The Expanding Cell Diversity of the Brain Vasculature

Endothelial Cells

Blood vessels are made up of a single layer of ECs that form a tubular structure (Zhao et al., 2015; Sweeney et al., 2019). The blood facing surface, or luminal surface, is covered by the glycocalyx—a gel-like covering comprised of glycoproteins, proteoglycans, and glycosaminoglycans (Ando et al., 2018; Kutuzov et al., 2018). Most brain ECs have a continuous cell membrane which lacks pores or fenestrations. Adjacent ECs are securely connected to one another by adherens junctional protein complexes and tight junction protein complexes occludes the intercellular cleft. Brain ECs have a high density of tight junction proteins which precludes significant paracellular flow and is a key feature regulating size-restricted transcellular passage of molecules (Zhao et al., 2015; Sweeney et al., 2019). Unlike other organs, ECs also display lower rates of pinocytosis or non-specific bulk-flow vesicular transport (Ben-Zvi et al., 2014; Andreone et al., 2017; Chow and Gu, 2017). The brain-facing or abluminal EC membrane is embedded in a protein-rich basement membrane (BM) serving as a cellular scaffolding essential for interactions with neighboring cells (Thomsen et al., 2017).

Low rates of pinocytosis and membrane permeability make brain ECs the anatomic site of the BBB. The BBB permits gases, e.g., carbon dioxide and oxygen, and lipophilic and small molecules (< 400 Da) to freely enter the brain, but limits circulating cells and other molecules from crossing the endothelium without regulated transport systems (Banks, 2009; Sweeney et al., 2019). ECs contain a number of different transmembrane transporters to regulate influx of circulating nutrients and proteins—including carrier-mediated, receptor-mediated, and active transporter proteins (Sweeney et al., 2019; Tjakra et al., 2019). Active efflux transporters or other transport proteins also help remove potentially toxic waste products from brain into the blood (Hladky and Barrand, 2018). ECs sense shear stress from blood flow or receive signaling cues from neurons or glia to secrete vasoactive substances to help modulate vasomotor responses in adjacent mural cells and titrate local blood flow (Chen et al., 2014; Iadecola, 2017; Chow et al., 2020).

Pericytes

In the microvasculature, e.g., capillaries, venules, and select arterioles, PCs are embedded in the shared BM and extend foot processes which cover much of the vessel wall (Winkler et al., 2012, 2014a; Grant et al., 2019). Unlike vSMCs which surround blood vessels circumferentially, PCs generally extend thin longitudinal processes with further delicate elaborations to cover more of the vascular surface. Interestingly, PCs cover the vasculature in a mutually exclusive pattern and do not overlap; upon experimental ablation of one pericyte, the neighboring pericytes will then grow their surface area to take over the newly absent territory (Berthiaume et al., 2018). Pericytes contribute to endothelial BBB properties through upregulation of tight junctional protein complexes and/or downregulation of transcytotic pathways—such as those mediated by plasmalemma vesicle associated protein (Plvap) or caveolin-1 (Hellstrom et al., 2001; Armulik et al., 2010; Bell et al., 2010; Daneman et al., 2010). They also secrete BM proteins to stabilize vessels (Thomsen et al., 2017). To further regulate brain molecular exchanges, PCs are enriched in multiple carrier- and receptor-mediated transporters, active transporter proteins, or ion channels (He et al., 2016; Vanlandewijck et al., 2018; Sweeney et al., 2019).

PCs modulate capillary diameter in response to neuronal or astrocytic cues (Peppiatt et al., 2006; Hall et al., 2014; Cai et al., 2018), but the functional significance of this is unclear. Some have shown that PCs regulate local CBF regulation and neurovascular coupling (Kisler et al., 2017b, 2020). Others have found this is principally the role of vSMCs, such as those in arterioles, or pre-capillary sphincters (Fernandez-Klett et al., 2010; Hill et al., 2015; Grubb et al., 2020). This discrepancy likely stems from a lack of clarity in regards to the exact definition of vSMCs vs. PCs. PCs supply trophic support to neurons and also modulate immune responses (Rustenhoven et al., 2017; Nikolakopoulou et al., 2019; Uemura et al., 2020). For example, PCs modulate expression of endothelial cell adhesion molecules, such as intercellular adhesion molecule-1 (Icam1), and may also play a direct role in the phagocytosis of extracellular proteins (Bell et al., 2010; Sagare et al., 2013; Ma et al., 2018). This has been suggested to increase after brain injury, such as ischemic stroke, and pericytes may assume a microglial-like phenotype (Ozen et al., 2014; Sakuma et al., 2016). Others have suggested pericytes may serve as multi-potent stem cells in vitro (Dore-Duffy et al., 2006; Crisan et al., 2008; Karow et al., 2018). However, lineage tracing has shown that pericytes do not significantly contribute to other cell lineages in vivo and do not differentiate into microglia following acute brain injuries (Guimaraes-Camboa et al., 2017; Huang et al., 2020).

Vascular Smooth Muscle Cells

vSMCs from concentric rings in larger arteries and become less layered and more sparse as vessels progressively branch to form pial and penetrating arterioles (Iadecola, 2017; Frosen and Joutel, 2018). In veins, vSMCs remain as discrete cells. Due to their location and composition, vSMCs contribute much of structural stability to the vessel wall and mediate synthesis and turnover of extracellular matrix proteins, such as collagen and elastin (Iadecola, 2017; Frosen and Joutel, 2018).

vSMCs serve as contractile cells and express a number of contractile proteins or associated regulatory proteins, such as smooth muscle alpha actin (ACTA2), and receptors to numerous vasoactive molecules, including adenosine, prostaglandins, or catecholamines, and to myogenic or flow-related stimuli (Koller and Toth, 2012; He et al., 2016; Kisler et al., 2017a; Chasseigneaux et al., 2018). Through these contractile properties, vSMCs contribute to regulation of cerebral blood flow and autoregulation (Fernandez-Klett et al., 2010; Hill et al., 2015; Iadecola, 2017). Arterial pulsations and/or oscillations in relaxation or contraction of vSMCs contribute to perivascular fluid flow via the glymphatic system (Iliff et al., 2013; Aldea et al., 2019; Wardlaw et al., 2020). Studies have supported that arteriolar vSMCs are the predominate site of regional CBF regulation (Hill et al., 2015; Grutzendler and Nedergaard, 2019), but the relative contributions of vSMCs and PCs to neurovascular coupling continues to be debated (Hamilton et al., 2010; Hall et al., 2014; Hill et al., 2015; Kisler et al., 2017b; Winkler et al., 2017).

Perivascular Fibroblast-Like Cells

A recent constellation of unbiased scRNAseq studies have identified PVFBs on all vessels except capillaries (Marques et al., 2016; Saunders et al., 2018; Vanlandewijck et al., 2018; Zeisel et al., 2018). PVFBs are located within the Virchow-Robin space and loosely adhere to vessels through small, fine cellular processes (Soderblom et al., 2013; Vanlandewijck et al., 2018; Rajan et al., 2020). PVFBs express platelet-derived growth factor receptor-α (Pdgfrα) and a number of extracellular matrix proteins—including fibrillar and non-fibrillar collagens, collagen-modifying enzymes, and small leucin-rich proteoglycans, such as lumican (lum) and decorin (Dcn) (Vanlandewijck et al., 2018; Rajan et al., 2020). The function of these new-defined cells has not been completely characterized. Emerging evidence early in zebrafish development in other vascular beds, such as intersegmental vessels, has suggested that PVFBs stabilize the vasculature prior to appearance of PCs, and lineage tracing has suggested a subpopulation of PVFBs may serve as PC progenitor cells (Rajan et al., 2020). In the mouse brain, however, collagen type 1 alpha 1 chain (Col1a1)-positive PVFBs are not detected until after the birth (Kelly et al., 2016). Therefore, it is presently unclear as to the role if any PVFBs play in the developing or adult mammalian brain vasculature under physiologic conditions. In response to traumatic injury, PVFBs have been suggested to contribute to scar formation (Soderblom et al., 2013). Others have ascribed scar formation to a subpopulation of PCs which may highlight difficulties in distinguishing perivascular cell types (Goritz et al., 2011; Dias et al., 2018).

Perivascular Macrophages

PVMs are myeloid cells distinct from microglia that reside in the Virchow-Robin space surrounding arterioles and venules (Lapenna et al., 2018; Yang et al., 2019). PVMs serve a vital role in the phagocytosis of blood-borne pathogens and antigen presentation to induce protective immune responses (Lapenna et al., 2018; Kierdorf et al., 2019). They also limit EC production of pro-inflammatory mediators suggesting a dual regulatory role in immune response (Serrats et al., 2010). PVMs promote EC BBB properties and tight junction protein expression (Zenker et al., 2003). Others have shown that PVMS modulate EC expression of nutrient receptors—such as solute carrier family 2 member 1 (Slc2a)—playing a homeostatic role in cerebral glucose metabolism (Jais et al., 2016). In brain regions devoid of a BBB, such as the circumventricular organs, PVM uptake restricts influx of blood-borne proteins and macromolecules providing a size selective barrier (Willis et al., 2007). Phagocytic properties are not limited to blood-borne molecules and may help clear other extracellular proteins, such as amyloid-β (Hawkes and McLaurin, 2009).

Endothelial Cells

For most brain regions, the EC membrane remains continuous without interruption (Zhao et al., 2015; Sweeney et al., 2019). Expression of the tight junction proteins remains constant throughout the arterial-venous axis in the brain, but show some ultrastructural differences—such as a greater overlap in arteries (Hanske et al., 2017). Higher permeability and reduced tight junction protein expression is reported for select CNS regions—such as the spinal cord (Winkler et al., 2012). In brain circumventricular organs—such as the subfornical organ, organum vasculosum of the lamina terminalis and area postrema—or choroid-plexus, ECs contain fenestrations or intracellular pores which permit high permeability (Ghersi-Egea et al., 2018).

Preservation of BBB integrity also occurs through suppression of caveolae and transcytosis (Ben-Zvi et al., 2014; Andreone et al., 2017; Chow and Gu, 2017). Classically low rates of vesicular transport were thought to be uniform. However, higher rates of vesicular transport have been observed in arteries/arterioles whereas receptor-mediated transcytosis is primarily detected in post-capillary venules (Hanske et al., 2017; Kucharz et al., 2020). A recent work has shown that suppression is not uniform and that abundant caveolae are selectively abundant in arteriolar ECs—which facilitates the signaling between neurons, ECs, and vSMCs underlying neurovascular coupling (Chow et al., 2020). Whether this phenomenon varies regionally has not been reported.

Mural Cells

Considerable variation is observed in mural cells—a term referring to both vSMCs and PCs in part to account for varying cell identity along the arterial-venous axis (Winkler et al., 2011, 2019; Figures 1B–E). In arteries with diameters > 100 μm, vSMCs are spindle shaped and form concentric rings ∼ 4–10 cells thick (Shiraishi et al., 1986). As arteries progressively branch and taper forming arterioles, vSMC cell layers become thinner and ultimately become discontinuous cells with rod-like or thin lateral processes in more terminal branches (Shiraishi et al., 1986; Iadecola, 2017; Figures 1C,D). In venules and veins, vSMCs remain discontinuous and are stellate in configuration (Hill et al., 2015; Smyth et al., 2018; Vanlandewijck et al., 2018; Figure 1E).

Whether a continuum of mural cell phenotypes exists and the point of transition between vSMC to pericyte remains controversial. Some have suggested that the transition from vSMCs to pericytes is discrete (Hill et al., 2015; Zeisel et al., 2015; Vanlandewijck et al., 2018). Others have described a transitional cell with shared attributes between both cell types (Hartmann et al., 2015; Grant et al., 2019; Grubb et al., 2020; Ratelade et al., 2020). Transitional cells have also been suggested in humans (Smyth et al., 2018; Ratelade et al., 2020). However, classification of these or closely related cells differs between groups with a number of other terms—including ensheathing PCs, pre-capillary PCs, vSMC-PCs hybrids, or precapillary vSMCs (Uemura et al., 2020). Despite these differences, most agree that the contractile cells in pre-capillary arterioles are responsible for regional CBF regulation and neurovascular coupling (Fernandez-Klett et al., 2010; Hill et al., 2015; Kisler et al., 2017b; Hartmann et al., 2020).

Brain PCs also display considerable morphologic heterogeneity—including ensheathing or transitional, mesh, thin-strand, or helical and stellate configurations (Hartmann et al., 2015; Arango-Lievano et al., 2018; Smyth et al., 2018; Grant et al., 2019; Uemura et al., 2020; Figure 1B). Ensheathing PCs—analogous to the transitional cells above—predominately localize to pre-capillary arterioles. Like other PCs, they have an ovoid cell body, but with cell processes that are larger and broader which enwrap and cover much of the endothelial wall (> 90%). They also express contractile proteins—such as smooth muscle α-actin (αSMA) (Hartmann et al., 2015; Smyth et al., 2018; Grant et al., 2019). Contractile protein expression terminates as arterioles branch further to form smaller capillaries. Throughout capillaries and post-capillary venules, PC cell processes assume a mesh-like or thin-strand appearance which covers ∼70 or ∼50% of the endothelial cell well, respectively (Hartmann et al., 2015; Smyth et al., 2018; Grant et al., 2019). The functional significance of these configurations is presently unknown but may reflect a facilitatory role in transport.

PC abundance and coverage of the EC wall is relatively constant in the adult brain—including the cortex, hippocampus, striatum, and cerebellum (Bell et al., 2010; Winkler et al., 2010, 2012). However, PCs are less abundant in the spinal cord and most reduced in the gray matter of the anterior horn (Winkler et al., 2012, 2013). Whether these alterations are the result of the relative abundance or presence of different pericyte subtypes is presently unknown. Much of this work has also been done in rodents and whether species-specific differences as with other cell types exists has also yet to be determined (O’Brown et al., 2018).

Perivascular Fibroblast-Like Cells

PVFBs share some cell marker expression with PCs—including N-aminopeptidase (also known as CD13, encoded by Anpep) and platelet-derived growth factor receptor-β (Pdgfrβ) (Soderblom et al., 2013). Unlike PCs, they are found along all vessels expect capillaries (Figures 1B,C,E). Morphologically they are more globular appearance with shorter and finer processes which enwrap the vessel wall. They are also more loosely associated with the vessels—described to be an “awkward hug” (Vanlandewijck et al., 2018; Rajan et al., 2020). Whether different morphologic subtypes exist along the arterial-venous axis or in different brain regions remains to be characterized.

Perivascular Macrophages

PVMs are confined to arterioles and venules and are one of several specialized macrophages known as “border associated macrophages”—including leptomeningeal, dural, and choroid plexus macrophages (Goldmann et al., 2016; Utz et al., 2020; Figures 1B,C,E). PVMs adopt a relatively simple ameboid morphology (Zeisel et al., 2015; Kierdorf et al., 2019). They are non-motile, but able to extend cellular processes along the Virchow-Robin Space (Goldmann et al., 2016). Other border associated macrophages are stellate or dendriform appearance (Goldmann et al., 2016; Kierdorf et al., 2019). Whether PVMs has regional or arterial-venous variation in morphology has not been reported.