Contractile pericytes determine the direction of blood flow at capillary junctions Albert L. Gonzalesa, Nicholas R. Kluga, Arash Moshkforoushb, Jane C. Leec, Frank K. Leec, Bo Shuic, Nikolaos M. Tsoukiasb,d, Michael I. Kotlikoffc, David Hill-Eubanksa, and Mark T. Nelsona,e,1 aDepartment of Pharmacology, University of Vermont, Burlington, VT 05404; bDepartment of Biomedical Engineering, Florida International University, Miami, FL 33199; cDepartment of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853; dSchool of Chemical Engineering, National Technical University of Athens, 157 72 Zografou, Greece; and eDivision of Cardiovascular Sciences, University of Manchester, M13 9PL Manchester, United Kingdom Contributed by Mark T. Nelson, September 2, 2020 (sent for review December 30, 2019; reviewed by William F. Jackson and Andy Shih) The essential function of the circulatory system is to continuously Pericytes are ubiquitous in the capillary microcirculation of all and efficiently supply the O2 and nutrients necessary to meet the vascular beds, reaching their highest densities within retinal and metabolic demands of every cell in the body, a function in which cerebral circulations (2). The defining morphological charac- vast capillary networks play a key role. Capillary networks serve teristics of a capillary pericyte, first depicted in meticulous hand an additional important function in the central nervous system: drawings in the early 20th century and later described in detail in acting as a sensory network, they detect neuronal activity in the electron micrographs (3, 4), are a prominent outward protruding form of elevated extracellular K+ and initiate a retrograde, propa- nucleus that aligns with the vessel lumen, extensions that span gating, hyperpolarizing signal that dilates upstream arterioles to the long axis, and projections that wrap around the endothelial rapidly increase local blood flow. Yet, little is known about how tube. This contrasts with all differentiated smooth muscle cells blood entering this network is distributed on a branch-to-branch described in the vasculature, which exhibit a morphology char- basis to reach specific neurons in need. Here, we demonstrate that acterized by a long, fusiform cell body that adopts a ring-shaped capillary-enwrapping projections of junctional, contractile peri- structure in vivo that encircles the endothelial cell layer in ar- cytes within a postarteriole transitional region differentially con- teries and arterioles, with its long axis oriented perpendicular to strict to structurally and dynamically determine the morphology of the direction of blood flow. capillary junctions and thereby regulate branch-specific blood The brain consumes a disproportionate share of the body’s PHYSIOLOGY flow. We further found that these contractile pericytes are capable energy resources and is highly sensitive to even brief disruptions of receiving propagating K+-induced hyperpolarizing signals prop- in blood flow. Because neurons lack the capacity to store sig- agating through the capillary network and dynamically channeling nificant energy reserves, the brain has evolved on-demand red blood cells toward the initiating signal. By controlling blood mechanisms for preferentially allocating blood flow to regions flow at junctions, contractile pericytes within a functionally dis- of higher neuronal activity. Such activity-dependent increases in tinct postarteriole transitional region maintain the efficiency and local blood flow (functional hyperemia) are mediated by an en- effectiveness of the capillary network, enabling optimal perfusion semble of mechanisms collectively termed neurovascular cou- of the brain. pling (NVC). The fact that NVC mechanisms exist suggests that simply oversupplying the entire brain is an inadequate evolution- functional hyperemia | cerebral blood flow | pericytes ary solution to the neuronal energy-resupply problem, implying that precision and efficiency are organizing principles that govern he fundamental purpose of the circulatory system is to pro- Tvide an uninterrupted supply of O2 and nutrients to all cells Significance of the body and to remove CO2 and other metabolic waste products. Capillaries, which constitute the vast majority of the Capillaries—the most abundant vessels in the circulatory — vasculature in terms of length, are the sites of gas and nutrient system deliver O2 and nutrients to all cells of the body. In the exchange between the blood compartment—including O2-carrying brain and retina, capillaries also act as a sensory web that de- red blood cells (RBCs)—and the surrounding tissue. Despite a tects neuronal activity. Here, we demonstrate that pericytes general appreciation of the relationship between the requirements localized at capillary junctions in a postarteriole transitional of tissues and the microvasculature that serves them, how RBCs region possess unique properties, notably including contrac- tility, that enable them to dynamically manipulate capillary and plasma are efficiently distributed throughout capillary net- branch diameters and exert fine control over the distribution of works so as to meet the needs of every cell remains poorly un- blood within the capillary network. In so doing, these con- derstood. Nowhere is our understanding of the mechanisms that tractile junctional pericytes fine tune the delivery of O2 and regulate the distribution of blood within capillary networks less nutrients and thus serve to meet the specific needs of neurons. complete than in the brain. Given these unique properties, pericytes represent a therapeutic The brain vasculature is composed of a network of inter- target for cardiovascular and neurodegenerative diseases. connected surface (pial) vessels that give rise to arterioles that penetrate orthogonally into the brain and feed a vast network of Author contributions: A.L.G. designed research; A.L.G. and N.R.K. performed research; capillaries. Arterioles are composed of an inner layer of endo- J.C.L., F.K.L., B.S., and M.I.K. contributed new reagents/analytic tools; A.L.G., A.M., and N.M.T. analyzed data; A.L.G., N.M.T., D.H.-E., and M.T.N. wrote the paper; A.M. wrote thelial cells (ECs), oriented in the direction of blood flow, sur- MatLab code for analysis; and M.T.N. was the principal investigator. rounded by a single layer of smooth muscle cells that wrap Reviewers: W.F.J., Michigan State University; and A.S., University of Washington. circumferentially around the endothelial cell layer and are sep- The authors declare no competing interest. arated from it by an internal elastic lamina (IEL) (1). Capillaries, Published under the PNAS license. on the other hand, are composed of endothelial cell tubes 1To whom correspondence may be addressed. Email: [email protected]. without a smooth muscle cell layer or IEL; instead, much of their This article contains supporting information online at https://www.pnas.org/lookup/suppl/ surface is covered over with perivascular mural cells (pericytes), doi:10.1073/pnas.1922755117/-/DCSupplemental. which are embedded in the basement membrane. www.pnas.org/cgi/doi/10.1073/pnas.1922755117 PNAS Latest Articles | 1of12 Downloaded by guest on October 1, 2021 the operation of these mechanisms. In fact, such precision is a containing both diverging and converging junctions, with seg- fundamental assumption underpinning brain-activity mapping based ments ultimately reuniting to drain blood back into the venous on measurements of blood oxygen level-dependent functional circulation. In the retina, arterioles radiating from the optical magnetic resonance imaging (BOLD-fMRI). Our recent work has disk, visualized using hydrazide staining to preferentially stain established a mechanistic basis for the neuron-to-microvascular the IEL (present only in arteries and arterioles), are analogous signaling necessary for efficient communication of neuronal meta- to the penetrating arterioles found in the cerebral circulation bolic demands to the cerebral vasculature, showing that activation (18) (Fig. 1A). Thus, in this formulation, all vascular elements of capillary endothelial cell inwardly rectifying potassium (Kir) downstream of these radiating arterioles are considered capil- + channels by K , a byproduct of neuronal activity, induces a prop- laries. Within these so-defined capillary regions, all perivascular agating electrical (hyperpolarizing) signal that causes upstream ar- cells with a protruding nucleus and cell body located atop the teriolar dilation and increased blood flow into the capillary network vessel are considered to be pericytes. (5). However, although this NVC mechanism provides a means of In addition to their classic “bump on a log” appearance, peri- communicating the need for increased blood flow toward a meta- cytes possess cytoplasmic extensions that spread along, and pro- bolically active anatomical region, it leaves open the question of jections that wrap around, the capillary tube (8) (Fig. 1B). These how blood flow is regulated within the capillary network. Whereas pial vessels are interconnected, and thus have con- siderable capacity for redirecting blood flow, a single parenchy- mal arteriole and associated capillary networks feed blood to a distinct cylindrical cortical volume (diameter, ∼500 μm) (6). In the absence of control mechanisms downstream of the arteriole, this arrangement predicts a stochastic distribution of blood within a volume of nonuniform neural activity, and thus has implications for the precision