Since the introduction of the term pericytes in 1923, their definition has been debated extensively regarding their function and marker expression, however, changed barely when focusing on location and morphology. Currently, a pericyte is defined as a cell with a protruding cell body that encapsulates the microvascular endothelial cells and is embedded in the vascular basement membrane (Dessalles et al., 2021). They have elongated, finger-like projections that embrace the capillary and are in contact with multiple endothelial cells (see back-side image) (Hirschi and D'Amore, 1996). The bulging cell body (soma) contains the nucleus surrounded by little cytoplasm. From here extend slender, primary processes with a diameter of only 0.05-0.4 μm arranged along the longitudinal vessel axis and are mainly built up of cytoskeletal elements (Bruns and Palade, 1968). Secondary processes branch from the primary ones and are transversely oriented, wrapping around the vessel circumference (Hartmann et al., 2015).
Pericytes are distributed frequently along the microvessels, but have been primarily observed to be present at endothelial cell branch points (Armulik et al., 2011).
Despite their regular presence and long extruding processes, pericytes are not in constant physical contact with each other. Opposed to VSMCs, they form a discontinuous layer, and their probing processes avoid contact, even retracting after reaching a process from a neighbouring pericyte (Berthiaume et al., 2018).
The traditional pericyte that contains an extruding soma (bump-on-log morphology) is still recognised as such for the capillary pericytes specifically. In addition, a continuum of distinct morphologies for pericytes along the microvasculature has been identified (Figure 1) (Grant et al., 2019). On pre-capillaries, there are so-called
‘ensheathing pericytes’ with short primary processes and longer secondary processes that completely encircle the capillary. Along the mid-capillary there are
‘thin-stranded pericytes’ with vastly long primary processes and extra short secondary processes that only partially encircle the vessel (Grant et al., 2019).
Thirdly, mesh pericytes are located on the post-capillaries and have a fractal-like organisation without the typical primary and secondary branching pattern (Grant et al., 2019; Hartmann et al., 2015). Recently, pre-capillary sphincters have been denoted as a type of pericytes at the first branching capillaries. Those pericytes have more circumferential processes around the capillaries and less longitudinal processes (Grubb et al., 2020).
Although pericytes and VSMCs are often mistaken for one another due their association with the vasculature, their morphology is profoundly different. The characteristic bump-on-a-log morphology of pericytes separates it from the more flattened cell body of VSMCs. In addition, VSMCs extend on a much shorter distance along a vessel, ∼20 µm, as compared to lengths of ∼40, 100, and 150 µm for ensheathing, mesh, and thin-stranded pericytes, respectively (Grant et al., 2019).
34
Figure 1. Continuum of pericyte morphology along microvessels.
In addition to an expansion of terminology on pericyte morphology, over the years pericytes have been ascribed multiple functions and interactive players. The next section describes the sophisticated signalling pathways delegated and received by pericytes to and from their surroundings, and the significant roles these pathways and interactions include.
Function, interactive players, and signalling pathways
Following pericyte discovery, the knowledge about their functions has elaborated immensely. From simple, scaffolding cells, pericytes transformed to pivotal, multifaceted cells that play an essential role in development, stability, integrity, and remodelling of blood vessels (Gerhardt and Betsholtz, 2003). Furthermore, these multi-functional cells regulate capillary blood flow, vascular permeability, and inflammatory responses (Armulik et al., 2011).
Vessel development, stability, and integrity
The primary cellular associates of pericytes are the endothelial cells. The proportion of pericytes to endothelial cells is tissue-dependent, with ratios varying from 1:1 in the retina and central nervous system (CNS) to 1:10 in the lung, and 1:100 in striated muscle (Shepro and Morel, 1993). Tight interaction between those two vascular cell types is crucial for vessel stability and integrity, as well as development of a mature microvasculature. Pericytes and endothelial cells are in direct physical contact with each other via several ways: peg-socket interdigitations, adhesion plaques, and gap junctions (Dessalles et al., 2021).
The intimate peg-socket connections are a very characteristic way of surface contact between pericytes and the underlying endothelium. They are micron-sized pericyte
‘fingers’ (pegs), that penetrate into invaginations (sockets) in the endothelial cells, and pericytes possibly use this contact to pull on the endothelial cells (Braverman and Sibley, 1990; Caruso et al., 2009). Adherence junctions consist out of N-cadherin interactions, and adherence plaques are fibronectin patches that anchor the pericyte to the endothelial cells (Gerhardt et al., 1999; Gerhardt et al., 2000). Gap
Arteriole Pre-capillary Capillary Post-capillary Venule
Thin-stranded pericyte
Mesh pericyte Ensheathing
pericyte
VSMC Mesh pericyte VSMC
junctions are specialized channels formed by connexxin 43 proteins (Cx43; also known as gap-junction protein alpha 1 (GJA1)) that connect the cytoplasm of pericytes with that of endothelial cells and allow for exchange of small molecules and ions (Cuevas et al., 1984; Fujimoto, 1995).
This intricate, physical contact between pericytes and endothelial cells is required for blood vessel maturation and stabilisation. Once pericytes make a stable interaction with endothelial cells they inhibit their proliferation and stimulate enforcement of inter-endothelial cell adherence junctions consisting out of VE-cadherin (Kruse et al., 2019; Orlidge and D'Amore, 1987). In addition, upon cell-cell contact, a tight adhesion establishes between Notch3 on pericytes with Jagged-1 (Jag-Jagged-1) on endothelial cells, which suppresses pericyte proliferation and promotes quiescence (Liu et al., 2009).
Besides physical contact with the endothelium, the pericytes have a tight connection with the non-cellular matrix, the vascular basement membrane. Pericytes are surrounded by this basement membrane, with exception of the previously described numerous focal points where they are in contact with endothelial cells. The microvascular basement membrane constitutes out of two major extracellular matrix components, collagen IV and laminin, which are linked together by heparin-sulphate proteoglycans (HSPGs) and nidogens (Nid1 and Nid2) (Leclech et al., 2020; Timpl, 1989). Formation of the vascular basement membrane is dependent on interaction of pericytes and endothelial cells. When pericytes are recruited around developing microvessels, extracellular deposition of collagen IV, fibronectin, HSPGs, and nidogens initiates assembly of the basement membrane (Stratman et al., 2009; Stratman et al., 2010). In addition, pericytes themselves contribute to the vascular basement membrane by deposition of collagen IV, fibronectin, and laminins (Jeon et al., 1996; Mandarino et al., 1993).
Pericyte-endothelial cell signalling
In addition to the physical interactions, pericytes and endothelial cells signal to each other through paracrine signalling that affects their growth and development positively and negatively (Figure 2). The major paracrine signalling is through the family of platelet-derived growth factors (PDGFs) and their receptors (PDGFRs).
During ongoing angiogenesis, the sprouting, tip endothelial cells release the dimer PDGF-BB protein into the surrounding extra cellular matrix (ECM), which is a major growth factor for the pericytes (Hellstrom et al., 1999). Specifically, a retention motif on the C-terminal of PDGF-BB binds with HSPGs in the ECM (Lindblom et al., 2003; Ostman et al., 1991). As a result, the nearby PDGFR- β-expressing pericytes sense the retained PDGF-BB protein and subsequently are attracted to the sprouting vessel (Lindblom et al., 2003). This PDGF-BB/PDGFR-β communication is essential for proper blood vessel formation and development of an organism, since complete knock-out of either Pdgfb or Pdgfrb during
36
embryogenesis, results in a non-viable outcome due to excessive haemorrhaging (Leveen et al., 1994; Lindahl et al., 1997; Soriano, 1994).
Besides PDGF-BB/PDGFR-β signalling, there are additional signalling factors that stimulate blood vessel growth, such as basic fibroblast growth factor (bFGF) (Nakamura et al., 2016) and vascular endothelial growth factors (VEGFs) (Darland et al., 2003). Especially VEGF-A and its binding to VEGFR-2 on endothelial cells are essential for vasculogenesis and angiogenesis; knock-out of either the ligand or the receptor results in embryonic lethality due to severe vasculature defects (Carmeliet et al., 1996; Shalaby et al., 1995). VEGF-A exists in four different isoforms that differ from each other in their affinity to bind HSPGs (Patel-Hett and D'Amore, 2011). Gradients of the smaller, more soluble VEGF-A isoforms guide migration of sprouting endothelial tip cells that express high levels of VEGFR-2.
The larger isoforms with higher HSPG affinity accumulate in the ECM and their concentration determines proliferation of sprout stalks (Gerhardt et al., 2003; Patel-Hett and D'Amore, 2011).
Where the PDGF-BB/PDGFR-β and VEGF-A/VEGFR-2 signalling stimulate blood vessel growth, there are several counteracting pathways that inhibit endothelial cell proliferation, stimulate quiescence, and induce vessel stabilisation. The major pathway through which pericytes negatively affect endothelial cell growth is TGF-β signalling. TGF-TGF-β is involved in suppressing endothelial cell proliferation. The latent, inactive form of TGF-β is activated when pericytes and endothelial cells make direct contact with each other, after which the latter gets inhibited in its proliferation by activated TGF-β (Antonelli-Orlidge et al., 1989). Similarly, reciprocal signalling from endothelial cell-derived TGF-β inhibits proliferation of pericytes (Yan and Sage, 1998). Concurrently with their differentiation and in response to TGF-β, pericytes secrete an isoform of VEGF-A that is retained in the ECM, thereby locally stimulating endothelial cell survival and promoting vessel stability (Darland et al., 2003). In addition, pericytes have been proposed to express the VEGF receptor VEGFR-1, which acts as a ligand-trap of VEGFs (Cao et al., 2010). Binding of VEGF-A to this receptor sequesters it from the endothelial-expressed VEGFR-2, hence, preventing initiation of angiogenesis in mature and quiescent blood vessels (Eilken et al., 2017; Fong et al., 1995). Thus, both pericytes and endothelial cells secrete TGF-β and express the corresponding receptors, which makes this a complex signalling pathway where both cell types are interdependent on one another.
In addition, two TGF-β receptors, activin-like kinase (ALK) 1 and 5, exert opposing cellular effects (Goumans et al., 2003; Goumans et al., 2002). Both receptors, as well as their TGF-β ligand, are essential for embryonic development, since deletion of either three genes results in embryonic lethality due to defective angiogenesis and impaired mural cell recruitment and differentiation (Dickson et al., 1995; Larsson et al., 2001; Oh et al., 2000). Endothelial cells express both ALK1 and ALK5, but
activation of ALK1 upon binding of TGF-β promotes cell proliferation and migration through Smad1/5, and activation of ALK5 stimulates vessel maturation through Smad2/3 (Goumans et al., 2002). ALK1 has been reported to be exclusive to endothelial cells, but ALK5 is broadly expressed by multiple cell types including pericytes (Aguilera and Brekken, 2014; Seki et al., 2006). Similar to ALK5 activation in the endothelium, ALK5 activation in pericytes stimulates quiescence and vessel stabilisation, partly through the Smad2/3 mediated transcription of FN1 (Aguilera and Brekken, 2014; Gaengel et al., 2009).
Angiopoietins are glycoproteins that interact with their receptors Tie1 and Tie2 to increase vessel stability. Angiopoietin-1 (Ang1) is secreted by pericytes and upon interaction with Tie2 on endothelial cells it maintains vessel integrity. Both proteins are very critical for proper blood vessel formation, since knock-out of either one results in angiogenic deficits and early embryonic lethality (Dumont et al., 1994;
Suri et al., 1996). Upon pericyte-secreted Ang1-binding to Tie2, the latter is phosphorylated and stimulates vessel stabilisation through regulation of transcription factor FOXO1 (Daly et al., 2004). Interestingly, Ang2, the antagonistic counterpart of Ang1, is stored in endothelial cells and swiftly released upon stimulation, after which it competes for Tie2 binding. Ang2 only weakly activates Tie2 and therefore counteracts Ang1 activity (Fiedler et al., 2004; Yuan et al., 2009).
Unexpectedly, it was later discovered that pericytes themselves also express Tie2, although in low levels, and that Tie2 activation in pericytes controls vessel maturation (Teichert et al., 2017).
A detailed balance among the aforementioned players is required for appropriate development of the vascular tree during embryogenesis and during adulthood processes that require blood vessel regeneration, such as wound healing and the female reproductive cycle (Gordon et al., 1995, female).
Figure 2. Pericyte-endothelial interactions.
38
Regulating capillary blood flow
Already in 1873 Rouget proposed that pericytes have contractile potential and possibly could mediate blood flow (Rouget, 1873). The contractility has been an ongoing debate (Hill et al., 2015) and is a highly investigated functional property of pericytes. The contractility varies between the type of pericyte and expression levels of alpha smooth muscle actin (αSMA, gene symbol: ACTA2), especially the ensheathing pericytes are accepted to possess contractile capabilities (Dessalles et al., 2021; Nehls and Drenckhahn, 1991).
The expression of contractile proteins, such as αSMA and tropomyosin, in stress fibres of pericytes (Alarcon-Martinez et al., 2018; DeNofrio et al., 1989; Joyce et al., 1985; Wallow and Burnside, 1980) is evidence for the contractile potential of pericytes. Especially, work in retinal and cerebral capillaries has shown contractility of these mural cells (Hall et al., 2014; Hamilton et al., 2010; Kornfield and Newman, 2014; Kureli et al., 2020). In addition, the continuing controversy includes those that agree that pericytes express αSMA, but that VSMCs are the significant cells with contractile capabilities (Fernandez-Klett et al., 2010), and others who disagree with any contractility of pericytes at all (Hill et al., 2015). Recently, it has been shown that pericytes form precapillary sphincters on the junction of the arteriole and the first branching capillary. These pericytes display high levels of αSMA and can alter cerebrovascular flow after constriction (Grubb et al., 2020).
Inflammatory response
Pericytes play many roles in immunoregulation, such as leukocyte trafficking and cytokine secretion (Rustenhoven et al., 2017). Mesh pericytes on post-capillaries have a morphology that is less suitable for contractile functions, and therefore these pericytes are thought to be responsible for regulating the immune functions. For instance, it was demonstrated that neutrophils crawl along venular walls guided by pericyte-expressed intercellular adhesion molecule 1 (ICAM-1) and enter the tissue between adjacent pericytes (Proebstl et al., 2012). Another cell adhesion molecule expressed by pericytes is melanoma cell adhesion molecule (MCAM, also known as CD146) (Crisan et al., 2008) and has been shown to contribute to transmigration (Bardin et al., 2009), and is therefore possibly another mechanism that pericytes could use for leukocyte extravasation. Moreover, pericytes along the mid-capillaries and arterioles have been observed to instruct neutrophils and macrophages to extravasate through the walls of the ensuing post-capillary venules, followed by additional interactions between these migratory innate immune cells and pericytes through ICAM-1 and macrophage migration-inhibitory factor (MIF) (Stark et al., 2013). To facilitate neutrophil extravasation, pericytes remodel the basement membrane around venules and allow for increased inter-pericyte gaps through their relaxation (Wang et al., 2012). In addition, pericytes respond to the inflammatory cytokine interleukin 17 (IL-17) by upregulating other
pro-inflammatory factors, such as CXCL1, CXCL8, and IL-6, which, in turn, recruit neutrophils and modulate neutrophil-mediated immunity (Liu et al., 2016).
Although most tissues contain relatively low numbers of pericytes, the brain and retinal vasculature have the highest pericyte density, with one pericyte for every single endothelial cell. Not surprisingly, pericytes are therefore significant players in these organs. The key roles of pericytes in the brain are the focus of the next section.
Pericytes in the brain
The average weight of an adult human brain is approximately 1.5 kg. It consumes 15-20% of both the body’s oxygen and glucose supply through its over 600 km long capillary network, consisting out of an estimated 100 billion capillaries. This organ is the best perfused of the human body with a capillary network so dense that every neuron is virtually supplied by its own capillary (Klein et al., 1986; Pardridge, 2005;
Wong et al., 2013). The brain parenchyma together with the fleeces around it (leptomeninges), and the cerebrospinal fluid (CSF), are defined as the CNS.
The CNS capillary network is distinct from the rest of the body in that it has a very dense pericyte coverage, with about one pericyte for every endothelial cell (Shepro and Morel, 1993). This tight barrier of strategically positioned pericytes around the brain endothelium is known as the blood-brain barrier (BBB), and provides a highly selective, semipermeable, physical interface between the brain parenchyma and the systemic circulation (Daneman and Prat, 2015; Keaney and Campbell, 2015). The BBB was demonstrated in 1885 by Nobel Prize-winning Paul Ehrlich when he injected a blue dye subcutaneously into a rabbit and took its (the rabbit’s) life 1.5 hour afterwards. He observed that all organs were blue permeated, except for the brain (Ehrlich, 1885). Interestingly, Ehrlich didn’t realise himself what his observations meant, and even when the unique property of the cerebral vasculature was proposed several years later, Ehrlich specifically rejected the idea that the endothelium could be structurally different between organs (Ehrlich, 1906;
Saunders et al., 2014). He concluded that with ‘his own long experience with the greatest variety of substances’, substances that did not permeate the brain were by means not neurotropic (Figure S4) (Ehrlich, 1906).
Vascularisation in the mouse brain and retina begins around E9.5 (Paredes et al., 2018). The formation of the BBB starts in humans during embryonic development, around 32 weeks gestation of the pre-term human infant (Semple et al., 2013). From E11.5-14.5 the PDGF-BB signalling from endothelial cells is driving the pericyte migration along the primitive vascular tubes. This PDGF-BB secretion by endothelial cells gets downregulated around E18.5 in the mature brain capillaries due to the prior pericyte investment between E14.5-17.5 (Hellstrom et al., 1999;
Lindahl et al., 1997). The pericytes in the CNS, as well as those from the retina and
40
thymus, originate from the neuroectodermal neural crest, in contrast to pericytes in the trunk of the body which are derived from the mesoderm (Armulik et al., 2011;
Etchevers et al., 2001; Trost et al., 2013).
The endothelial cells that are tightly connected by tight junctions in combination with the dense coverage of pericytes make that the BBB is a highly selective and partly permeable barrier to the blood stream. In order to regulate the influx and efflux of oxygen and nutrients, the endothelial cells contain a multitude of specialised transporters to allow for transcellular metabolite transport (Villasenor et al., 2019). These properties of the CNS microvasculature create an immune-privileged environment that protects the brain from toxins and pathogens.
The PDGF-BB/PDGFR-β signalling is crucial for pericyte recruitment to the nascent vessels in BBB integrity and formation, since deletion of Pdgfrb results in vascular permeability, due to a lack of pericytes and endothelial tight junctions (Daneman et al., 2010). Similarly, a mouse model with a diminished pericyte coverage demonstrates increased vascular permeability due to altered CD71 expression in endothelial cells and polarisation of astrocyte end feet (Armulik et al., 2010).
In addition to the BBB, the pericytes take a central position in the so-called neurovascular unit (NVU). In this cellular complex the pericytes are sandwiched between tightly connected endothelial cells including their shared basement membrane and astrocyte end feet processes (Figure 3) (Sweeney et al., 2016).
Together they synergistically regulate neurovascular coupling, BBB permeability, and maintain homeostasis in the CNS. Although the BBB components are the main structure of the NVU, the interactions with the surrounding astrocytes, microglia, and neurons are also critical for its functions and maintenance (Daneman and Prat, 2015).
Figure 3. The neurovascular unit. The pericyte (grey) occupies a central position in the neurovascular unit, encircling endothelial cells on one side and being in touch with astrocyte end feet on the other.
Especially the pericytes play a pivotal role in the functions of the BBB and NVU (Sweeney et al., 2016). Their central location is excellent for relaying the crosstalk between the endothelial cells and the cerebral surroundings. The contractile ability of pericytes allows them to regulate capillary blood flow in response to local metabolic demand towards regions that have increased neuronal activity, a process called neurovascular coupling (Hall et al., 2014) (a mechanism that was already observed in the mid-19th century (Donders, 1851)).
The tight BBB not only assures that the brain is protected from unwanted molecules, but also makes it an immune-privileged environment by restricting passage of leukocytes and pathogens. However, during inflammation, pericytes can facilitate leukocyte extravasation in response to inflammatory cytokines such as tumour necrosis factor alpha (TNF-α) and IL-1β, by secreting cytokines that attract them, such as IL-8 (Pieper et al., 2013). In addition, TNF-α and IL-1β stimulate increased expression of ICAM-1 and VCAM-1, molecules that both aid leukocyte crawling.
In addition to instructing immune cells, brain pericytes exert macrophage functions themselves. Pericytes isolated from rat brain capillaries express the macrophage markers CD11b and ED-2 and were able to perform phagocytosis (Balabanov et al., 1996). The ED-2 antigen is part of the CD163 receptor that has been associated with the M2 macrophages, which have a proangiogenic and immunosuppressive phenotype (Kwiecien et al., 2019). In addition, human brain pericytes were demonstrated to express the classical pan-macrophage marker CD68 (Holness and Simmons, 1993; Sasaki et al., 1996).
Heterogeneity
As described in the previous sections, there is ample diversity among pericytes regarding morphology and function. Moreover, there has been an ongoing debate on the heterogeneity of molecular markers expressed by pericytes; a major challenge is that neither of these markers is exclusive to nor comprehensive of all pericytes.
The following section discusses the spectrum of antigens that has been used to identify pericytes, as well as the speculative pericyte subtypes.
Pericyte markers
Two common pericyte markers were discussed in the previous sections, PDGFR-β and αSMA. Especially, PDGFR-β is used extensively and this surface antigen seems to be expressed by the majority of pericytes. However, this protein is expressed by perivascular fibroblasts as well (Soderblom et al., 2013). The contractile protein αSMA has been under decennia-long debate if it is expressed by pericytes and has recently been confirmed as such, but only in certain morphological subtypes (ensheathing pericytes) or conditions (tumour pericytes). However, this protein is mainly expressed by the other type of mural cells, VSMCs, and
42
myofibroblasts. In addition, pericytes express the surface antigens proteoglycan neural/glial antigen 2 (NG2; gene symbol: chondroitin sulphate proteoglycan, CSPG4), Endosialin (CD248), and MCAM, as well as several intracellular proteins;
regulator of G-protein signalling 5 (RGS5), Desmin, Nestin, and alanine aminopeptidase (ANPEP, also known as CD13). None of these markers are exclusive to pericytes, or encompass all pericytes, and their expression pattern differs on the tissue, developmental state, and type of vessel (Armulik et al., 2010;
Murfee et al., 2005; Yotsumoto et al., 2015).
Pericyte subtypes
Investigating pericytes is challenging due to their heterogeneity both among and within organs. A specific gene expression pattern has been observed between brain and lung pericytes (Vanlandewijck et al., 2018), and stellate cells and mesangial cells are the pericyte equivalents in the liver and kidney, respectively (Hellerbrand, 2013; Popik et al., 2019). As a result of the plethora of markers available, pericytes have been identified by different constellations of markers in different studies, and sometimes only a single marker is used to visualise a pericyte. The lack of specific biological markers to identify pericytes feeds the ongoing debate on their definition, function, and significance (Attwell et al., 2016).
A possible explanation for the absence of unique molecular markers for pericytes has been speculated as that there exist distinct functional pericyte subsets (Cortez et al., 2014). Several studies claim to have identified such populations of pericytes. In skeletal muscle, for instance, two types of pericytes were detected based on their Nestin expression, with ‘type-1’ pericytes being Nestin-negative and ‘type-2’
Nestin-positive, while both types expressed NG2 (Birbrair et al., 2013b). Type-1 pericytes were involved in tissue fibrosis and type-2 pericytes displayed angiogenic potential (Birbrair et al., 2013c; Birbrair et al., 2014b). Another study noticed that spinal cord pericytes that were Desmin+/αSMA+ differed from pericytes that in addition were positive for glutamate aspartate transporter (GLAST; gene symbol:
Slc1a3), the latter being involved in scar formation after spinal cord injury (Goritz et al., 2011).
To elucidate the functional diversity among pericytes remains challenging.
However, recent advances in the field of transcriptomics benefited greatly to the exploration of pericyte heterogeneity. A selection of studies that investigated the microvasculature of the brain through single-cell transcriptomics is discussed in the next section.
Single cell sequencing studies on brain pericytes
The technique of single-cell RNA sequencing (scRNA-seq) enables to acquire a transcriptomic profile of each single cell of a given cell suspension. In contrast to
bulk RNA sequencing, scRNA-seq allows for a detailed dissection of the heterogeneity within a tissue, and for its potential to resolve tissue complexity it was awarded the Method of the year 2013 by Nature (2014). Rare cell populations, such as pericytes, can be obscured when analysing an average gene expression profile of a tissue which happens with bulk RNA sequencing. Therefore scRNA-seq is a powerful tool to investigate pericytes within a cellular ensemble.
Several transcriptome studies attempted to unravel the complex composition of the brain or the genetic expression profile of pericytes, anticipating to detect pericyte subtypes (Table 1). A study on whole mouse cortex detected endothelial cells and mural cells, besides several CNS specific cell types, such as neurons, oligodendrocytes, microglia, and astrocytes (Zeisel et al., 2015). Mural cells contained both pericytes and VSMCs and were enriched for Acta2. While the study mostly focused on dissecting heterogeneity of all cell types besides those of the vasculature, exploration of pericyte-enriched genes in their online available database, demonstrates that Acta2 was primarily expressed among annotated VSMCs. Rgs5, Pdgfrb, and Cd248 appear to have similar expression levels between VSMCs and pericytes, while Kcnj8 and Abcc9 are mainly expressed in pericytes.
Interestingly, the expression levels of any of these genes was relatively low in the annotated pericytes compared to VSMCs, suggesting that this study did not capture many pericytes or that the mural cell clusters deserved more attention in the annotation between VSMCs and pericytes. In a more elaborate, second study from the same group on mouse CNS combined with the peripheral nervous system, three pericyte groups (PER1, PER2, PER3) were detected besides VSMCs. All three pericyte clusters shared expression of Kcnj8, Abcc9, Cspg4, Atp13a5, and Vtn, while the single VSMC cluster expressed Acta2 and Tagln (Zeisel et al., 2018). A third single-cell transcriptomic study on whole mouse brain identified mural cells, endothelial cells, and fibroblast-like cells, among the typical CNS cell types. Seven mural cell clusters could be separated that represented pericytes (Kcnj8, Abcc9, Vtn), VSMCs (Acta2), and cells that shared pericyte and VSMC markers (Rgs5, Acta2) (Saunders et al., 2018).
In addition to these scRNA-seq studies on whole mouse brain, several studies focused specifically on the brain vasculature. One study described the mural cell gene profile as a zonation, with arterial and arteriole VSMCs enriched for Acta2 and Tagln on one end, and pericytes and venous VSMCs on the other, displaying weak Acta2 and Tagln expression in combination with high expression of Abcc9. All mural cells expressed Pdgfrb and Cspg4, and these genes can therefore not be used to distinguish between pericytes and VSMCs (Vanlandewijck et al., 2018).
Although this study concluded the absence of pericyte subtypes, a continuation of this study detected gene expression diversity between pericytes of different organs (He et al., 2018; Vanlandewijck et al., 2018). Brain pericytes abundantly expressed genes of membrane transporters, such as Atp13a5, and uniquely expressed Anpep, when compared to pericytes from lung tissue. Both lung and brain pericytes shared
44
the expression of Higd1b, Vtn, Mcam, S1pr3, Ifitm1, Baiap3, and Ehd3 (Vanlandewijck et al., 2018). A study in rat brain also focused specifically on isolating the vasculature. They demonstrated the isolation of mid-capillary mural cells and those from arterioles through a mild enzymatic digestion. Differential bulk RNA sequencing analysis of the two mural cell groups was in concordance with the mural cell zonation; VSMCs overexpressed Acta2 and Tagln, as well as Cnn1 and Myh11, compared to mid-capillary pericytes. Both mural cell types expressed Pdgfrb, Cspg4, and Rgs5 (Chasseigneaux et al., 2018).
Comparably to the studies in mouse tissue, transcriptome studies in human brain analysed the composition of whole brain tissue and its vasculature with different approaches (Table 1). Although not all studies were able to detect pericytes (Darmanis et al., 2015; Zhong et al., 2018), the majority did capture several types of vascular cells, including endothelial cells and pericytes. Single-cell transcriptome analysis of human prefrontal cortex detected a pericyte cluster that was enriched for PDGFRB, CD248, and RGS5.
Remarkably, they observed that CD19, a B-cell marker, was highly expressed among the pericytes as well (Parker et al., 2020). Another RNA sequencing study compared the human and mouse brain vasculature by specifically isolating microvessels with laser-capture microdissection (Song et al., 2020). They detected human pericytes with high expression of PDGFRB, ABCC9, and RGS5, while they lacked enrichment of KCNJ8, ANPEP, and CSPG4, all of which had increased expression in mouse pericytes. In addition, mouse expression of Vtn and Atp13a5 was enriched over their expression in human microvessels. Similarly, human pericytes highly expressed ANGPT2 and PTGDR2, as compared to a minimal expression in mouse pericytes, while NOTCH3, COLEC12, NDUFA4L2, EHD2, and NES had a high expression in human pericytes and medium expression levels in mouse (Song et al., 2020).
Besides the noticeable differences among pericytes in healthy tissue and the speculated existence of pericyte subtypes, pericytes differ depending on the pathological condition of the tissue. Regarding the key role of pericytes in proper functioning of the CNS, it is not surprising that dysfunction of pericytes is involved in numerous neuropathological conditions. Degeneration of pericytes leads to a compromised BBB that allows for leukocyte infiltration and subsequent neuroinflammation. One of the most notable neurological diseases due to BBB breakdown is Alzheimer disease, where increased BBB permeability causes elevation of ameloid β and neuronal death (Sagare et al., 2013). Other neurological complications with an altered BBB are stroke, Huntington disease, diabetic retinopathy, and multiple sclerosis (Cheng et al., 2018; Hammes, 2005;
Hirunpattarasilp et al., 2019). Above all, brain tumours are an impacting neurological condition where the opposing roles of pericytes have been speculated.
Table 1. Transcriptomics in brain or vasculature.
Overview of single-cell transcriptome studies in mouse and human brain or vasculature. Song et al. (2020) is an exception in the list and performed bulk RNA-seq instead of scRNA-seq. AC: Astrocytes. EC: Endothelial cells. EpC:
Ependymal cells. FB: Fibroblasts. FL: Fibroblast-like cells. LC: Lymphoid cells. LCM: Laser-capture microdissection.
MES: Mesenchymal cells. MG: Microglia. Mϕ: Macrophages. N: Neurons. NPC: Neural progenitor cells. OD:
Oligodendrocytes. PC: Pericytes. PPG: Peripheral glia (includes Schwann cells). PVM: Perivascular macrophages. RG:
Radial glia cells (progenitors of the glia lineages, including astrocytes and oligodendrocytes). VSMC: Vascular smooth muscle cells. VLMC: Vascular leptomeningeal cells.
Reference Samples (n) Cells1 Method Selection Dissociation Cell types Zeisel et al.,
2015 Mouse primary
somatosensory cortex and hippocampus
3005 Fluidigm
C1 None. Papain
dissociation system (Worthington)
AC, EC, MG, N, OD, PC, PVM, VSMC.
Darmanis et
al., 2015 Human cortical tissue; adult (8), embryo (4)
466 Fluidigm
C1 CD45—. Papain
dissociation system (Worthington)
AC, EC, MG, N, OPC.
Zeisel et al.,
2018 Mouse brain
and spinal cord 160,796 Fluidigm
C1, 10X None. Papain
dissociation system (Worthington)
AC, EC, EpC, MG, N, OD, OPC, PC, PPG, PVM, VSMC, VLMC.
Saunders et
al., 2018 Mouse brain2 690,000
Drop-Seq None. Papain
dissociation system (Worthington)
AC, EC, FL, MG/Mϕ, N, OD, OPC, PC, VSMC.
He et al., 2018;
Vanlandewijck et al., 2018
Mouse brain
and lung 49403
Smart-seq2 Cldn5+, Pdgfrb+, Cspg4+/Pdgfrb+, Tagln+, Pdgfra+.
Neural tissue dissociation kit (Miltenyi) + Myelin removal
EC, PC, PVF, VSMC.
Zhong et al., 2018; Parker et al., 2020
Human embryonic prefrontal cortex (9)
2309
Smart-seq2 None. Collagenase
IV + papain + DNase I
AC, EC, LC, MG, N, NPC, PC, OPC.4 Song et al.,
2020
Human (3) and mouse (3) brain microvessels
RNA-seq LCM of
microvessels (≤10 μm).
RNeasy microkit (Qiagen)
EC, PC.
Muhl et al., 2020
Mouse heart, skeletal muscle, colon, bladder (24)
61585
Smart-seq2 Pdgfra+, Acta2+, Pdgfrb+, Cspg4+.
Miltenyi + collagenase IV (Sigma)
FB, PC, VSMC.
Maë et al.,
2020 Mouse brain
(3) 1187
Smart-seq2 Cldn5+/Cspg4—. Neural tissue dissociation kit (Miltenyi) + Myelin removal
EC.
Eze et al., 2021
Human brain of foetus in 1st trimester (10)6
289,000 10X None. Papain
dissociation system (Worthington)
EC, MG, MES, N, PC, RG.
1. Total of cells that passed after qualtiy control analysis. 2. Nine brain regions were dissected: frontal cortex, striatum, hippocampus, thalamus, cerebelleum, posterior cortex, substantia nigra and ventral tegmental area, entopeduncular and subthalamic nucleus, and globus pallidus externus and nucleus basalis. 3. Brain: 3436 cells; lung: 1504 cells. 4.
Zhong et al. (2018) performed original sequencing, Parker et al. (2020) detected additional clusters in further analysis, i.e. EC, LC, and PC. 5. Heart: 1279 cells; skeletal muscle: 1754 cells; colon: 1646 cells; bladder: 1479 cells. 6. When it was identifiable at that developmental stage the following sections were dissected: telencephalon, diencephalon, midbrain, hindbrain, cortex, cerebellum, thalamus, and ganglionic eminences.