Brain Microvascular Pericytes in Vascular Cognitive Impairment and Dementia

1Pericytes are mural cells, embedded within the basement membrane, and surrounding microvessels as illustrated in Figures 1 and 2. These cells were originally described in late 19th century and initially named “pericytes” in 1923 by Zimmermann in accordance with their location enveloping the endothelium, and their being embedded in the basement membrane outside the microvessels. Although pericytes were considered to contribute to architectural maintenance and contraction of capillaries, little had been known about their multifunctional characteristics and roles in neurological disorders until late 20th century. In the last 20 years, however, using a combination of markers and advancing technologies, a variety of functions of pericytes in health and disease have been revealed. Especially, microvascular pericytes in the central nervous system (CNS) have come into focus as they contribute to the maintenance of blood–brain barrier (BBB), regulation of cerebral blood flow (CBF), and clearance of toxic waste products from the brain as well as other multifunctional properties.

2The brain constitutes ∼2% of the adult human body weight but receives ∼20% of the cardiac output through CNS vascular network. In the brain, small vessels can be largely classified as three different types by their size and constituent cell types: (1) arterioles, (2) capillaries, and (3) venules. There are gradual transitions between these vessel types; the transitions between arterioles and capillaries are called as pre-capillary arterioles while those between capillaries and venules are called as post-capillary venules.

3The arterioles branch from large arteries and follow the outer rim of the brain via the meninges. They penetrate perpendicularly into the cortex (penetrating arterioles), and upon entering the white matter, they begin to coil, loop, and spiral. Running through the brain parenchyma, the arterioles further split into smaller arterioles. As their diameters and constituent cell types are changed, the vessels make a transition to capillaries. The capillaries then increase their diameter again and transition into the post-capillary venules, which join to form collecting venules that collect into larger veins. In the small vessels (from the arterioles to venules), there are two types of mural cells separately located outside of endothelial layer: (1) vascular smooth muscle cells (SMCs) and (2) pericytes (Figure 1).

4Smooth muscle cells and pericytes express shared mural cell markers including neuron-glial antigen 2 (NG2, or transmembrane chondroitin sulfate proteoglycan; CSPG4) platelet derived growth factor receptor beta (PDGFRβ), alanyl aminopeptidase (ANPEP, or CD13), vimentin, regulator of G protein signaling 5 (RGS5). Other mural cell markers such as α-smooth muscle actin (αSMA, or actin alpha 2, smooth muscle; ACTA2), transgelin (or smooth muscle protein 22-α; SM22α), calponin1 (CNN1), desmin, and melanoma cell adhesion molecule (MCAM, or CD146) are expressed more in SMCs than pericytes. On the other hand, pericytes, but not SMCs, express ATP binding cassette subfamily C member 9 (ABCC9) and preferentially internalize or take up the fluoroNissl dye NeuroTrace 500/525 when applied to the brain surface. Most of the gene expression of pericytes, however, overlaps between SMCs and a certain subtype of pericytes. Furthermore, the expression of all these markers changes during growth and development, and may be up- or down-regulated in pathological conditions. Therefore, cell morphology and anatomical position should be taken into consideration to distinguish SMCs and pericytes. Table 1 provides anatomical differences in the cerebral small vessels and mural cell markers.

5The classification of small vessels is sometimes complicated and controversial because of the definition of constituent mural cells. Although there is a consensus that SMCs are located in arterioles and venules as well as larger arteries and veins, the classification and nomenclature of pericyte-surrounding vessels have been greatly debated mainly due to the heterogeneity of pericytes. Pericytes in the capillaries gradually transition to SMCs in the arterioles; drawing a clear line between those vessels is quite difficult. Originally, Zimmermann defined pericytes including their transition form to SMCs, residing on the three consecutive vessels, namely, (1) pre-capillary arterioles, (2) capillaries, and (3) post-capillary venules. Zimmermann therefore differently named the pericytes on each vessel: (1) pre-capillary pericytes, at the last arterial ends that merge into the capillary system; (2) capillary pericytes, at the capillaries in the narrowest sense; and (3) post-capillary pericytes, on post-capillary venules up to veins showing regular, fusiform smooth muscle fibers.

6As techniques such as three-dimensional live imaging have been developed, the branching order coming off penetrating arterioles has also been taken into consideration to define the vessels in rodent brains. The definition of the vessels, however, has varied depending on the studies. While some studies have defined all vessels including proximal and distal branches coming off penetrating arterioles as capillaries, others have defined proximal branches as pre-capillary arterioles. To make matters more complicated,, have asserted that the mural cells on the proximal branches coming off arterioles should be called as SMCs, which have provided confusion in the field with the result that different members of the field use different terminologies and definitions about pericytes and pericyte-residing vessels. Nowadays, to avoid confusion, the researchers have claimed that the capillaries should include transition to the arterioles, and the mural cells on those capillaries should be called as “pericytes”. In this review, we will describe the differences of mural cells in the small vessels, namely, (1) SMCs on the arterioles and venules and (2) pericytes on the capillaries and post-capillary venules, introducing the different terminology of pericytes. Thereafter, we will focus on the pericyte function and dysfunction in health and diseases.

7In the arterioles, SMCs continuously enwrap the abluminal side of endothelial cell layer and make myoendothelial gap junction. The SMCs in the arterioles have an inconspicuous soma and extend broad processes that strongly express αSMA transgelin, desmin, CD146, and CNN1 as well as shared mural cell markers including NG2, PDGFRβ, CD13, vimentin, and RGS5. Outside of the SMCs is perivascular space with fibroblast-like cells, surrounded by collagen layer and endfeet of astrocytes.

8In the capillaries, the vessel size further decreases, and the endothelial layer is intermittently surrounded by pericytes. Compared to the peripheral vascular beds, CNS capillaries have higher pericyte-to-endothelial cell ratios (1:1 to 1:3) and around 70–80% of the capillary surface area is covered with pericyte cell processes. Capillary pericytes have a conspicuous protruding ovoid cell body with long thin processes that course along the capillary for longer distances and are embedded within the basement membrane.

9The role of capillary pericytes in CBF control has long been debated. Using mouse models, some researchers have reported that arteriolar SMCs but not capillary pericytes regulate CBF in response to neuronal activities or ischemic stress. Others, however, have shown that capillary pericytes also change the vessel diameter and CBF by stimuli, neuronal activation, or ischemia, suggesting capillary pericytes also contribute to CBF regulation.

10Because pericytes are morphologically and functionally heterogeneous, pericytes are sometimes subclassified according to their topology, morphology, and the protein expression levels.

11The studies of mouse brain cortices using two-photon microscopy have revealed the morphological and functional distinction of the pericytes on the proximal branches (mostly up to 2nd or 4th order) coming off penetrating arterioles from those on the higher branch-order capillaries or larger arterioles. The pericytes located in this point of transition possess highly visible and protruding ovoid soma with thin and circumferential processes enveloping the vessels. These cells express more αSMA than pericytes in mid-capillaries, but not as much as SMCs in the penetrating arterioles. Aside from αSMA, the cells also express desmin and transgelin, but hardly express CNN1.

12Because these pericytes are positioned at the transition between arterioles and capillaries, and have shared some characteristics with SMCs, the terminology and classification of these mural cells have been hotly debated. The cells have been variably called as ensheathing pericytes, transition/transitional pericytes, pre-capillary pericytes, simply ‘pericytes’ or ‘capillary pericytes’, smooth muscle-pericyte hybrids, arteriole SMC (aaSMC), or pre-capillary SMCs.

13At the same time, the vessels of proximal branches coming off penetrating arterioles are also differently defined as pre-capillary arterioles, post-arteriole capillaries or a part of capillaries.

14The cells in this proximal branches (especially, 1st to 2nd branches) coming off arterioles has drawn attention as they highly contribute to neurovascular coupling (NVC) and no-reflow phenomenon after acute ischemia. Although referring to these mural cells as a subtype of “pericytes” has become almost a consensus, the shifting nomenclature of these cells and vessels has been the root of recent controversies on pericyte roles as regulators of CBF (discussed below).

15In the mouse brain, pericytes in the mid-capillaries are divided into two subtypes according to the morphology of their processes, namely, (1) mesh pericyte and (2) thin-strand pericyte or helical pericyte. The mesh pericytes adopt a mesh-like appearance and are located on the proximal side of a capillary with higher coverage area than thin-strand pericytes. The thin-strand pericytes or helical pericytes extend thin, meandering processes that run along the vessel lumen. These two types of pericytes express NG2, CD13, and PDGFRβ, and slightly express CD146 and αSMA, but hardly express desmin, transgelin, nor CNN1. Instead, pericytes express ABCC9 and preferentially take up the fluoroNissl dye NeuroTrace 500/525. The pericytes in these capillaries play vital roles for BBB maintenance and small molecule transport.

16In the post-capillary venules, different shaped mesh pericytes, also called stellate/stellate-shaped pericytes, surround the endothelial layer. These cells have many slender and shorter branching processes than capillary pericytes. They express αSMA, ABCC9, cysteine sulfinic acid decarboxylase (P-selectin), and endomucin. The expression level of αSMA is lower than the mural cells in the arterioles and proximal capillaries. The pericytes in the post-capillary venules are thought to regulate immune cell entry to the brain parenchyma like those in other tissues. Outside of the pericytes are astroglial end-feet forming glia limitans. Between the endothelial basement membrane and astrocytic basement membrane is perivascular space, where antigen presenting cells reside. Fibroblast-like cells are within the astrocytic basement membrane.

17Post-capillary venules are collected to form ascending venules. In the venules, the endothelial cell layer is surrounded by stellate-shaped SMCs with broad leaf-like processes. Venous SMCs express NG2, CD13, PDGFRβ, αSMA, transgelin, ABCC9, but not CNN1. And the expression level of αSMA and transgelin in the venous SMCs is lower than the mural cells in the arterioles. SMCs in brain venules express NG2, which is different from the venules of peripheral tissues. Outside of SMCs are perivascular space and fibroblast-like cells.

18The CNS vascular system possess a highly selective semipermeable border formed by the BBB wherein tight junctions and adherens junctions created by endothelial cells strictly regulate the movement of ions, molecules, and circulating cells between the blood and the brain. The tight and adherens junctions are controlled by various types of cells surrounding the endothelium, such as pericytes, astrocytes, perivascular oligodendrocyte precursor cells (OPCs), interneurons, perivascular macrophages, microglia, and other immune cells (Figure 2).

19Capillary pericytes play especially crucial roles in the function of the BBB. Pericyte ablation leads to breakdown of the BBB in the mouse brain. Pericytes control protein expression in the tight junctions, their alignment with endothelial cells, and the bulk-flow transcytosis of fluid-filled vesicles across the BBB.

20Pericytes also play a key role in the generation of new blood vessels. During angiogenesis, a complex web of bidirectional signaling pathways between endothelial cells and pericytes is essential for forming and stabilizing new blood vessels. The signaling molecules involved in these processes include platelet-derived growth factor B (PDGFB)/PDGF receptor beta (PDGFRβ), transforming growth factor beta (TGFβ), Notch, vascular endothelial growth factor (VEGF), sphingosine-1 phosphate (S1P)/S1P receptor 1 (S1PR1 or EDG), and angiopoietin 1 and 2 (ANGPT1, ANGPT2)/TEK receptor tyrosine kinase (TEK, or TIE2) all of which differentially contribute to these signaling activities. During angiogenesis, pericytes are reported to be recruited from the bone marrow as well as brain parenchyma in response to the PDGF-BB secreted from endothelial cells. On the other hand, pericytes induce endothelial cell sprouting and stabilization via secreting TGFβ, VEGF, and ANGPT1. Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis including microaneurysm. When a single brain pericyte is ablated, the processes from neighboring pericytes are extended to contact uncovered regions of the endothelial cells and maintain the vessel diameter and vessel stability.

21Cerebral blood flow is dynamically altered in response to changes of transient neuronal activity, which is referred to as NVC. It is controlled by the cells within the neurovascular unit (NVU) including endothelial cells, pericytes, SMCs, astrocytes, OPCs, and neurons. In response to the different neurotransmitters, pericytes dilate capillaries and increase local CBF. In pathological conditions such as ischemic stroke and AD, brain capillaries are constricted by pericytes. In ischemic stroke mouse brains, damaged and dead pericytes squeeze the capillaries and sustain the reduction of CBF even after recanalization of the larger vessels, causing the no-reflow phenomenon. The burden of amyloid beta (Aβ) oligomer causes pericyte contraction and capillary stenosis, which decreases CBF in the AD brains.

22The role of pericytes in regulation of vessel diameter has been heatedly debated. When Rouget first describe the branched cells on the capillary wall, which is nowadays called as pericytes, he regarded them as contractile cells. Thereafter, the studies which supported or objected to the pericyte contractility were successively reported. In terms of CBF regulation, SMCs located at arterioles were traditionally thought to control CBF. This view of CBF dynamics was revolutionized by the findings that capillary diameter also changes with neural activity and ischemia. Furthermore, the loss of pericytes has been reported to lead to diminishing CBF in response to functional hyperemia in pericytes-deficient mice. However, refuted that pericytes are involved in the regulation of CBF, and put forward the view that arteriolar SMCs may be the key players regulating CBF. also showed pre-capillary and penetrating arterioles, but not pericyte in capillaries are responsible for the CBF increase induced by neural activity. These controversial reports most likely stem from the different definitions of pericytes in the proximal capillaries. Some of these reports concur that the mural cells in the proximal branches coming off penetrating arterioles respond to the stimulations outlined above and change vessel diameters accordingly. Pericytes residing at the proximal capillaries possess both characteristics of pericytes and SMCs, which may lead to discrepant interpretations by different investigators. Some groups showed stimulation-evoked increases in synaptic activity and capillary dilation starting mostly at the first- or second-order capillary then propagating along arterioles and downstream capillaries, which may position the pericytes in the proximal capillaries as the major regulators of CBF. In addition, reported that a pre-capillary sphincter, at the junction between the penetrating arteriole and first order branch, modulated capillary flow while protecting the downstream capillary bed from adverse pressure fluctuations. Taken together, the proximal branches coming off arterioles seem to be the gatekeeper that controls CBF in the capillary beds.

23Pericytes internalize small molecules and neurotoxic blood-derived products which enter the breached BBB (i.e., immunoglobulins, fibrin and albumin) through receptor-mediated endocytosis or non-specific pinocytosis. Pericytes also internalize large solid substance through phagocytosis. Engulfed molecules are transported to lysosomes for enzymatic degradation or possibly transported to the blood circulation. While tumor necrosis factor alpha (TNFα) and interferon-γ (IFNγ) enhance phagocytic uptake, TGFB1 attenuates phagocytic uptake in pericytes.

24Pericytes may clear substances derived from the brain parenchyma as well as around vessels. Pericyte loss aggravates Aβ deposition in transgenic mice. Aβ clearance by pericytes is mainly performed through receptor-mediated endocytic pathways, especially low-density lipoprotein receptor-related protein 1 (LRP-1).

25Another clearance system which might be related to pericytes is the CNS lymphatic drainage system. In the CNS, there are two major extracellular fluids, namely, (1) cerebrospinal fluid (CSF) and (2) interstitial fluid (ISF). CSF drains to cervical lymph nodes via the cribriform plate and nasal lymphatics, as well as via dural lymphatics and along cranial nerves. ISF containing metabolic products of the brain as well as Aβ and tau drains to lymph nodes by the shared or distinct pathways from CSF. Two different pathways for draining ISF to the periphery — the perivascular and paravascular pathways — are controversially proposed. In the perivascular pathway, ISF and solutes from CNS parenchyma enter the basement membranes of capillaries, where pericytes are embedded, and drain directly via tunica media of arterioles and arteries out of the brain to cervical lymph nodes. Paravascular pathway, also known as glymphatic system, denotes the moving of CSF into the brain along arterial perivascular spaces and successively into the interstitium to mix with ISF, which then guides flow toward the venous perivascular spaces, removing metabolic waste of ISF to the CSF via convective bulk flow. In the glymphatic system, astrocytes are important for ion buffering and fluid exchange between the CSF and ISF. The exchange through the glymphatic system is suggested to be dependent on the water channel aquaporin-4 (AQP4) located in astrocytic endfeet. However, it remains enigmatic whether or not AQP4 is solely responsible for this fluid transport or not. Recently, insufficient PDGFB signaling in the Pdgfbret/ret mice has shown decreased pericyte coverage of the vessels with decreased AQP4 polarization to astrocyte endfeet, which impairs maturation of the glymphatic function. The focal absence of pericytes correlates with relocation of AQP4 from astrocytic endfeet to the soma of astrocytes. Pericytes express laminin-α2 (LAMA2), laminin-β1, and laminin-γ1, which encode the subunits of laminin 211. Laminin 211 deposits in the vascular basement membrane and interacts with dystrophin in astrocytes, which acts as a molecular bridge to AQP4 to keep it in the astrocyte endfeet. Indeed, Lama2 knockout in mice results in BBB abnormalities in association with loss of AQP4 polarization to astrocyte endfeet. The above referenced reports suggest that pericytes might influence the development of the glymphatic system through deposition of laminin 211 in the vascular basement membrane, which maintains the polarization of AQP4 at astrocytic endfeet. However, there are critical assessments of the proposed glymphatic system. Several observations or simulations do not support the glymphatic mechanism nor convective fluid flow of CSF. Hence, the existence of the paravascular pathway as a CNS drainage system is still under debate.

26Brain pericytes have many properties of immune regulating cells such as (1) responding to and expressing pro-inflammatory and anti-inflammatory molecules, (2) regulating leukocyte extravasation and trafficking, and (3) controlling immune cell activation including T cells, macrophages, and microglia. In the mouse brain, pericytes function as the initial sensor of systemic inflammation and relay the infection signal to neurons by secreting chemokine CC chemokine ligand 2 (CCL2, also known as monocyte chemotactic protein-1, MCP1).

27Pericytes express and release several mediator molecules that enhance leukocyte extravasation. Although the endothelial cells are well known to induce leukocyte crawling and extravasation, pericytes also contribute to leukocyte transmigration. In vivo observation of mouse skin vessels have demonstrated that leukocyte extravasation occur only post-capillary venular pericytes. After inflammation stimuli, neutrophils exhibited transendothelial migration (TEM) and sub-endothelial cell crawling along pericyte processes, which was supported by pericyte-derived intercellular adhesion molecule-1 (ICAM-1) and its leukocyte integrin ligands, macrophage-1 antigen (Mac-1) and lymphocyte function–associated antigen-1 (LFA-1). Then, the leukocytes transmigrated to the interstitium through the gaps between adjacent pericytes. After extravasation, the leukocytes interact with capillary pericytes as well. Pericyte-monocyte interaction is mediated mainly by macrophage migration-inhibitory factor (MIF) and CCL2, whereas neutrophil migration involves MIF and C-X3-C motif chemokine ligand 1 (CXCL8, also known as interleukin 8, IL8).

28Exposure of pericytes to cytokines such as interleukin 1 beta (IL1β) and TNFα triggers the release of inflammatory molecules and matrix metalloprotease 9 (MMP9), leading to BBB breakdown in vitro. The immunomodulatory factors secreted by pericytes including IL1β, TNFα, IFNγ, and interleukin 6 (IL6) induce a proinflammatory state in astrocytes, microglia, and endothelial cells, and cause apoptotic neuronal death.

29Conversely, pericytes can also secrete several anti-inflammatory substances such as interleukin 33 (IL33) and C-X3-C motif chemokine ligand 1 (CX3CL1), both of which are shown to promote anti-inflammatory microglial phenotype in mouse models. Furthermore, depletion of pericytes induced inflammatory responses in endothelial cells and perivascular infiltration of macrophages in mouse retinal vessels, suggesting pericytes exerts an anti-inflammatory effect on endothelial cells under normal conditions.

30Pericytes display some similarities to mesenchymal stem cells. Responding to the stimuli and environmental changes, pericytes may transform into multipotent stem cells and differentiate into various cells including neural, vascular, and glial cells. Pericytes extracted from ischemic mouse brain and human brain pericytes under oxygen-glucose deprivation states develop stem properties in vitro. Pericytes under ischemic condition in vivo and in vitro are also reported to acquire a microglial phenotype corresponding with increased phagocytic property.

31These phenotype changes of pericytes under stimulation can be beneficial for the compensatory remodeling after brain injury and ischemia, rapid response to infection and inflammation, and clearing compromised cells or neurotoxic substances breaching an impaired BBB. However, no multipotency of pericytes in aging and injury in vivo has been reported, challenging the current view of pericytes as tissue-resident multipotent progenitors.

32Central nervous system injury evokes the recruitment of astroglia and scar formation. Pericytes and OPCs as well as astrocytes are observed within glial scars. After spinal injury or ischemic stroke, pericytes proliferate and migrate to the injured region and form a glial scar. Extracellular matrix proteins, such as periostin have shown to be expressed in the extracellular space of the injury region, which induces pericyte proliferation and leads to scar formation. The glial scar around the injury site forms a barrier between the injured and the non-injured tissue to prevent further neuronal loss, which eventually hinders the axonal regeneration in the scarred area. Recent evidence has demonstrated that the glial scar can also promote CNS regeneration after injury, suggesting a dual function. The complexity and heterogeneity of the glial scar derived from different cell types (i.e., astrocytes, pericytes, and OPCs) at various phases in CNS diseases remains to be elucidated.

33Pericytes and endothelial cells are connected to a shared basement membrane by several types of integrin molecules. In areas lacking the basement membrane, interdigitations of pericytes and endothelial cell membranes, called peg and socket contacts, form direct connections by N-cadherin and connexin 43. The crosstalk between pericytes and endothelial cells is indispensable for angiogenesis, vascular stability, and BBB formation. For CNS pericytes and endothelial cells, PDGFB/PDGFRβ, TGFβ, Notch, VEGF, and S1P/S1PR1 signaling events are well investigated. ANGPT signaling is investigated in the retinal endothelial cells and pericytes.

34In the angiogenesis of the mouse brain, PDGF-BB secreted by endothelial cells recruits PDGFRβ-positive pericytes and progenitor cells. PDGFB signaling also stimulates pericyte proliferation, and sustained PDGF-BB–PDGFRβ signaling in the adult CNS is required for pericyte cell survival.

35TGFβ signaling is vital for microvessel stability affecting both endothelial cells and pericytes. Endothelially secreted TGFβ regulates differentiation of pericyte progenitors and induces pericyte contractile protein expression and extracellular matrix production and facilitates proper pericyte attachment in coordination with Notch signaling. Pericyte-derived TGFβ contributes to endothelial maturation through SMAD signaling.

36Vascular endothelial growth factor produced by pericytes and endothelial cells also shows reciprocal interaction. Pericyte-derived VEGF in the mouse brain promotes endothelial sprouting and cell survival. VEGF treatment enhances pericyte coverage of brain endothelial cells with increased N-cadherin production. VEGF induces proliferation and migration of pericytes as well as endothelial cell stabilization.

37S1P is originally described as secreted by endothelial cells. Its receptor, S1PR1 is expressed in mural cells including pericytes. S1P secreted by endothelial cells is essential for pericytes coverage in the mouse brain and stabilizes endothelial/pericyte cell adhesion through N-cadherin and maintains the BBB. Human pericytes secrete S1P, which induces the expression of adhesion proteins in human retinal endothelial cells in vitro.

38ANGPT1 and ANGPT2 differently contribute to angiogenesis. The ANGPT1 is mainly expressed in pericytes and ANGPT2 is mainly expressed in endothelial cells. The ligand of ANGPT, TEK is mainly expressed in endothelial cells. Pericyte-derived ANGPT1 activates endothelial TEK and promotes endothelial survival. TEK is also expressed at lower levels by pericytes and its downstream signaling in pericytes is essential for angiogenesis. In angiogenesis, ANGPT2 was thought to antagonize ANGPT1, but later was found to act as both agonist/antagonist of TEK signaling in the endothelium. ANGPT2 expressed by mouse endothelial cells leads to the dissociation of TEK expressing pericytes from vessels, which initiates endothelial cell sprouting.

39Crosstalk between pericytes and endothelial cells is also mediated by circular RNA. Diabetes-related stress up-regulates a circular RNA, cPWWP2A (PWWP domain containing 2A) expression in pericytes, which inhibit microRNA-579 and regulate vascular integrity.

40The crosstalk between pericytes and astrocytes contributes to BBB maintenance, NVC, and white matter attenuation under chronic hypoperfusion.

41Pericytes facilitate the attachment of astrocyte endfeet to the BBB and pericyte-deficient mice lose AQP4 in the endfeet of astrocytes. On the other hand, astrocytes control pericyte migration, differentiation, and the juxtaposition of pericytes to endothelial cells. Astrocyte-derived apolipoproteins differently regulate cyclophilin A (CypA) signaling in pericytes, which controls BBB integrity. In NVC, astrocytic calcium signaling mediates capillary dilation via pericytes.

42Oligodendrocyte precursor cells (OPCs) have recently emerged as one of the contributors to BBB. According to their regional differences, OPCs can be divided into two subtypes, namely, perivascular OPCs and parenchymal OPCs. In the human and mouse brain, perivascular OPCs are attached to cerebral endothelial cells and pericytes through basal lamina, and thereby are thought to become novel components of the BBB. OPC-specific TGFβ1 depleted mice exhibited cerebral hemorrhage and loss of BBB function, showing the role of OPCs for BBB maintenance through TGFβ1 signaling. In vitro experiments have shown that OPC-derived factors increase pericyte proliferation whereas pericyte-derived factors support OPC self-renewal and differentiation. In the developing mouse forebrain, pericyte-derived TGFβ family proteins contribute to the migration and distribution of OPCs in brain parenchyma, while perivascular OPC migration to the vessels in the developing CNS requires interaction with endothelium but not pericytes.

43In the adult mouse brain, pericytes respond to toxin-induced demyelination in the brain and stimulate OPC differentiation during remyelination through Lama2. Pericyte-derived Lama2 also instructs neuronal stem cells to an oligodendrocyte fate.

44A recent report has shown that odor triggers rapid Ca2+ elevations in OPC processes before pericytes and SMCs dilate the vessels responding to synaptic activation, suggesting possible relationship between OPCs and pericytes in the NVC.

45Microglia have been regarded as the main executor of inflammation after acute and chronic CNS disorders. The interaction between microglia and vascular cells – including pericytes – has important roles for vascular inflammation, angiogenesis, and BBB integrity. Although endothelial cells are thought to be the main source of cytokines and chemokines which trigger microglial activation upon vascular inflammation, pericytes are also known to be key mediators in this process. In response to TNFα, rat brain pericytes in vitro produce IL6 and macrophage inflammatory protein 1 (MIP1), which trigger microglial activation. Activated microglia disrupt the BBB, which triggers angiogenesis. In the mouse brain, pericytes initially respond to the systemic inflammation within 2 h and secrete CCL2 before the response of astrocytes or microglia. Given that CCL2 is also known to activate microglia and microglial process motility dynamics are altered 48 h after systemic infection, pericytes might modulate microglial process motility and physical dynamics around the vessels in response to infection. Furthermore, pericytes themselves acquire a microglial phenotype after ischemic stroke as mentioned above. Conversely, pericytes also secrete several anti-inflammatory substances such as IL33 and CX3CL1, both of which has shown promote anti-inflammatory microglial phenotype in mouse models.

46In the human and mouse brain, perivascular macrophages lie under the basement membrane alongside pericytes. Perivascular macrophages maintain tight junctions between endothelial cells and limit vessel permeability, phagocytose potential pathogens before they enter tissues from the blood and restrict inappropriate inflammation. Although pericytes and perivascular macrophages are localized close to each other and possess shared functions including regulation of vascular permeability and phagocytosis, little is known about how pericytes interact with perivascular macrophages in the vascular niche.

47Blood–brain barrier breakdown and microvessel dysfunction has been observed in various CNS disorders such as small vessel disease (SVD), ischemic acute stroke, intracerebral hemorrhage, Alzheimer’s disease (AD), traumatic brain injury (TBI)/chronic traumatic encephalopathy (CTE), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Lewy body diseases (LBD), and epilepsy. In particular, pericyte dysfunction is thought to be a critical factor for aggravating dementing diseases such as vascular cognitive impairment/dementia and AD.

48Cognitive impairment/dementia related to vascular pathology is classified according to the causative vessel size, that is, ‘small vessel’ disease and ‘large vessel’ disease, although their crosstalk would be essential for the pathogenesis of both disorders. Cerebral SVD contributes to a wide range of pathological processes, which affect the small vessels including small arteries, arterioles, venules, and capillaries in the brain. In contrast, large vessel disease in the brain may result in stroke and hemorrhage, which affect various type of arteries. Table 2 provides the roles of CNS pericytes in health and disease focusing on cerebrovascular diseases and AD.

49SVD is characterized by pathological changes in the small vessels with a diameter < 100 μm, with concentric smooth muscle thickening in arterioles, as well as pericyte degeneration, basal membrane thickening, endothelial, and astrocyte end-feet swelling in capillaries. The slowly progressive worsening of microcirculatory structure and function results in white matter changes, which can be detected by magnetic resonance imaging (MRI).

50Small vessel disease is commonly known to be co-morbid brain pathology in a wide range of neurodegenerative diseases including CTE, MS, LBD, and diverse tauopathies including AD. In sporadic SVD, pericytes play pivotal roles as they reside in the small vessels and contribute to maintenance of the BBB, vascular integrity, inflammation, and angiogenesis. Chronic hypoperfusion in the rodent brain results in degeneration of pericytes and decreased pericyte coverage in brain blood vessels, and increased BBB permeability followed by white matter attenuation. Pericyte-deficient mice also cause circulatory failure in the brain which can trigger white matter functional deficits and neuronal loss.

51A leaky BBB allows for the extravasation of toxic-blood derived products such as fibrinogen, which accumulates around the vasculature as insoluble fibrin. Fibrinogen/fibrin infiltration results in clustering and activation of macrophages and microglia as well as chemokine- and antigen presentation-mediated recruitment and activation of T cells, causing axonal degeneration. Pericytes produce numerous pro-inflammatory mediators including reactive oxygen/nitrogen species (ROS/RNS), which induces neurons to undergo stress-induced apoptosis. This pro-inflammatory status in the vessels induces leukocyte adhesion and microglial activation. Under chronic hypoperfusion, bone morphogenetic protein 4 (BMP4) expression is increased by pericytes, which induces astrogliogenesis and aggravates white matter attenuation.

52Chronic hypoperfusion induces compensatory angiogenesis by increasing the expression of angiogenetic factors such as VEGF, ANGPT1/2, and MMP9. VEGF and ANGPT1 promote sprouting and proliferation of endothelial cells, and recruitment of pericytes. MMP9 regulates the detachment of pericytes from vessels thereby triggering angiogenesis.

53The importance of pericytes in SVD may be emphasized by the fact that one of the most common inherited cerebral SVD, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), shows aggregation of mutant Notch3 protein around capillary pericytes as well as arteriolar SMCs. Pericytes express Notch3 and are first affected by Notch3 aggregation in Notch3R169C mice, suggesting pericytes might be a main contributor in the pathogenesis of CADASIL. A recent study, however, has shown no change in pericyte coverage in the white matter lesion of CADASIL patients nor Notch3R169C mice, arguing against the prevailing hypothesis that pericyte loss is the primary driver of white matter lesions. The jury is still out on the contribution of pericytes to the white matter damage.

54Residing in the microvessels, pericytes have a great influence on the condition of the brain following acute ischemic stroke caused by thrombosis or embolism affecting larger vessels. The biological roles of pericytes, such as regulation of CBF, BBB maintenance, inflammation and immunological properties, angiogenesis, and scar formation are all involved in the status of the ischemic brain.

55During arterial obstruction, pericytes positioned on the proximal capillaries constrict the vessels and impede capillary blood flow, which lasts even after arterial recanalization, developing a no-reflow phenomenon. The debate about whether these cells are pericytes or SMCs was discussed above.

56During stroke, BBB permeability is increased, and sustained ischemia leads to increased BBB disruption. Severe BBB disruption during stroke increases the risk of hemorrhage in patients treated with intravenous tissue-type plasminogen activator. During ischemia, as in SVD, ROS production and MMP9 up-regulation in pericytes contribute to BBB breakdown. An enzymatic source of ROS production, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), is highly up-regulated by pericytes in the peri-infarct region of the mouse brain subjected to middle cerebral artery occlusion (MCAO), and overexpression of NOX4 in pericytes induces BBB breakdown by up-regulating MMP9. Pericytes also directly release MMP9 during ischemia, which interrupts the tight junctions between endothelial cells and the binding of astrocyte endfeet to the vascular wall. VEGF up-regulation by pericytes under ischemic conditions has also been reported to disrupt the BBB in vitro and in vivo, and this triggers further angiogenesis. However, a report has shown that prolonged exposure to VEGF enhances post-ischemic BBB integrity and reduces infarct volume in mice subjected to transient MCAO. Thus, VEGF might have a pluripotent role in BBB integrity according to the dosage and timing of its release.

57Pericytes might also play a role in regulating ischemia-induced leukocyte infiltration as pericytes express cell surface adhesion molecules and induce leukocyte transmigration in response to inflammatory mediators. Pericytes express ICAM-1, which guide leukocyte migration through gaps between pericytes by interacting with the integrin ligands on leukocytes.

58Aside from these detrimental roles, pericytes play a beneficial role in ischemic stroke via promoting angiogenesis and scar formation. During MCAO-inducing ischemia in the mouse brains, pericytes are recruited from the periphery as well as parenchyma, and are involved in angiogenesis and blood vessel stabilization. Pericyte migration to the infarcted area forming the core of the scar, which is distinct from the astroglial scar surrounding the core. Consistent with that, Pdgfrb+ ⁣/− mice demonstrated decreased fibrosis in the ischemic area and enlarged infarct volume. Although glial scar formation is beneficial to prevent toxic substances from spreading, it should be noted that excessive or long-lasting glial scar formation inhibits axonal regeneration and stalls the recovery process. Furthermore, detachment of pericytes from capillaries allows them to migrate toward ischemic region thereby causing further leakage of the BBB. The same is true of angiogenesis. While angiogenesis increases the blood supply to the peri-infarct area, insufficient angiogenesis results in leaky blood vessels leading to brain hemorrhage.

59AD is the most prevailing dementia among the elderly and is defined pathologically by the presence of Aβ accumulation in brain parenchyma as Aβ plaque and aggregation of hyperphosphorylated tau as neurofibrillary tangles as well as neuritic plaques and neuropil threads. Aβ also accumulate in the vessels as cerebral amyloid angiopathy (CAA). Recently, it has been increasingly recognized that the decreased CBF and white matter attenuation associated with BBB breakdown correlates with the accumulation of AD pathology, and contributes to the onset and progression of dementia. CBF reduction, BBB breakdown in the hippocampus, and an increase in PDGFRβ level in the CSF occur even in the very early stages of cognitive impairment as well as later stages of AD. In AD patient brains, microvessels are frequently narrowed and irregular in diameter especially in the vicinity of the senile plaques, which is accompanied by decreased capillary bed densities. Some vessels in these area are collapsed with lacking endothelial cells, and do not carry blood flow, called string vessels. In the mouse brain, infusion of Aβ caused endothelin-1 (ET1) upregulation in cerebral vasculature through receptor for advanced glycation end products (RAGE), which contributes to Aβ-induced CBF reduction. CBF reduction accompanied by increased vascular RAGE and ET1 is also observed in Tg2576 mice, which is ameliorated by blocking Aβ and RAGE binding. A recent study has shown that capillaries in the AD brains are constricted by pericytes, which causes a decrease in CBF. In rat brains, Aβ oligomer-induced ROS triggers the release of ET1 to stimulate pericytes contraction and CBF reduction. AD patients also show a decrease in pericyte coverage with an increase in extravascular immunoglobulin G and fibrin deposition. The apolipoprotein E4 genotype, which is a major genetic risk factor for late-onset AD, leads to pericyte loss and enhances CypA-MMP9 pathway of BBB degradation. Pericytes express LRP1 and other Aβ-binding receptors such as the low density lipoprotein receptor (LDLR), RAGE, and CD36 in brains with AD pathology including CAA. Aβ accumulation in pericytes is observed in human AD brains and in the brains of APPsw/0 mice. At the ultrastructural level of AD brains, pericytes are disorganized and exhibit mitochondrial abnormalities, pinocytotic vesicles, and accumulation of osmophilic material.

60Loss of BBB integrity caused by pericyte deterioration may induce an influx of immune cells into the brain, driving inflammation, and CBF stagnation and thereby impairing Aβ clearance, all of which aggravate AD pathology. Indeed, both the depletion of pericytes in the APPsw/0 mice (APPsw/0; Pdfgfb+ ⁣/−) and chronic cerebral hypoperfusion in the APPSwInd Tg mice aggravate AD pathology such as increasing Aβ deposition and tau phosphorylation followed by neuronal loss. Notably, a high-fat diet, which leads to vascular related diseases, exacerbates AD pathology accompanied by pericyte dysfunction in the APPsw/PS1 mice.

61Although Aβ is toxic to the pericytes, pericytes basically take an active part in the clearance of Aβ by phagocytosis and translocation through BBB. Pericytes clear Aβ aggregates via an LRP1/ApoE isoform-specific mechanisms, suggesting a potential therapeutic target for controlling Aβ clearance in AD.

62Since pericytes have multifunctional properties and contribute to the various neurological disorders, pericytes as a therapeutic target, can be approached from various aspects: (1) prevention of BBB dysfunction, (2) promoting angiogenesis and vascular stability, (3) reduction of pericyte constriction under pathological condition, (4) up-regulation of Aβ clearance, (5) control of inflammation, (6) implantation therapy through multipotential stem cell properties, and (7) regulation of proper scar formation.

63As the BBB tightly restricts the passage of substances into the CNS, it is challenging to deliver drugs from blood circulation into the brain. Therefore, the delivery methods which enable the drugs to pass through BBB may be beneficial as exemplified by encapsulating drugs in liposomes or nanoparticles.

64Loss of pericytes and BBB dysfunction are common in a variety of neurological disorders including cerebrovascular diseases and AD. Thus, promoting the interaction of pericytes and endothelial cells by regulating PDGFB/PDGFRβ, TGFβ, Notch, ANGPT/TEK, and VEGF signaling could be therapeutic by preventing BBB dysfunction and facilitating proper angiogenesis and vascular stability. For instance, increasing PDGF-BB in the endothelial cells and/or PDGFRβ in the pericytes could boost pericyte proliferation and migration to microvessels while increasing TGFβ signaling could promote pericyte proliferation and attachment to the vessels. Indeed, administration of TGFβ showed increased BBB formation under ischemic condition in vitro and in vivo. VEGF treatment was also shown to enhance post-ischemic BBB integrity and reduce infarct volume in rodent models of MCAO. Recently, regulating RNA for maintaining the BBB has been investigated and overexpression of cPWWP2A or silencing microRNA-579 expression promoted pericyte-endothelial cell crosstalk and microvascular stability. Administration of microRNA-149-5p attenuated BBB permeability and improved the outcomes of rat subjected with transient MCAO.

65Reduction of ROS and MMP9 should also prevent pericyte-mediated BBB breakdown. A recent study observed that MMP9 inhibitors reduced pericyte-associated BBB leakage. A free radical scavenger edaravone has been sown to ameliorate brain damage after ischemia via pericyte-mediated angiogenesis and vessel stability. Further, cilostazol – a phosphodiesterase 3 inhibitor – promoted angiogenesis through pericyte proliferation with inhibition of the MMP9, which maintained vascular integrity in spontaneously hypertensive stroke prone (SHR-SP) rat while cilostazol also ameliorated cerebral hemorrhage in mice by protecting the BBB, suggesting cilostazol has additional effects of vascular stability aside from antithrombosis.

66Since the no-reflow phenomenon hampers the tissue recovery after recanalization in the arteries, researchers have tried to find clues to prevent capillary constriction by pericytes after ischemia. These ischemia-induced pericyte contraction have shown to be relieved by suppressing ROS/RNS, removal of external Ca2+, or administration of adenosine and sodium nitroprusside. Capillary constriction by pericytes and CBF reduction are also observed in AD brains, which may aggravate cognitive decline. Aβ infusion into mouse brain causes RAGE-ET1 mediated CBF reduction, similar to the CBF reduction observed in aged Tg2576 mice. Furthermore, a RAGE-specific inhibitor recovered CBF and lowered the Aβ burden in APPsw/0 mice. Aβ oligomers induce pericyte constriction by ROS mediated ET1 release. This Aβ-evoked constriction was reversed by applying the vasodilator C-type natriuretic peptide and could be halted by blocking NOX4 or ET1 receptors, suggesting potential therapeutic target for CBF reduction in AD.

67Because pericytes take up Aβ, then degrade or excrete it into the circulation, boosting pericyte function could be therapeutic target for Aβ clearance in AD. Consistent with that, pericyte loss in the APPsw/0 mice showed increased Aβ accumulation and tau phosphorylation. Pericytes internalize and clear aggregated Aβ by LRP1-dependent ApoE isoform-specific mechanism, highlighting up-regulation of LRP1 as a therapeutic target for Aβ clearance.

68Neuroinflammation is present in almost all neurological diseases. Pericytes release both pro-inflammatory and anti-inflammatory mediators and regulate recruitment of immune cells from the blood to the brain parenchyma. Although inflammation may have some positive aspects such as immunoprotection against pathogens, clearance of toxic substances, and support of angiogenesis, excessive inflammation causes BBB leakage, tissue damages and neuronal loss.

69Targeting ROS-mediated inflammation is a feasible therapeutic target in terms of suppressing release of pro-inflammatory cytokines by pericytes with preventing pericyte loss and BBB dysfunction. A free radical scavenger edaravone has been sown to ameliorate brain damage after ischemia. Targeting receptor or downstream signaling stimulated by pericyte-secreted cytokines may also be potential therapeutic target. Blocking pericyte-derived BMP4 by its receptor antagonist noggin treatment was shown to suppress astrogliogenesis and alleviate white matter damage resulting from chronic cerebral hypoperfusion. Targeting transcription factors that regulate immune functions following inflammatory insults might be another option to suppress the detrimental effects of inflammation.

70Implantation of mesenchymal stem cell-derived pericytes in mice that model AD plaque pathology reduces the Aβ burden, demonstrating the possibility of cell-based therapy for AD treatment. Easy accessibility of pericytes for autologous transplantation highlights their capabilities for future therapeutic studies. Since pericyte-like cells derived from induced pluripotent stem cells (iPSC) acquire BBB properties and are incorporated with iPSC-derived endothelial cells, astrocytes and neurons, iPSC-derived pericytes might also be promising for implantation therapy of AD and other neurological disorders. Pericytes themselves have been shown to acquire stem cell-like and microglial properties after ischemia, which offers another potential therapeutic target for recovery form CNS diseases.

71As discussed above, scar formation in the ischemic brain and brain/spinal cord injury has pluripotent effects on diverse CNS conditions. While the scar formation by pericytes and glia play fundamental roles in promoting angiogenesis and tissue remodeling, reducing pericyte-derived scar formation has been reported to promote axonal regeneration and recovery from spinal cord injury. Administration of periostin-neutralizing antibody, which suppresses pericyte-induced scar formation, ameliorates functional recovery after spinal cord injury, but further studies are needed to demonstrate the therapeutic potential of these strategies including the appropriate timing and degree of intervention.

72All CNS cells and tissues need a blood supply coming from outside the brain. Located at the interface between CNS tissue and blood circulation and having multi-functional properties, pericytes play a variety of fundamental roles in the healthy CNS. As a result, pericytes offer many opportunities for therapeutic intervention in a broad range of neurological disorders, including cerebrovascular disorders and AD. Vascular cognitive impairment/dementia and AD account for more than 3/4 of dementing diseases, and vascular pathology is often observed in a various neurodegenerative disease, especially in AD, where pericytes are thought to contribute. With increasing knowledge about the molecular mechanisms operating in pericytes and their crosstalk with neighboring cells, the targeting pericytes as a therapeutic strategy has become increasingly important and research on this topic is likely to accelerate more in the future.

73MU conceptualized the study, designed and drafted the manuscript and figures, and handled the funding. TM supervised and critically revised the manuscript for important intellectual content. MI, VL, and JT handled the funding, supervising, and making critical revision of the manuscript for important intellectual content.

74The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.