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Capillary pericytes mediate coronary no-reflow after myocardial ischaemia

Research Article
Cite this article as: eLife 2017;6:e29280 doi: 10.7554/eLife.29280
3 figures, 1 table and 1 additional file

Figures

Cardiac pericyte morphology is appropriate for regulating capillary diameter.

(A) Coronary capillaries in rat left ventricle (dashed arrows in left panel show longitudinal capillaries, solid arrows show connector capillaries) labelled for basement membrane (FITC-isolectin B4) and for pericytes (example somata labelled with arrows in right panel) with antibody to the proteoglycan NG2. (B) A larger percentage of parallel capillaries (in rat,) receive pericyte contacts (<1 μm away) than do connector capillaries running between the parallel capillaries. (C) Mouse pericytes labelled with antibody to PDGFRβ (example somata labelled with arrows) and with NG2-DsRed. (D) Percentage of 2874 NG2-expressing pericytes (left) that also express PDGFRβ and of 3047 PDGFRβ−expressing pericytes (right) that also expressed NG2 (averaged over 36 confocal stacks each, from 4 mice, 2–9 months old) in the left ventricle of NG2-dsRed mice. (E) Pericyte in NG2-DsRed mouse showing soma (white arrow) and circumferential and longitudinal processes (yellow arrows). (F) The mean rat pericyte inter-soma distance is similar in the lateral wall of the left ventricle (LLV), the septal wall of the left ventricle (SLV), and anterior and posterior walls of the left ventricle (APLV). (G) Mouse pericyte circumferential processes can extend over much of the capillary surface between pericyte somata. (H) Labelling of tyrosine hydroxylase (blue) shows a close association of sympathetic axons (white arrows) with rat pericytes (cyan arrows). (I) At higher magnification, tyrosine hydroxylase labelled axon varicosities (putative transmitter release sites, cyan) can be seen apposed to pericyte somata and processes. (J) Both capillaries and pericytes (including soma and processes) frequently have sympathetic axon varicosities within 1 μm (in rat). (K–M) Examples of pericytes labelled with Alexa647-isolectin B4 (red) that also labelled (green) for α-SMA (K), β-actin (L) or γ1-actin (M). White arrows indicate somata; yellow arrows indicate actin-labelled processes. (N) Percentage of pericytes expressing the 3 actin isoforms in three hearts. Data are mean ± s.e.m. Numbers on bars are of capillaries (panels B, J), images (D), intersoma distances (F) or pericytes (J, N).

https://doi.org/10.7554/eLife.29280.003
Ischaemia and reperfusion lead to no-reflow mediated by capillary block.

(A, B) Low power view of sham-operated heart (A) and a heart after LAD coronary artery occlusion and reperfusion (B), with perfusion volume assessed as intensity of FITC-albumin (green). In (B) vessels are also labelled with isolectin B4 - Alexa Fluor 647 (purple) to define location of unperfused tissue. Regions of interest (ROIs) for analysing the intensity of FITC-albumin fluorescence are shown in yellow. (C) Perfused volume (assessed from mean FITC-albumin intensity), in ROIs indexed with numbers starting at the interventricular septum and proceeding clockwise around the left ventricle (as seen from above), for five sham-operated hearts (control), six hearts made ischaemic and reperfused (ischaemia), and eight hearts made ischaemic and exposed to adenosine starting 5 min before reperfusion (isch + ado). (D) Percentage of capillaries blocked in the anterior wall of the left ventricle for the three experimental conditions (numbers on bars are of ‘capillaries examined, image stacks examined’). Data are mean ± s.e.m. P values are corrected for multiple comparisons.

https://doi.org/10.7554/eLife.29280.004
Figure 3 with 1 supplement
No-reflow reflects blockage by pericyte constriction.

(A) Image of perfused and non-perfused capillaries in post-ischaemic left ventricle. Isolectin B4 labelling (white) defines positions of all vessels, while FITC-albumin labelling (green) shows vessels that are perfused. Bottom left capillary is completely non-perfused; top green capillary is fully perfused; lower green capillary is blocked halfway across the image. (B, C): NG2-labelling of pericytes (B) and merge (C) of the images (A) and (B) show pericyte processes constricting vessel at block site. (D–F) Another example set of images as in (A–C), showing two capillaries blocked near pericyte somata. (G) Normalised intensity of (background-subtracted) FITC-albumin (green) labelling along the centre of the capillary lumen across 20 block sites. (H) Cumulative probability distribution for the distance from capillary blockage sites to the nearest pericyte soma (black) and for the position expected (see Figure 3—figure supplement 1) if blocks occurred at positions independent of pericyte locations (significantly different, p=3.9×10−5). Comparing the experimental distribution with a theoretical distribution increasing linearly to one at a distance of 30 μm (see Image Analysis in Materials and methods) also showed a significant difference (p=7.6×10−8). (I) Ratio of capillary diameter at pericyte somata to the diameter at positions ~ 10 μm upstream after ischaemia, after ischaemia with adenosine (ado), and for sham-operated hearts (Con). (J) Diameter at pericyte somata after ischaemia, after ischaemia with adenosine, and for sham-operated hearts (Con) (all pericyte locations were measured, not just those associated with capillary blockages, for which the mean diameter after ischaemia was smaller: 3.19 ± 0.24 μm, n = 30). (K) Capillary blockage in an area of the heart with a neutrophil (labelled for neutrophil elastase, NE, bottom left) present outside the capillaries. (L) Pericyte (labelled with isolectin B4) near a blockage constricting a vessel with two red blood cells (RBCs, labelled for glycophorin A) trapped in the constriction. (M) Blockage-associated pericyte (labelled for NG2) that also labels for α-SMA. Numbers on bars are of pericytes. Data are mean ± s.e.m. P values in I-J are from Mann-Whitney tests and are corrected for multiple comparisons.

https://doi.org/10.7554/eLife.29280.005
Figure 3—figure supplement 1
Calculation of probability distribution for distance from a randomly placed block to a chosen pericyte soma.

(A) Diagram showing the calculation of the probabilities on the right of the panel. (B) Cumulative probability distribution for the position of a randomly placed block on this capillary. Figure 3H was calculated by averaging over 42 such distributions for capillaries where block was observed.

https://doi.org/10.7554/eLife.29280.006

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Rattus norvegicus (Sprague Dawley, male)RatUCL Biological Services
genetic reagent (Mus musculus/spretus, male and female)NG2-DsRed micedoi: 10.1242/dev.004895JAX 008241
antibodyanti-NG2 (rabbit polyclonal)Merck MilliporeAB5320(1:200)
antibodyanti-NG2 (mouse monoclonal)AbCamab50009(1:200)
antibodyanti-PDGF receptor beta (rabbit polyclonal)Santa Cruz Biotechnologysc-432(1:200)
antibodyanti-tyrosine hydroxylase (sheep polyclonal)Merck MilliporeAB1542(1:500)
antibodyanti-alpha smooth muscle actin (rabbit polyclonal)AbCamab5694(1:100)
antibodyanti-beta actin (mouse monoclonal)Abbiotec251815(1:100)
antibodyanti-gamma actin (mouse monoclonal)AbCamab123034(1:100)
antibodyanti-glycophorin A (mouse monoclonal)AbCamab9520(1:2000)
antibodyanti-neutrophil elastase (goat monoclonal)Santa Cruzsc9521(1:50)
antibodyanti-ICAM1 (mouse monoclonal)AbCamAb171123(1:100)
antibodyAlexa Fluor 405 goat anti-rabbit (polyclonal)Life TechnologiesA31556(1:500)
antibodyAlexa Fluor 555 donkey anti-rabbit (polyclonal)Life TechnologiesA31572(1:500)
antibodyAlexa Fluor 555 donkey anti-mouse (polyclonal)Life TechnologiesA31570(1:500)
chemical compoundisolectin B4 - Alexa Fluor 647Molecular ProbesI32450(1:200)
chemical compoundisolectin B4 - FITCSigma-AldrichL2895(1:200)
chemical compoundadenosineSigma-AldrichA9251
chemical compoundgelatinSigma-AldrichG26255% in PBS
chemical compoundFITC-albuminSigma-AldrichA97711:200 in 5% gelatin
chemical compoundDAPIMolecular ProbesD1306
chemical compoundterazosinSigmaT4680
softwareSpike2www.ced.co.ukin vivo data acquisition
softwareImageJhttps://imagej.nih.gov/ij/image analysis
softwareOriginProwww.originlab.com/Originstatistical analysis

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