Contractile pericytes determine the direction of blood flow at capillary junctions

Abstract

The essential function of the circulatory system is to continuously and efficiently supply the O₂ and nutrients necessary to meet the metabolic demands of every cell in the body, a function in which vast capillary networks play a key role. Capillary networks serve an additional important function in the central nervous system: acting as a sensory network, they detect neuronal activity in the form of elevated extracellular K⁺ and initiate a retrograde, propagating, hyperpolarizing signal that dilates upstream arterioles to rapidly increase local blood flow. Yet, little is known about how blood entering this network is distributed on a branch-to-branch basis to reach specific neurons in need. Here, we demonstrate that capillary-enwrapping projections of junctional, contractile pericytes within a postarteriole transitional region differentially constrict to structurally and dynamically determine the morphology of capillary junctions and thereby regulate branch-specific blood flow. We further found that these contractile pericytes are capable of receiving propagating K⁺-induced hyperpolarizing signals propagating through the capillary network and dynamically channeling red blood cells toward the initiating signal. By controlling blood flow at junctions, contractile pericytes within a functionally distinct postarteriole transitional region maintain the efficiency and effectiveness of the capillary network, enabling optimal perfusion of the brain.

Keywords: cerebral blood flow; functional hyperemia; pericytes.

Fig. 1.

The morphology of pericytes confers asymmetry on capillary junctions within retinal capillary networks. (A) Representative low-magnification image identifying the sharp boundary (*) between arteriole and capillary vessels of the retinal vascular network stained with FITC-conjugated lectin (green) and Texas Red-hydrazide (red). (Scale bar, 50 µm.) (B) Schematic depiction (Left) and fluorescence images (Right) of the retinal vasculature of NG2-dsRed mice stained with FITC-conjugated lectin showing arterioles and transitioning pericyte morphology from ensheathing to mesh and thin-strand type. (Scale bars, 5 µm.) (C, Left) Representative low-magnification image of the retinal vascular network from an NG2-dsRed mouse stained with FITC-conjugated lectin (green). (Scale bar, 50 µm.) (Right) Summary data showing the percentage of capillary junctions containing cells positive for the pericyte marker NG2 and the ratio of daughter branch diameters, expressed as Diameter_Small/Diameter_Large. The presence of NG2-positive cells was determined from 526 junctions from nine confocal stacks. The ratio of daughter branch diameters was determined from 102 junctions from 11 vascular trees (n = 3 to 4 mice).

Fig. 2.

Cytoskeletal elements within a feeding retinal arteriole and capillary networks. (A to E) Stitched together montage of high resolution fluorescence images (Top) and whisker-box plot (Bottom) showing the retinal vasculature stained with FITC-conjugated lectin (gray) and labeled with phalloidin, a pan-specific stain for filamentous actin (from 5 vascular trees) (A); immunostained for α-actin (n = 11 vascular trees) (B); immunostained for calponin (n = 12 vascular trees) (C); stained with Tubulin Tracker, for filamentous microtubules (n = 5 vascular trees) (D); and imaged in SMMHC (Myh11)-tdTomato mice (n = 5 vascular trees) (E). Capillary junctional fluorescence was calculated as described in Methods. (*P ≤ 0.05 vs. smooth muscle cells [SMC], ^#P ≤ 0.05 vs. first junction, ^$P ≤ 0.05 vs. second junction, and ^ϮP ≤ 0.05 vs. third junction). (Scale bars, 10 µm.)

Fig. 3.

U46619-induced dynamic contraction of pericytes in the postarteriole transitional region. (A and B) Representative images (Left), time course (Middle), and summary data (Right) showing capillary constriction following administration of U46619 (100 nM; red) or membrane depolarization with 60 mM KCl (blue), and immediate vessel relaxation to application of zero extracellular Ca²⁺ ([Ca²⁺]_o = 0, green) mediated by pericytes in the postarteriole transitional region (proximal) (A) and at locations deeper in the capillary bed (distal) (B). Average starting baseline diameter indicated by dotted line (n = 30 junctions, n = 5 to 6 mice; *P ≤ 0.05 vs. U56619 and ^#P ≤ 0.05 vs. 60 mM KCl). (Scale bars, 5 µm.) (C and D) Summary data showing U46619 (100 nM)-induced pericyte-mediated capillary constriction in the presence of cytocholasin D (5 µM) and latrunculin B (1 µM; orange) (n = 27 to 38 junctions from n = 5 mice) (C) or the MLCK inhibitor ML-7 (5 µM, purple) (n = 17 to 23 junctions from n = 4 mice) (D). (E) Summary data showing relaxation of arterioles and pericytes in the postarteriole transitional region (proximal) and more distal locations (distal), following microtubule depolymerization with nocodazole (10 µM, light blue) (n = 8 vascular trees from n = 4 mice; *P ≤ 0.05 vs. nocodazole and U46619 treatment).

Fig. 4.

L-type Ca²⁺ channels and IP₃Rs, but not RyRs, contribute to the contractile dynamics of pericytes in the postarteriole transitional region. (A and B) Representative images and traces (A) and summary data (B) showing Ca²⁺ events in pericytes (P1, P2, and P3) following administration of 100 nM U46619 (n = 40 ROIs from 12 cells, n = 3 mice; *P ≤ 0.05 vs. baseline). (C) Summary data showing the frequency (freq.) of Ca²⁺ events at baseline, and following 60 mM K⁺-induced membrane depolarization in the absence and presence of 100 nM nimodipine (n = 36 ROIs from 12 cells, n = 4 mice; *P ≤ 0.05 vs. baseline and ^#P ≤ 0.05 vs. 60 mM KCl). (D) Summary data showing contraction of projections in response to K⁺-induced membrane depolarization (60 mM KCl) in the absence and presence of 100 nM nimodipine (n = 9 cells, n = 3 mice. *P ≤ 0.05 vs. 60 mM KCl). (E) Summary data showing the frequency of Ca²⁺ events in the presence of nimodipine (n = 30 ROIs from 10 cells, n = 4 mice. *P ≤ 0.05 vs. nimodipine), without and with added U46619. (F) Summary data showing U46619-induced contractions in the absence and presence of nimodapine (100 nM) (n = 14 cells, 4 mice; *P ≤ 0.05 vs. U46619). (G) Summary data showing the frequency of Ca²⁺ events in the presence of cyclopiazonic acid (CPA, 10 µM), without and with added U46619. (H) Summary data showing contraction of projections in the presence of CPA (10 µM), without and with added U46619 (n = 7 cells, n = 3 mice; *P ≤ 0.05 vs. U46619). (I) Summary data showing the contribution of ER Ca²⁺ to Ca²⁺ events. (Left) Ca²⁺ event frequency at baseline and following administration of Bt-IP₃ (10 µM) (n = 28 ROIs from 5 cells, n = 3 mice; *P ≤ 0.05 vs. baseline). (Right) Ca²⁺ event frequency in the presence of 1 µM xestospongin C (Xesto C), without and with added U46619 (n = 25 ROIs from 6 cells, n = 3 mice). (J) Summary data showing Ca²⁺ event frequency in the presence of 100 nM tetracaine, without and with added U46619 (n = 47 ROIs from 12 cells, n = 6 mice; *P ≤ 0.05 vs. baseline). (K) Representative image (Left) and trace (Right) of Ca²⁺ in smooth muscle (arteriole, red) and pericytes in the postarteriole transitional region (blue) following administration of caffeine (5 mM) and ionomycin (10 µM). The trace shows the average and SE of five arteriole/pericyte preparations (n = 5 vascular trees, n = 4 mice).

Fig. 5.

Junctional pericytes are capable of independently controlling Ca²⁺ and contraction of capillary branches. (A) Representative images of a single proximal junctional pericyte from a retina isolated from an acta2-GCaMP-GR transgenic mouse showing projections wrapping around all capillary branches. (Scale bars, 5 µm.) (i) Schematic showing placement of ROIs for recording Ca²⁺ events (boxes) and changes in luminal diameter (dashed lines) for eight cellular projections from a single pericyte. (ii–iv) Representative image of fluorescence intensities relative to baseline (F/F_o) for Ca²⁺ events restricted to one side (ii) or occurring on both sides (iii and iv) of a pericyte projection wrapping around capillary branches. (B) Representative traces of Ca²⁺ fluorescence (upward deflection) and contraction events (shaded downward deflection) from the eight ROIs depicted in i. (C) Pearson’s correlation coefficient (Corr.) matrix of the average correlation coefficients for all possible combinations of ROIs within (diagonal elements) and between projections 1 and 8. Heat map depicts the degree of positive (red) and negative (blue) correlation. (D) Whisker-box plot from junctional pericytes depicting average correlation coefficients for Ca²⁺ events within a projection or between projections located on the same or on different capillary branches. Data from n = 5 pericytes (n = 36 projections; 114 ROIs) revealed a higher correlation for Ca²⁺ events in ROIs within a projection (137 ROI pairs in 36 projections; Corr. = 0.46 ± 0.04) or across projections within the same capillary branch (323 ROI pairs between 36 projection pairs; Corr. = 0.26 ± 0.03) compared to ROIs from different capillary branches (861 ROI pairs between 84 projection pairs; Corr. = 0.07 ± 0.02).

Fig. 6.

Pericyte Ca²⁺ events determine capillary branch diameter in vivo. (A) Cerebral circulation in an anesthetized acta2-GCaMP-GR transgenic mouse injected with TRITC (red)-dextran to illuminate the vasculature, visualized through a cranial window using 2PLSM. (Scale bars, 50 µm.) (B, Left) Representative in vivo images showing the fluorescence intensity of GCaMP (Ca²⁺ events) and mCherry, and the geometry of capillary branches. (i) mCherry fluorescence; arrowheads indicate pericytes. (ii) GCaMP fluorescence intensity relative to baseline (F/Fo). (iii) Branch angle. (iv) Branch diameter. (Middle) Representative GCaMP traces; mCherry trace shown for comparison. (Right) Correlation between Ca²⁺ event frequency (event/s) and branch angle and diameter in the two daughter branches (d₁ and d₂) of individual junctional pericytes. Different daughter branches are denoted by triangles and circles. (C–K) Disruption of blood flow following U46619-induced constriction of junctional pericytes in the postarteriole transitional region in vivo. (C) Representative images showing pipette placement and RBC flux before and after picospritzing U46619 (100 nM) onto the targeted proximal pericyte. (D and E) Representative traces and summary data at 60 s showing the effects of U46619 on RBC flux (cells/s) down daughter branches d₁ (purple) and d₂ (orange). Flow of RBCs was completely, but transiently, halted in both branches (30%) (i), differed between the two branches (30%) (ii), or decreased in both branches (40%) (n = 10, n = 7 mice; *P ≤ 0.05 vs. baseline) (iii). (F–H) Blood flow remained symmetrical following application of aCSF (control) onto junctional pericytes in the postarteriole transitional region in vivo. (F) Representative images showing pipette placement and RBC flux (line scans) through daughter branches d₁ and d₂ of a proximal pericyte, before and after picospritzing aCSF. (G) Representative traces showing the running average of RBC flux (cells/s) down each daughter branch, d₁ (purple) and d₂ (orange). RBC flux remained relatively constant (i and ii) throughout imaging; when changes in flux occurred, they were symmetrical between branches (iii). (H) Summary data showing RBC flux during baseline and 30 s after picospritzing aCSF (n = 10 pericyte junctions, n = 5 mice). (I–K) Blood flow is unaffected by stimulation of distal, noncontractile pericytes with U46619. (I) Representative images showing pipette placement and RBC flux (line scans) through the daughter branches of distal pericytes before and after picospritzing U46619. (J) Representative trace and summary data showing the effects of picospritzing aCSF onto distal pericytes (n = 19 pericyte junctions, n = 10 mice). (K) Representative trace and summary data showing the effects of picospritzing U46619 (100 nM) onto distal pericytes (n = 10 pericyte junctions, n = 6 mice). (Scale bars, 10 µm.)

Fig. 7.

Branch-specific dilation in response to retrograde hyperpolarization. (A) Proposed mechanism by which K⁺-induced retrograde hyperpolarization increases cerebral blood flow (5). Increases in local K⁺ around capillaries activates K_IR channels, generating local hyperpolarization that propagates upstream to the feeding arteriole. Membrane hyperpolarization causes arterial and pericyte relaxation, promoting an increase in blood flow into the capillaries. (B) Schematic of a pressurized retina preparation. The ophthalmic artery of an isolated mouse retina is cannulated and the retina is pinned down en face. (C, Left) Representative image of the feeding arteriole and capillary branch most proximate to the arteriole. Picospritzing pipette is targeted downstream of one branch (Stim. branch). (C, Right) Ca²⁺ and branch diameters recorded over time. (D) Representative traces showing average pericyte Ca²⁺ and diameter in stimulated and unstimulated branches following picospritzing 15 mM K⁺ and TRITC-dextran tracer downstream of one branch of the monitored postarteriole transitional segment. (E and F) Summary data showing the increase in branch diameter and decrease in Ca²⁺ in pericyte projections after picospritzing 15 mM K⁺ downstream of one branch (n = 18, n = 5 mice; *P ≤ 0.05 vs. Stim. branch).

Fig. 8.

Branch-specific increases in capillary blood flow in response to retrograde hyperpolarization. (A) Representative images showing pipette placement and RBC flux (line scans) through daughter branches d₁ and d₂ enwrapped by a junctional pericyte in the postarteriole transitional segment, before and after picospritzing 15 mM K⁺ downstream of one branch of the monitored junction. (B) Representative traces showing the running average of RBC flux (cells/s) down each daughter branch, d₁ (purple) and d₂ (orange), following administration of 15 mM K⁺ downstream of one branch. (i–iii) K⁺-dependent increases in RBC flux through the stimulated branch reduced flux (67%; i and ii) or had no effect (33%; iii) on flow in the unstimulated branch. (C) Summary data showing RBC flux at baseline and 30 s after stimulation by picospritzing 15 mM K⁺ downstream of one branch of the monitored junctional pericyte in the postarteriole transitional segment (n = 11 pericyte junctions, n = 6 mice; *P ≤ 0.05 vs. baseline). (D and E) Representative images, trace, and summary data showing the effects of picospritzing 15 mM KCl downstream of a distal pericyte (n = 12 pericyte junctions, n = 6 mice; *P ≤ 0.05 vs. baseline). (Scale bars, 10 µm.)