Advanced age decreases local calcium signaling in endothelium of mouse mesenteric arteries in vivo

In endothelial cells of mesenteric arteries controlling splanchnic blood flow in anesthetized mice, spontaneous Ca²⁺ signals occur far less frequently with advanced age. The internal elastic lamina also has fewer holes enabling direct contact between endothelial and smooth muscle cells. These changes with advanced age can contribute to vascular dysfunction.

Abstract

Aging is associated with vascular dysfunction that impairs tissue perfusion, physical activity, and the quality of life. Calcium signaling in endothelial cells (ECs) is integral to vasomotor control, exemplified by localized Ca²⁺ signals within EC projections through holes in the internal elastic lamina (IEL). Within these microdomains, endothelium-derived hyperpolarization is integral to smooth muscle cell (SMC) relaxation via coupling through myoendothelial gap junctions. However, the effects of aging on local EC Ca²⁺ signals (and thereby signaling between ECs and SMCs) remain unclear, and these events have not been investigated in vivo. Furthermore, it is unknown whether aging affects either the number or the size of IEL holes. In the present study, we tested the hypothesis that local EC Ca²⁺ signaling is impaired with advanced age along with a reduction in IEL holes. In anesthetized mice expressing a Ca²⁺-sensitive fluorescent protein (GCaMP2) selectively in ECs, our findings illustrate that for mesenteric arteries controlling splanchnic blood flow the frequency of spontaneous local Ca²⁺ signals in ECs was reduced by ∼85% in old (24–26 mo) vs. young (3–6 mo) animals. At the same time, the number (and total area) of holes per square millimeter of IEL was reduced by ∼40%. We suggest that diminished signaling between ECs and SMCs contributes to dysfunction of resistance arteries with advanced age.

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NEW & NOTEWORTHY

In endothelial cells of mesenteric arteries controlling splanchnic blood flow in anesthetized mice, spontaneous Ca²⁺ signals occur far less frequently with advanced age. The internal elastic lamina also has fewer holes enabling direct contact between endothelial and smooth muscle cells. These changes with advanced age can contribute to vascular dysfunction.

endothelium-dependent vasodilation is governed by a rise of intracellular Ca²⁺ concentration ([Ca²⁺]_i), which stimulates the production of autacoids (e.g., nitric oxide, prostacyclin) and initiates endothelium-derived hyperpolarization (EDH). Whereas relaxation of smooth muscle cells (SMCs) via autacoids is manifest in conduit arteries, EDH prevails in resistance arteries and arterioles (6, 13). With advanced age, these respective signaling pathways, and therefore vasodilation, are impaired (2, 7, 17, 27). In vitro preparations of resistance arteries have revealed localized Ca²⁺ signals in endothelial cells (ECs) that arise from both intracellular (14) and extracellular (21, 30) sources. These discrete Ca²⁺ events occur spontaneously as well as through complementary signaling pathways (32), thereby activating nearby small- and intermediate-conductance Ca²⁺-activated K⁺ channels (K_Ca2.3 and K_Ca3.1, respectively). The ensuing hyperpolarization is transmitted to SMCs through gap junctions located at endothelial projections through holes (i.e., fenestrae) in the internal elastic lamina (IEL) that enable direct physical contact with SMCs (8, 11, 13, 23, 32). Thus signaling through gap junctions localized to myoendothelial junctions (MEJs) provides a unique intercellular signaling microdomain whereby local increases in EC [Ca²⁺]_i lead to SMC relaxation and vasodilation (14, 30, 34).

Previous studies of local Ca²⁺ signals in ECs of isolated vessel preparations have relied upon the addition of fluorescent Ca²⁺-sensitive indicators, opening the vessel to expose the endothelium, or combinations thereof (14, 15, 19, 25, 30, 34). Whereas intravital microscopy has been used to image global [Ca²⁺]_i signaling of arteriolar ECs in vivo (1, 31), these techniques have not been applied to resolve local Ca²⁺ signals of resistance arteries controlling tissue blood flow. Advancing age is associated with impaired vascular function attributed primarily to the endothelium (7, 16, 27); however, the effect of aging on EC Ca²⁺ signaling at MEJs is unknown. Thus a key goal of the present study was to adapt our intravital preparation of mesenteric arteries (MAs) (35) for confocal imaging, using mice that express the fluorescent Ca²⁺ indicator GCaMP2 selectively in ECs (31) to evaluate the effect of advanced age on spontaneous EC Ca²⁺ signals during blood flow control. While the number and size of IEL holes vary with vessel caliber and location (22, 23), the effect of aging on IEL holes is unknown. Therefore, the second goal of this study was to determine whether (and if so, how) the number and size of IEL holes differ between MAs of young and old mice. We tested the hypothesis that local EC Ca²⁺ signaling is impaired with advanced age along with a reduction in IEL holes that enable MEJ formation.

METHODS

Ethical approval and animal use.

All procedures were approved by the Institutional Animal Care and Use Committee of The University of Missouri and performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Intravital experiments were performed on young (3–6 mo) and old (24–26 mo) Tg (RP24-25504-GCaMP2)1Mik (GCaMP2) mice bred on a C57BL/6J background at The University of Missouri and genotyped at weaning. Males and females positive for the GCaMP2 transgene were studied under identical conditions, with order randomized across age and sex. Experiments examining the IEL were performed on young (3–6 mo) and old (24–28 mo) C57BL/6J mice bred at colonies maintained by the National Institute of Aging at Charles River Laboratories (Wilmington, MA). Experimental protocols included MAs from three to seven mice of each age group, typically utilizing at least two vessel segments (studied independently) per mouse. Each mouse was anesthetized with pentobarbital sodium (60 mg/kg, ip injection), and abdominal fur was removed by shaving. After experimental procedures, the anesthetized mouse was euthanized with an overdose of pentobarbital via intracardiac injection followed by cervical dislocation.

Surgical preparation for studying mesenteric arteries in vivo.

After initial injection of pentobarbital, the mouse was given supplemental injections (15 mg/kg) as needed to maintain stable anesthesia as confirmed by lack of withdrawal to toe or tail pinch. The mouse was positioned on a heated aluminum plate to maintain esophageal temperature at 37°C. A midline laparotomy was performed to exteriorize a loop of jejunum with associated mesenteric vasculature. Exposed tissue was superfused (∼5 ml/min) with bicarbonate-buffered physiological salt solution (in mM: 131.9 NaCl, 4.7 KCl, 2 CaCl₂, 1.17 MgSO₄, 18 NaHCO₃) equilibrated with 5% CO₂-95% N₂ (pH 7.4, 36°C). Arterial arcades chosen for study were standardized across experiments and contained second-order MAs with each branch >500 μm long [diameter 120–150 μm (35)]. The loop of intestine and vascular arcade were spread over a transparent pedestal (Sylgard 184, Dow Corning, Midland, MI) for imaging. Exposed intestine was secured with pins through the edges of mesentery, with care taken to avoid trauma to the intestine or its vascular supply. The intestine was covered with plastic wrap to prevent evaporation. To visualize vessels for Ca²⁺ imaging, we carefully dissected periarterial fat away from the vessel of interest while viewing through a stereomicroscope (35).

Intravital confocal imaging and analyses of EC Ca²⁺ signaling events.

The completed preparation was moved to an intravital imaging system based on an Olympus BX51WI microscope (Center Valley, PA) and allowed to equilibrate for 30 min. Superfusion at 36°C was maintained from a temperature-controlled (SW-60, Warner Instruments, Hamden, CT) 50-ml reservoir fed continuously by a supply of physiological salt solution. For fluorescence imaging, GCaMP2 was excited at 491 nm with a Cobalt Calypso laser (Cobalt, Solna, Sweden). Each field of view (FOV) contained ∼15 ECs, and images were acquired at 500–550 nm through a ×20 water immersion objective (numerical aperture 1.3) at 30 frames/s with a spinning disk confocal scanner unit (CSU-X1, Yokogawa, Tokyo, Japan) coupled to a CCD camera (XR-TURBO EX 620, Stanford Photonics, Palo Alto, CA). Images were acquired with the same laser power and gain settings for both age groups and recorded with Piper Control software (Stanford Photonics) on a personal computer. A technical limitation of our in vivo approach was the inability to consistently maintain recordings of Ca²⁺ events and IEL holes in the same focal plane with a ×60 immersion objective, as otherwise done in isolated MAs studied en face (14, 19, 30). We therefore focused our in vivo studies on resolving Ca²⁺ events with the ×20 objective and performed complementary studies of IEL holes in isolated MAs (below).

Analyses of EC Ca²⁺ signals were performed with SparkAn software provided by Dr. Adrian Bonev (University of Vermont). A 10 × 10-pixel region of interest (ROI; 6.25 × 6.25 μm) was placed over each site exhibiting a Ca²⁺ event during the 10-s recording period applying a boxcar average of 3 frames. Events with amplitudes > 1.2 F/F_o (where F = signal and F_o = baseline fluorescence) were analyzed, and these data were imported into Microsoft Excel. Statistical analyses were performed with GraphPad Prism 5 (La Jolla, CA). Differences were accepted as statistically significant with P ≤ 0.05.

Fluorescence staining and imaging the IEL.

Second-order MAs from young and old mice were isolated, cut open longitudinally, and pinned en face (endothelium exposed) to a 12-well plate coated with Sylgard 184 with pins made from tungsten wire (diameter = 25 μm; Goodfellow, Huntingdon, UK). Arteries were fixed in 4% paraformaldehyde for 20 min, washed in phosphate-buffered saline (PBS; Sigma catalog no. P5368), incubated in Alexa Fluor 633 hydrazide (10⁻⁶ M; catalog no. A-30634, Life Technologies, Carlsbad, CA) for 60 min to label the IEL, and then washed with PBS and mounted on slides with ProLong Gold (Life Technologies) and a coverslip. Slides were imaged with a Leica SP5 confocal laser-scanning microscope (Leica Microsystems, Buffalo Grove, IL). Images of fluorescent staining of the IEL were acquired with a HCX PL APO ×60 glycerol immersion objective (numerical aperture 1.3; Leica) and an ×8 line average. For all slide sets, young and old MAs were imaged with similar laser power and gain settings. Two MA segments from each of four mice per age group were used for analysis of IEL holes. For each vessel 3–5 images were acquired, for a total of 30 images from young MAs and 34 from old MAs. Holes in the IEL were counted and analyzed with a custom macro in ImageJ. Each image (3,750 μm²) was adjusted to increase contrast and then converted to binary form with the “threshold” command such that only the holes remained black. The “Particle Analyzer” function was then used to count and measure holes that fell within defined size and circularity ranges. The resulting data were exported to a spreadsheet for analyses.

Chemicals and reagents.

All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. Solutions were prepared fresh for each day's experiments.

RESULTS

In vivo EC Ca²⁺ signaling events in old and young MAs.

To determine whether advanced age is associated with impaired local EC Ca²⁺ signaling in MAs, we exposed the mesentery in anesthetized mice to observe the vascular supply of the small intestine in vivo (35). Confocal imaging detected spontaneous local Ca²⁺ signaling in ECs of MA segments. For recordings of 10-s duration in a FOV that contained ∼15 ECs, local Ca²⁺ signaling events in young MAs occurred in 4.3 ± 0.5 active sites and exhibited the following properties (Fig. 1): frequency: 0.31 ± 0.03 Hz; amplitude (F/F_o): 1.31 ± 0.01; full duration at half-maximal amplitude (FDHM): 0.11 ± 0.01 s. In old MAs, local Ca²⁺ signaling events occurred in 0.6 ± 0.2 sites (P < 0.05 vs. young MAs). Each of the properties of local Ca²⁺ signals was also altered significantly in old vs. young MAs, with frequency 0.19 ± 0.03 Hz, amplitude 1.61 ± 0.16, and FDHM 0.08 ± 0.01 s (Fig. 1). Thus advanced age was associated with a ∼85% reduction in the number of active Ca²⁺ signaling sites in ECs, a ∼40% reduction in the frequency of these localized signaling events, a ∼20% increase in their amplitude, and a ∼25% reduction in duration.

Fig. 1.

Advanced age decreases spontaneous local endothelial Ca²⁺ signals in vivo. A: representative image depicting placement of regions of interest (ROIs; 1–6) over active sites on MA endothelium. Each FOV segment contained ∼15 ECs and was recorded for 10 s. Scale bar, 100 μm. B: graph depicting Ca²⁺ signal amplitudes over time from ROIs in A. This record was selected to illustrate the range of signal amplitudes within a FOV; representative traces include boxcar averaging of 3 frames. C–F: analysis of local Ca²⁺ signal properties from MAs in young and old mice. Summary data are means ± SE for active signaling sites per observed FOV (C); frequency of Ca²⁺ events per active site in each FOV (D); amplitude of Ca²⁺ signaling events (E); and full duration at half-maximal amplitude of Ca²⁺ events (FDHM; F). These data represent 88 active sites in 20 MAs of 9 young mice and 9 active sites in 14 MAs of 6 old mice. *P < 0.05, old vs. young.

Effect of aging on IEL holes.

Compared with MAs from young mice (Fig. 2A), MAs from old mice (Fig. 2B) had ∼40% fewer IEL holes per square millimeter (young: 14.1 ± 0.8, old: 8.2 ± 0.4; P < 0.05; Fig. 2C). However, the average area of individual holes did not change significantly with age (young: 1.8 ± 0.1 μm², old: 2.0 ± 0.1 μm²; Fig. 2D), nor did distribution of hole sizes change significantly between ages (Fig. 2F). Thus with advanced age the percentage of total IEL area occupied by holes also decreased by ∼40% in old vs. young MAs (young: 2.6 ± 0.3%, old: 1.6 ± 0.1%; P < 0.05; Fig. 2E).

Fig. 2.

Decreased IEL holes with advanced age. A and B: representative images of IEL in MAs of young (A) and old (B) mice stained with Alexa Fluor 633 hydrazide. Scale bars, 20 μm. C–E: summary data are means ± SE for average number of holes per area (mm²) of IEL (C); individual hole areas (D); and % IEL area occupied by holes (E). F: frequency histograms summarizing distribution of IEL hole areas across all MAs studied from young and old mice. Old MAs: 1,080 holes analyzed from 30 vessel segments; young MAs: 1,634 holes analyzed from 34 vessel segments. For clarity, F excludes holes > 8 μm² (26 holes for young, 20 holes for old). These data represent 8 MAs from 4 mice per age group. *P < 0.05, old vs. young.

DISCUSSION

This is the first study to utilize high-speed confocal microscopy to image local Ca²⁺ signals of MAs in vivo. The presence of spontaneous localized Ca²⁺ events is consistent with in vitro studies of murine MAs showing local Ca²⁺ pulsars from inositol 1,4,5-trisphosphate (IP₃) receptors (14) and sparklets from transient receptor potential vanilloid 4 (TRPV4) channels (30) associated with MEJs. Local EC Ca²⁺ signals are also integral to the regulation of vasomotor function through the activation of TRPA1 (21) and K_Ca3.1 (14) channels, which are both concentrated in EC projections through IEL holes. Because such local events can be amplified into larger Ca²⁺ signals within ECs to evoke vasodilation (30), their diminution with advanced age is functionally relevant in the context of impaired endothelium-dependent vasodilation and restricted blood flow in the aging vascular supply (7, 17).

The dramatic reduction in number of sites for local EC Ca²⁺ signaling events in MAs from old mice (Fig. 1C) is a major finding of this study and likely reflects a reduction in endothelial projections as well as the number of IEL holes. Nevertheless, the properties of Ca²⁺ signaling events were also altered by advanced age. Thus the frequency as well as the duration of individual Ca²⁺ events were decreased significantly in old vs. young ECs, while their amplitude increased (Fig. 1, D–F). These changes in Ca²⁺ signal morphology with advanced age likely reflect changes in calcium content within the endoplasmic reticulum together with the expression and localization of relevant receptors and ion channels. Such changes having been identified here, further studies are required to determine the cellular and molecular adaptations in the signaling pathways underlying these adaptations, particularly in light of their effects on vasomotor control.

This study is the also the first to investigate the effect of aging on IEL holes in resistance arteries. Our results in MAs highlight that while the relative distribution of IEL hole sizes was not altered with advanced age, their density and total area were markedly lower in MAs of old mice compared with MAs of young mice (Fig. 2). A study of pig thoracic aortas (9) found no age-related changes in the size of IEL holes at 6–9 mo compared with those at 6–8 wk. However, the 6–9 mo age group does not reflect changes in IEL structure with advanced age, as it approximates human adolescents. In contrast, mice at 24–26 mo approximate humans at 65–70 yr. Furthermore, MEJs are more prevalent in small resistance arteries compared with larger conduit arteries (22, 23). Thus an aging-related decline in myoendothelial communication may be of more functional significance in resistance vessels, which rely on EDH-mediated vasodilation to a greater extent than conduit arteries, where endothelium-derived autacoids act as a primary mediator of SMC relaxation (24, 28, 33). The decrease in IEL holes and total hole area seen here with advanced age in mouse MAs is consistent with findings in MAs of hypertensive rats, where vascular and EC dysfunction were associated with smaller holes in the IEL compared with normotensive rats (10). Hypertensive stroke-prone rats also had smaller IEL holes in carotid arteries vs. normotensive control rats (3). We therefore propose that the age-related decrease in IEL holes contributes to vascular dysfunction via decreased myoendothelial signaling between ECs and SMCs at MEJs in MAs of old mice.

In light of myoendothelial communication during vasomotor control, a decline in both local EC Ca²⁺ signals and IEL holes may also impact the effects of sympathetic nerve activity, which increases with advanced age (26). The release of norepinephrine from perivascular sympathetic nerves acts on G_q protein-coupled α₁-adrenoreceptors of SMCs, leading to formation of IP₃ and (via Ca²⁺ release from the sarcoplasmic reticulum) a global rise in SMC [Ca²⁺]_i to evoke vasoconstriction (20). In turn, diffusion of these second messengers through MEJs (11, 12, 14) evokes local Ca²⁺ signals in endothelial projections, which provide negative feedback to SMCs through the activation of K_Ca channels (12, 19, 30, 34) as well as endothelial nitric oxide synthase (eNOS), which are both enriched at MEJs (18, 34). In this manner, bidirectional signaling between SMCs and ECs is integral to the physiological regulation of vascular resistance and blood flow control. With advancing age, the increase in sympathetic nerve activity may help to compensate for the desensitization of adrenoreceptors observed in old MAs (35). Nevertheless, the reduction in spontaneous Ca²⁺ signaling (Fig. 1) and loss of IEL holes (Fig. 2) would attenuate negative feedback from ECs to SMCs, thereby elevating vascular resistance and restricting tissue blood flow. Furthermore, loss of local EC Ca²⁺ signaling is consistent with a reduction in eNOS activity in rats (4, 5) as well as humans (29) with advanced age. Also contributing to endothelial dysfunction with advanced age are a reduction in eNOS cofactor availability and an increase in reactive oxygen species that effectively reduce nitric oxide bioavailability (16, 27). In accord with the present findings, we propose that the reduction in spontaneous local Ca²⁺ signals with advanced age reflects impaired myoendothelial communication in association with a reduction in IEL holes.

It should be recognized that the occurrence of IEL holes need not be proportional to the occurrence of MEJs, as there are far more IEL holes than there are endothelial projections through them at any given time (23). However, this association has only been studied in young animals, where IEL holes are likely to remain relatively consistent. On the basis of the present findings, we suggest that the significant reduction in the number and total area of IEL holes with advanced age reduces the probability for MEJ formation and can thereby impair both the initiation of local EC Ca²⁺ signals and the bidirectional signaling between ECs and SMCs, including EDH and its role in modulating sympathetic vasoconstriction. A limitation of the present study is that neither the source of local EC Ca²⁺ events nor their approximation to IEL holes containing MEJs was resolved definitively in vivo. Further studies are also needed to determine whether the observed age-related changes in IEL hole numbers impact MEJ formation and signaling events that have been localized to this microdomain (14, 19, 30, 34). Nevertheless, the reduction in both spontaneous EC Ca²⁺ signaling events and IEL holes reported here for MAs of old vs. young mice suggests that vascular dysfunction with advanced age is associated with a decrease in the capacity for myoendothelial signaling in resistance arteries.

GRANTS

This research was supported by National Heart, Lung, and Blood Institute Grants R37-HL-041026 and R01-HL-086483 to S. S. Segal and Grant F32-HL-118836 to E. M. Boerman.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s). The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

AUTHOR CONTRIBUTIONS

Author contributions: E.M.B. and S.S.S. conception and design of research; E.M.B. and J.E.E. performed experiments; E.M.B., J.E.E., and S.S.S. analyzed data; E.M.B. and S.S.S. interpreted results of experiments; E.M.B. prepared figures; E.M.B. drafted manuscript; E.M.B. and S.S.S. edited and revised manuscript; E.M.B., J.E.E., and S.S.S. approved final version of manuscript.