IKCa channel function for producing hyperpolarization remained stable regardless of age group and MßCD treatment (ΔVm: ∼-33 mV). However, as deficient during AD, KIR channel function was restored (ΔVm: -9±1 mV) versus pre-MßCD conditions (-5±1 mV); a progressive effect that reached -14±1 mV hyperpolarization at 60 min following MßCD washout. CONCLUSION: In female animals, MßCD treatment of brain endothelium selectively restores KIR versus SKCa/IKCa channel function during AD. Thus, the endothelial cholesterol-KIR channel interface is a novel target for ameliorating perfusion of the AD brain.

Development of Alzheimer's Disease Progressively Alters Sex-Dependent KCa and Sex-Independent KIR Channel Function in Cerebrovascular Endothelium.

BACKGROUND: Development of Alzheimer's disease (AD) pathology is associated with impaired blood flow delivery of oxygen and nutrients throughout the brain. Cerebrovascular endothelium regulates vasoreactivity of blood vessel networks for optimal cerebral blood flow. OBJECTIVE: We tested the hypothesis that cerebrovascular endothelial Gq-protein-coupled receptor (GPCR; purinergic and muscarinic) and K+ channel [Ca2+-activated (KCa2.3/SK3 and KCa3.1/IK1) and inward-rectifying (KIR2.x)] function declines during progressive AD pathology. METHODS: We applied simultaneous measurements of intracellular Ca2+ ([Ca2+]i) and membrane potential (Vm) in freshly isolated endothelium from posterior cerebral arteries of 3×Tg-AD mice [young, no pathology (1- 2 mo), cognitive impairment (CI; 4- 5 mo), extracellular Aß plaques (Aß; 6- 8 mo), and Aß plaques + neurofibrillary tangles (AßT; 12- 15 mo)]. RESULTS: The coupling of ΔVm-to-Δ[Ca2+]i during AßT pathology was lowest for both sexes but, overall, ATP-induced purinergic receptor function was stable throughout AD pathology. SKCa/IKCa channel function itself was enhanced by ∼20% during AD (Aß+ AßT) versus pre-AD (Young + CI) in males while steady in females. Accordingly, hyperpolarization-induced [Ca2+]i increases following SKCa/IKCa channel activation and Δ[Ca2+]i-to-ΔVm coupling was enhanced by ≥two-fold during AD pathology in males but not females. Further, KIR channel function decreased by ∼50% during AD conditions versus young regardless of sex. Finally, other than a ∼40% increase in females versus males during Aß pathology, [Ca2+]i responses to the mitochondrial uncoupler FCCP were similar among AD versus pre-AD conditions. CONCLUSION: Altogether, AD pathology represents a condition of altered KCa and KIR channel function in cerebrovascular endothelium in a sex-dependent and sex-independent manner respectively.

Aging Alters Cerebrovascular Endothelial GPCR and K+ Channel Function: Divergent Role of Biological Sex.

Age-related dementia entails impaired blood flow to and throughout the brain due, in part, to reduced endothelial nitric oxide signaling. However, it is unknown whether sex affects cerebrovascular Gq-protein-coupled receptors (GPCRs) and K+ channels underlying endothelium-derived hyperpolarization (EDH) during progressive aging. Thus, we simultaneously evaluated intracellular Ca2+ ([Ca2+]i) and membrane potential (Vm) of intact endothelial tubes freshly isolated from posterior cerebral arteries of young (4-6 mo), middle-aged (12-16 mo), and old (24-28 mo) male and female C57BL/6 mice. Purinergic receptor function (vs. muscarinic) was dominant and enhanced for [Ca2+]i increases in old females versus old males. However, Ca2+-sensitive K+ channel function as defined by NS309-evoked Vm hyperpolarization was mildly impaired in females versus males during old age. This sex-based contrast in declined function of GPCRs and K+ channels to produce EDH may support a greater ability for physiological endothelial GPCR function to maintain optimal cerebral blood flow in females versus males during old age. As reflective of the pattern of cerebral blood flow decline in human subjects, inward-rectifying K+ (KIR) channel function decreased with progressive age regardless of sex. Combined age-related analyses masked male versus female aging and, contrary to expectation, hydrogen peroxide played a minimal role. Altogether, we conclude a sex-based divergence in cerebrovascular endothelial GPCR and K+ channel function while highlighting a previously unidentified form of age-related endothelial dysfunction as reduced KIR channel function.