Calcium Signal Profiles in Vascular Endothelium from Cdh5-GCaMP8 and Cx40-GCaMP2 Mice

Chen, Yen-Lin; Baker, Thomas; Lee, Frank; Shui, Bo; Lee, Jane C.; Tvrdík, Petr; Kotlikoff, Michael I.; Sonkusare, Swapnil K.

doi:10.1159/000514210

Cited by 12 publications

(8 citation statements)

References 44 publications

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“…These mice possess the sequence to produce the high dynamic range and fast kinetics genetically-encoded Ca 2+ indicator GCaMP8 under control of the endothelial cell specific promoter cdh5 . 23 cdh5:Gcamp8 transgenic mice were used for all Ca 2+ imaging experiments in this study.…”

Section: Methodsmentioning

confidence: 99%

Amyloid-β disrupts unitary calcium entry through endothelial NMDA receptors in mouse cerebral arteries

Peters

Gee

Pawlowski

et al. 2021

J Cereb Blood Flow Metab

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

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Section: Methodsmentioning

confidence: 99%

Amyloid-β disrupts unitary calcium entry through endothelial NMDA receptors in mouse cerebral arteries

Peters

Gee

Pawlowski

et al. 2021

J Cereb Blood Flow Metab

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“…The lung slices were stretched to 1.5 times their resting width and mounted on Sylgard inserts using tungsten micropins (Scientific Instrument Services, Ringoes, NJ) as previously described with mouse mesenteric arteries (13). The tissues were then inverted and placed in a 35mm glass-bottom chamber containing HEPES physiological solution (pH 7.45).…”

Section: Ca 2+ Imaging In Mounted Lung Slicesmentioning

confidence: 99%

“…Regulation of endothelial Ca 2+ signaling in the pulmonary microvasculature has been crucially linked with endothelial dysfunction associated with respiratory diseases (3,12). In order to examine these signaling activities, recent technological advances have focused on development of endothelial specific genetically encoded Ca 2+ indicators in various biological systems (13). As these fluorescence-based indicators are capable of reporting Ca 2+ signals in the endothelial cells throughout the circulatory system, we were able employ this model to study the lung circulation.…”

Section: Introductionmentioning

confidence: 99%

Dynamic region of interest histometric analysis of endogenous calcium activity in intact lung tissue

Sen

Knighten

Aziz

et al. 2023

Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XXI

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Calcium (Ca 2+ ) signaling in endothelial cells plays an important role in regulation of wide range of physiological processes in the pulmonary microcirculation. A dysregulation in the intracellular Ca 2+ concentration can serve as an initiator of different pathological conditions like lung endothelial barrier disruption, edema, inflammation, etc. Normal physiologic signaling is localized spatio-temporally and signaling 'signatures' define the specificity of outcomes within the cells. However, analyzing the signaling dynamics acquired through imaging of live tissues has been challenging owing to the intricate patterns of the distinct signals. Moreover, signal analysis tools based on whole-field or static region of interest (ROI) assessments may under-or overestimate measurements of signaling parameters with respect to event origination, spread and duration. In the current study we designed an algorithm for detection and analysis of these biological signaling events based on dynamic ROI tracking, where time-dependent polygonal ROIs are automatically assigned to the changing perimeters of detected signaling events. This approach allowed for robust tracking of signals and quantification of the signaling event parameters over time We next applied this algorithm on image sequences of lung slices isolated from genetically encoded mice expressing the endothelial specific cdh5GCaMP8 (GFP-based) calcium indicator, and observed an inherent dynamic Ca 2+ signaling profile within the pulmonary microvascular endothelium. To investigate the versatility of our algorithm, we further treated the lung slices with acetylcholine (ACh) or subjected them to Ca 2+ -free (CAF) medium, to examine the emerging change in patterns of the profiles from the inherent basal dynamics. Under both these conditions, our software revealed distinct changes in event parameters with respect to event amplitude, duration and spread of the signaling events. Thus, our algorithm allowed us to identify distinct Ca 2+ signaling patterns associated with various stimuli, thereby enabling identification of these signatures under a wide variety of sub-cellular/pathologic challenges.

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“… 77 The most commonly used GECIs for studying

dynamics are members of the GCaMPs series, from GCaMP2 to various forms of GCaMP5, and GCaMP6 77 – 79 that can be targeted to specific cell types for noninvasive acute and chronic imaging using the Cre-loxP system. 80 Although new forms, jGCaMP7 and jGCaMP8 have been developed and shown to have improved sensitivity and kinetics, to our knowledge, they have only been tested in neurons 81 and vascular cells. 80 The majority of published studies on astrocytes have used GCaMP6 sensors ( Fig.…”

Section: Synthetic and Genetically Engineered Indicatorsmentioning

confidence: 99%

“… 80 Although new forms, jGCaMP7 and jGCaMP8 have been developed and shown to have improved sensitivity and kinetics, to our knowledge, they have only been tested in neurons 81 and vascular cells. 80 The majority of published studies on astrocytes have used GCaMP6 sensors ( Fig. 1 ).…”

Section: Synthetic and Genetically Engineered Indicatorsmentioning

confidence: 99%

Toolbox for studying neurovascular coupling in vivo, with a focus on vascular activity and calcium dynamics in astrocytes

Tran

2022

Neurophoton.

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. Significance: Insights into the cellular activity of each member of the neurovascular unit (NVU) is critical for understanding their contributions to neurovascular coupling (NVC)—one of the key control mechanisms in cerebral blood flow regulation. Advances in imaging and genetic tools have enhanced our ability to observe, manipulate and understand the cellular activity of NVU components, namely neurons, astrocytes, microglia, endothelial cells, vascular smooth muscle cells, and pericytes. However, there are still many unresolved questions. Since astrocytes are considered electrically unexcitable, signaling is the main parameter used to monitor their activity. It is therefore imperative to study astrocytic dynamics simultaneously with vascular activity using tools appropriate for the question of interest. Aim: To highlight currently available genetic and imaging tools for studying the NVU—and thus NVC—with a focus on astrocyte dynamics and vascular activity, and discuss the utility, technical advantages, and limitations of these tools for elucidating NVC mechanisms. Approach: We draw attention to some outstanding questions regarding the mechanistic basis of NVC and emphasize the role of astrocytic elevations in functional hyperemia. We further discuss commonly used genetic, and optical imaging tools, as well as some newly developed imaging modalities for studying NVC at the cellular level, highlighting their advantages and limitations. Results: We provide an overview of the current state of NVC research, focusing on the role of astrocytic elevations in functional hyperemia; summarize recent advances in genetically engineered indicators, fluorescence microscopy techniques for studying NVC; and discuss the unmet challenges for future imaging development. Conclusions: Advances in imaging techniques together with improvements in genetic tools have significantly contributed to our understanding of NVC. Many pieces of the puzzle have been revealed, but many more remain to be discovered. Ultimately, optimizing NVC research will require a concerted effort to improve imaging techniques, available genetic tools, and analytical software.

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Calcium Signal Profiles in Vascular Endothelium from Cdh5-GCaMP8 and Cx40-GCaMP2 Mice

Cited by 12 publications

References 44 publications

Amyloid-β disrupts unitary calcium entry through endothelial NMDA receptors in mouse cerebral arteries

Amyloid-β disrupts unitary calcium entry through endothelial NMDA receptors in mouse cerebral arteries

Dynamic region of interest histometric analysis of endogenous calcium activity in intact lung tissue

Toolbox for studying neurovascular coupling in vivo, with a focus on vascular activity and calcium dynamics in astrocytes

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