Research ArticleVASCULAR BIOLOGY

Spatially structured cell populations process multiple sensory signals in parallel in intact vascular endothelium

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Science Signaling  18 Dec 2018:
Vol. 11, Issue 561, eaar4411
DOI: 10.1126/scisignal.aar4411
  • Fig. 1 CCh-evoked Ca

    2+ signals in the endothelium of intact arteries. (A) A representative image frame showing ~200 endothelial cells from an imaging field with automatically identified regions of interest (ROIs; red outlines) superimposed onto the imaging field of view. Ca2+ imaging (green) illustrates the endothelial response to treatment with CCh (100 nM) overlaid on a basal (no treatment) Ca2+ image. (B) Expanded view of the endothelium from the area highlighted in the red box in (A). Seven different ROIs are noted with different color outlines. (C) Baseline-corrected Ca2+ signals (F/F0) obtained from the fluorescence within the ROIs shown in (B). The color for each trace matches that for the ROI in (B). (D) Overlaid Ca2+ signals from all individual cells identified in (A). The black line shows the mean response. (E) A representative single endothelial cell Ca2+ signal and (F) the corresponding images with time points indicated by the roman numerals. Scale bars, 50 μm. Data are representative of n = 50 independent experiments from different artery preparations.

  • Fig. 2 Concentration dependence of CCh-evoked endothelial Ca

    2+ signaling. (A) Representative composite images showing endothelial Ca2+ activity in rat carotid artery endothelium in response to increasing concentrations of CCh. The three images show illustrative data from a single experiment in which the endothelium was stimulated with increasing concentrations of CCh consecutively. The full CCh concentration series applied in each experiment was 10 increments from 1 nM to 30 μM. Scale bars, 50 μm. (B) Average Ca2+ responses (F/F0) from all cells for each concentration of the complete CCh concentration series. The individual traces are color-coded on the basis of the concentration of CCh applied. (C) Ca2+ traces from a single cell. The color code is the same as that in (B). (D) Percentage of cells activated by increasing concentrations of CCh. The x axis is log10 of the CCh concentration. M, molar. (E) Ca2+ traces from all 129 individual cells from the dataset shown in (A) to (D). The black line represents the mean response. Different colors were assigned to the traces based on the amplitude of the initial response to CCh in each cell at the highest CCh concentration (far right). The color of each specific cell’s trace was maintained throughout subsequent additions of agonist, thus allowing for direct comparison of the same cells between different CCh concentrations. Data are representative of n = 5 independent experiments from different artery preparations from different animals.

  • Fig. 3 CCh activates M3 receptors in the endothelium.

    (A) Representative experiment showing the effect of the selective M3 antagonist 4-DAMP on the average maximal CCh-evoked Ca2+ activity across all cells in an endothelial sample. Two responses to CCh were measured before the addition of 4-DAMP. CCh and 4-DAMP applications are indicated by the lines above the traces. After CCh and 4-DAMP were washed out, cells were stimulated with ATP to test for viability. (B) Composite Ca2+ images showing the cells responding to two consecutive applications of CCh (green), followed by an application of CCh plus 4-DAMP. Cell viability was assayed by measuring the Ca2+ response to ATP (red). All images are from the same field of endothelium. Scale bars, 50 μm. (C) Ca2+ signals from each cell in (B), including 30 s of baseline readings and 60 s of activation. Each individual cell’s response is color-coded and overlaid. The black line represents the average. (D) Paired peak Ca2+ (F/F0) responses from individual cells (circles) matched for each treatment (gray lines) from a single experiment. The average response is indicated by white circles and matched across each treatment by a black line. The right y axis (plotting density) shows the color code used on each of the plotted data points. The axis color codes the frequency of occurrence of specific peak F/F0 values. Red indicates a higher frequency of occurrence of a particular peak F/F0 value, and blue indicates a low frequency of occurrence of a peak F/F0 value. (E) Paired summary data illustrating changes in peak F/F0 response values averaged across all cells (n = 5). *P < 0.05. Data are representative of n = 5 independent experiments from different artery preparations from different animals.

  • Fig. 4 Concentration dependence of ATP-evoked endothelial Ca

    2+ signaling. (A) Representative composite images showing Ca2+ activity in rat carotid artery endothelium in response to stimulation with increasing concentrations of ATP. The three images show illustrative data from a single experiment in which the endothelium was stimulated with increasing concentrations of ATP (from 100 nM to 1 mM) consecutively. Scale bars, 50 μm. (B) Average Ca2+ responses (F/F0) responses from all cells for each concentration of the complete ATP concentration series. The individual traces are color-coded on the basis of the concentration of ATP applied. (C) Ca2+ traces from a single cell. The color code is the same as that in (B). (D) Percentage of cells activated by increasing concentrations of ATP. The x axis is the log10 of the ATP concentration. (E) Ca2+ traces from all 153 individual cells from the data shown in (A) to (D). The black line represents the mean response. Different colors were assigned to each trace based on the amplitude of the initial response to ATP in each cell at the highest ATP concentration (far right) to allow for direct comparison of the same cells between different ATP concentrations. Data are representative of n = 5 independent experiments from different artery preparations from different animals.

  • Fig. 5 Responses to ATP persist in the absence of extracellular Ca

    2+. (A) Composite Ca2+ images showing Ca2+ responses (red) to two consecutive applications of ATP (EC25) in Ca2+-containing buffer (control and repeat 1), followed by application of ATP in Ca2+-free buffer [physiological saline solution (PSS)]. Ca2+ was then restored to the bathing solution for a final application of ATP (repeat 2). All images are from the same field of endothelium. (B) Ca2+ signals from the individual responding cells in (A). Twenty seconds of baseline measurements and 60 s of recording after ATP activation are shown. All traces from individual cells are overlaid, and the black line represents the average. Time of activation is shown above the trace. (C) Peak Ca2+ response (F/F0) from individual cells (circles) matched for each treatment (gray lines) from a single experiment. The average response is indicated by white circles and matched across each treatment by a bold black line. The plotting density color coding indicates the distribution of peak F/F0 values. (D) Late (steady state) Ca2+ response (F/F0) to ATP (EC25) measured at 45 s in controls, after removal and replenishment of extracellular Ca2+. (E) Average peak and steady-state responses to ATP (EC25) in control (red) and after removal of external Ca2+ (blue); n = 5, P > 0.05. (F) Composite Ca2+ images showing the Ca2+ responses to a maximal concentration of ATP (100 μM). Stimulations, removal of extracellular Ca2+, and restoration of extracellular Ca2+ were performed as in (A). All images are from the same field of endothelium. (G) Ca2+ signals from the individual responding cells in (F). All traces from individual cells are overlaid, and the black line represents the average. (H) Peak Ca2+ response (F/F0) from each individual cell (circles) matched for each treatment (gray lines) from a single experiment. Average response indicated by white circles and matched across each treatment by bold black line. The plotting density color coding indicates the distribution of peak F/F0 values. (I) Late (steady state) Ca2+ response (F/F0) to ATP (maximal concentration; 100 μM) measured at 45 s in controls, after removal of external Ca2+, and after the return of extracellular Ca2+. (J) Average peak and steady-state responses to ATP (maximal concentration; 100 μM) in control (red) and after removal of external Ca2+ (blue); P > 0.05 for the peak response; *P < 0.05 for the late response. All scale bars, 50 μm. Data are representative of n = 5 independent experiments from different artery preparations from different animals.

  • Fig. 6 ATP activates P2Y2 receptors in the endothelium.

    (A) Representative experiment showing the effect of the P2Y1 antagonist MRS2179 and P2Y2 antagonist ARC118925 on the maximal Ca2+ response to ATP. The experiments were conducted consecutively on the same sample, and the traces show the average responses from all cells in the field. At the end of the experiment, CCh was applied to confirm cell viability. (B) Composite images showing the Ca2+ responses of cells to ATP in the presence of MRS2179, ARC118925, or both (red) and the subsequent responses to CCh (green) at the end of the experiment. All images are from the same field of endothelium. Scale bars, 50 μm. (C) Ca2+ signals from each cell in (B), including 30 s of baseline and 60 s of activation. (D) Peak Ca2+ response (F/F0) from each individual cell (circles) matched for each treatment (gray lines) from a single experiment. The average response is indicated by white circles and matched across each treatment by a bold black line. The plotting density y axis shows the color code used on each of the plotted data points. The axis color codes the frequency of occurrence of specific peak F/F0 values. Red indicates a higher frequency of occurrence of a particular peak F/F0 value, and blue indicates a low frequency of occurrence of a peak F/F0 value. (E) Paired summary data illustrating changes in peak F/F0 response values averaged across all cells. Each color represents a different animal and is maintained across the different treatments (n = 5). *P < 0.05. Data are representative of n = 5 independent experiments from artery preparations from different animals.

  • Fig. 7 Different agonists activate spatially distinct clusters of cells.

    (A) Representative composite images of Ca2+ activity in the endothelium in response to the EC25 and EC75 concentrations of CCh and ATP in a single artery. The cells that responded within the first 4 s after agonist application (first responding) and the entire complement of cells responding throughout the 60 s of agonist application (full response) are shown. The boxes outline a region of cells responding to CCh but not to ATP in the same field of endothelium. Images are scaled to show all activated cells. Scale bars, 50 μm. (B) The number of first-responding cells in (A) and their spatial relationships were calculated from Voronoi neighbor analysis and presented as “star maps.” The plot shows the cells that responded to each agonist within the first 4 s of activation (dots) among the total population of cells (gray crosses). Lines connect responding cells to their nearest responding neighbors. (C and D) The number of neighboring cells activated by the EC25 and EC75 of ATP and CCh in the first 4 s (C) and total cells activated in the full response (D). The total number of neighbors (6) was determined for each cell from the Voronoi neighbor analysis (black circles), as well as the number of neighbors activated for each of the conditions (red circles) and the number of neighbors predicted to be activated from a random model (blue circles). The number of neighbors activated for each of the conditions is significantly greater (P < 0.05) from that predicted from a random model of activated cells. The expectation of the number of neighbors activated randomly was determined as the (mean number of neighbors) * (activating fraction) for each agonist. (E) The number of cells activated at the concentrations shown on the x axis as a percentage of the total population of cells in the field (red) and as the percentage of precisely the same cells that are activated by each of the other conditions (blue). (F) The number of first-responding clusters (red, left) and the number of cells in a first-responding cluster (blue, right) that respond at the EC25 and EC75 of CCh and ATP. Data are representative of n = 5 independent experiments from artery preparations from different animals.

  • Fig. 8 Heterogeneous distribution of muscarinic and purinergic receptors.

    (A) Representative fluorescence images showing the endothelial cell surface marker PECAM-1 (green), purinergic P2Y2 receptors (red), and muscarinic M3 receptors (blue) in the endothelium. PECAM-1 and P2Y2 receptors were visualized by immunofluorescence, and M3 receptors were visualized using a fluorescently tagged M3 receptor ligand. (B) Expanded view of the boxed region in (A). (C) Negative controls for P2Y2 and M3 fluorescence staining. PECAM-1 labeling shows cell boundaries. The negative control for P2Y2 immunostaining omitted the primary antibody (Ab), and the negative control for M3 staining was generated by labeling with the fluorescent M3 receptor ligand in the presence of an excess of the M3 receptor competitive antagonist 4-DAMP. (D) Summary of fluorescence imaging data showing the amount of M3 staining in the presence and absence of the antagonist 4-DAMP (blue), the amount of labeling in the presence and absence of the P2Y2 primary antibody (red), and the extent of overlap of specific M3 and P2Y2 staining (gray). (E) Summary data showing the percentage of cells with M3 receptor staining (blue), P2Y2 receptor staining (red), and the percentage of cells with both (gray) (n = 3 independent experiments from artery preparations from different animals; *P < 0.05, unpaired t test). Scale bars, 50 μm.

  • Fig. 9 Signal integration and parallel processing.

    (A) Representative composite Ca2+ images showing cells that respond in the first 4 s after application of the EC25 concentrations of CCh (green), ATP (red), and both agonists applied simultaneously (cyan). Images are of the same field of endothelium. Scale bars, 50 μm. (B) Ca2+ responses from all activated cells in the field of endothelium shown in (A). Agonists were present for the duration indicated by the line above each trace. (C) Examples of responses from three separate cells to CCh, ATP, and both agonists applied together. In each panel, traces are from the cells indicated by the numbered white dots in (A). (D) Mean peak responses (black circles) to the EC25 of CCh and ATP separately and when both were applied together. The red line shows the calculated mean of peak response when both agonists were added separately, and the red-shaded region indicates the SEM for this measure. The blue line shows the sum of the peak responses when both agonists were added separately, and the blue-shaded region indicates the SEM for this measure. (E) Mean steady-state responses (black circles) to the EC25 of CCh and ATP separately and when both were applied together. The red line shows the calculated mean of the steady-state response when both agonists were added separately, with the red-shaded region indicating the SEM. The blue line shows the sum of the steady-state responses when both agonists were added separately, with the blue-shaded region indicating the SEM. Data are representative of n = 5 independent experiments from artery preparations from different animals.

  • Fig. 10 Signal integration and communication across cells.

    (A) Composite Ca2+ images showing cells that respond in the first 4 s after the application of the EC25 and EC75 concentrations of CCh (green) or ATP (red) and to the EC25 concentrations of CCh and ATP applied simultaneously (cyan). All images are from the same field of endothelium. (B) Ca2+ responses from the cells in (A), including 30 s of baseline measurements before agonist application and 60 s after agonist application. (C) Higher magnification view of the boxed area in (A) of an example cell (yellow outline) showing its responses to low (EC25) and high (EC75) concentrations of CCh and ATP. (D) Ca2+ responses of the individual cell shown in (C). Scale bars, 50 μm. Data are representative of n = 3 independent experiments from artery preparations from different animals.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/561/eaar4411/DC1

    Fig. S1. CCh-evoked Ca2+ signaling in the endothelium.

    Fig. S2. Flow-induced endothelial Ca2+ signaling.

    Fig. S3. Ca2+ dependence of CCh- and ATP-evoked endothelial relaxation.

    Fig. S4. Concentration dependence of the CCh-evoked endothelial Ca2+ response.

    Fig. S5. Concentration dependence of the ATP-evoked endothelial Ca2+ response.

    Fig. S6. The sequence of agonist addition does not alter the pattern of cell activation.

    Fig. S7. Specificity of the antibody recognizing the P2Y2 receptor.

    Fig. S8. Heterogeneity in NO production.

    Movie S1. Regional Ca2+ signals evoked by 100 nM CCh.

    Movie S2. Spatially distinct cell clusters activated by the EC25 concentrations of CCh and ATP.

  • The PDF file includes:

    • Fig. S1. CCh-evoked Ca2+ signaling in the endothelium.
    • Fig. S2. Flow-induced endothelial Ca2+ signaling.
    • Fig. S3. Ca2+ dependence of CCh- and ATP-evoked endothelial relaxation.
    • Fig. S4. Concentration dependence of the CCh-evoked endothelial Ca2+ response.
    • Fig. S5. Concentration dependence of the ATP-evoked endothelial Ca2+ response.
    • Fig. S6. The sequence of agonist addition does not alter the pattern of cell activation.
    • Fig. S7. Specificity of the antibody recognizing the P2Y2 receptor.
    • Fig. S8. Heterogeneity in NO production.
    • Legends for movies S1 and S2

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Regional Ca2+ signals evoked by 100 nM CCh.
    • Movie S2 (.avi format). Spatially distinct cell clusters activated by the EC25 concentrations of CCh and ATP.

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