The BBB is composed of ECs, basal lamina, astrocytes, and pericytes. it maintains homeostasis of the brain microenvironment by controlling the entry and exit of certain substances in the brain (Bergers and Song, 2005; Keaney and Campbell, 2015). Hence, BBB destruction is an important pathological change that occurs in IS (Keaney and Campbell, 2015). In the development of cerebral microcirculation, pericytes contribute to the formation and maintenance of BBB. A low count of pericytes greatly increases the permeability of BBB, further confirming that pericytes are key to maintaining its integrity (Bell et al., 2010). Winkler et al. demonstrated that platelet-derived growth factor-B/platelet-derived growth factor receptor-B (PDGF-B/PDGFR-B) can mediate pericyte recruitment to the vessel walls in the embryonic brain and regulate the development of cerebral microcirculation and the BBB. They used mouse models lacking PDGF-B and PDGFR-B to investigate whether the loss of pericytes can influence brain capillary density, and found that loss of pericytes can disrupt cerebral microcirculation and BBB integrity, which occur in neurodegenerative and neurovascular disorders (Winkler et al., 2010). In addition, pericytes synergize with other components of the BBB such as ECs, basal lamina, and astrocytes (Gundersen et al., 2014). In mice with defective pericyte generation, structural abnormalities in tight junctions were observed using electron microscopy. Further, the permeability of BBB also increased (Daneman et al., 2010). Trans-endothelial electrical resistance (TEER) is a widely accepted quantitative technique for measuring the integrity of tight junction dynamics in endothelial monolayer cell culture models. It is an indicator that can provide a measure of pore access through ECs and the integrity of cell-to-cell junctions. TEER can serve as an excellent tool to elucidate endothelial barrier functions, such as responses to cytokines, pathological stimuli, and potential therapeutic agents (Ramnath and Satchell, 2020). It is a strong indicator often used to evaluate BBB integrity, and an increase in TEER usually indicates a decrease in BBB permeability (Srinivasan et al., 2015; Vatine et al., 2019). Glial cell line-derived neurotrophic factor (GDNF) facilitates neuronal or axonal regeneration, and increases the expression of claudin-5 and the TEER value of ECs. Shimizu et al. demonstrated that GDNF secreted from pericytes of the brain and peripheral nerves upregulated the expression of claudin-5, the main protein that constitutes the tight junction of ECs, which increased the TEER value of microvascular ECs (Shimizu et al., 2012). This result confirmed that pericytes are essential for maintaining BBB permeability, as claudin-5 is now considered to be one of the most important components involved in maintaining BBB function (Nitta et al., 2003; Dithmer et al., 2017). Hence, targeting GDNF secreted by pericytes may be a new therapeutic strategy to alter the function of the BBB and promote nerve regeneration.

Another study illustrated that in a gel matrix, pericytes and astrocytes migrate together and adhere to capillary-like structures to form glial vascular complex-like structures. Compared with astrocytes alone, pericytes in combination with astrocytes inhibited the degradation of the capillary-like structures to a greater extent (Itoh et al., 2011). Furthermore, various studies have shown that pericytes are involved in the formation and degradation of extracellular matrix (ECM) in the basal layer. Pericytes produce degrading proteases, such as matrix metalloproteinase (MMP)-2 and MMP-9, which can promote the degradation of ECM in the early stage of angiogenesis. This leads to the detachment of pericytes from basal lamina and their migration to newly formed vascular, helping repair the damaged BBB after IS (Zozulya et al., 2008; Takata et al., 2011; Jullienne et al., 2016). During the vascular stabilization process, pericytes express inhibitors of MMP such as the metalloproteinase-3 tissue inhibitor to inhibit the degradation of ECM and promote vascular maturation and BBB repair (Dalkara et al., 2011; Winkler et al., 2011). In contrast, the structural and functional changes in pericytes, such as the occurrence of oxidative stress in the acute stage of brain ischemia, can also lead to the destruction of the BBB and accumulation of neurotoxic substances. Oxidative stress also activates pericytes to secrete MMP-9 and other factors. Activated MMP-9 induces pericyte damage, shedding, and migration. These factors further aggravate adhesion damage (Nishimura et al., 2016). Thus, in the early stage of ischemia, pericytes and the factors secreted by them may lead to disruption of the BBB. This evidence illustrated that pericytes undergo a two-way regulation. Therefore, inhibiting the secretion of MMP-9 by pericytes in the acute stage of IS and promoting the expression of MMP inhibitors secreted by pericytes during vascular stabilization process can be considered to be possible therapeutic strategies of IS.

Pathological changes in smooth muscle cells after IS can interrupt cerebral blood flow (CBF). Studies have shown that pericytes have potential contractility and can regulate the blood supply to terminal vessels with damaged smooth muscle cells (Liu et al., 2021; Mariana et al., 2021). Pericytes express the contractile protein α-SMA, which dilates and contracts blood vessels, participates in neurovascular coupling, and regulates CBF (Cheng et al., 2018). Therefore, α-SMA is a commonly used pericyte marker, and higher expression of α-SMA indicates stronger contractility (Kelly-Goss et al., 2014). Some studies have indicated that transforming growth factor-β (TGFβ) increases the expression of α-SMA (Shih et al., 2003), whereas fibroblast growth factor-2 (FGF-2) has the opposite effect (Papetti et al., 2003). Regulating the contractility of pericytes and microvascular blood flow by regulating the expression of α-SMA could be considered as a potential strategy for IS treatment. Notably, the hyperconstriction of blood vessels is pathological and is facilitated by smooth muscle and pericytes. Pericyte contraction is more pronounced during reperfusion after IS. Reperfusion may cause more damage to the injured tissue than ischemia itself. Among them, oxidative stress is an effective contraction inducer of pericytes and is a particularly prominent damage mediator in ischemia-reperfusion injury (O’Farrell et al., 2017). Yemisci et al. (2009) reported contractility changes in pericytes during reperfusion in IS in vivo. Pericyte contraction occurs 1 h after reperfusion in a stroke middle cerebral artery occlusion model. It has been shown that pericyte contraction is induced by oxidative and nitrifying free radicals before reperfusion relieves pericyte contraction (Yemisci et al., 2009). This study demonstrates the importance of pericyte contractility in stroke pathology and suggests that persistent contraction of pericytes may be responsible for the poor outcomes of thrombolytic therapy in stroke.

Pericytes also contribute to the cerebral no-reflow phenomenon in which microcirculation cannot be fully restored after the occluded blood vessel is reopened (Kloner et al., 2018). Electron microscopic examination of the cerebral microvascular system in the ischemic area showed capillary damage manifested as swollen ECs and astrocytic ends, intravascular platelets and fibrin, and white blood cell blockage (McCabe et al., 2018). These changes compress capillaries, leading to the persistence of microcirculation disorders (Paljärvi et al., 1983; Taskiran-Sag et al., 2018). Pericytes remained contracted during ischemia even after the main blood supply artery was successfully reopened, which may be the main reason for the non-reflux phenomenon (Yemisci et al., 2009; Dalkara and Alarcon-Martinez, 2015). Hall et al. (2014) suggested that cerebral ischemia-induced pericyte contraction and the subsequent death of pericytes. The contraction of pericytes is regulated by intracellular Ca²⁺ concentration. Upon cerebral ischemia, a large Ca²⁺ influx triggers calcium overload and contraction of pericytes, causing cerebral microvasculature contraction. The subsequent death of pericytes leaves the microvasculature in a continuous state of contraction, which causes microcirculation failure. During reperfusion, pericytes produce a large amount of reactive oxygen species (ROS) and calcium overload is aggravated, whereas suppressing the reperfusion-induced increase in ROS can promote microcirculation reperfusion (Taskiran-Sag et al., 2018). Therefore, preventing the contraction and death of pericytes as soon as possible after IS may help in the recovery of microvascular CBF, promote microcirculation reperfusion, and improve the outcome of IS.

Pericytes have various properties of immune regulatory cells, including leukocyte recruitment, reactive secretion of inflammatory factors, antigen presentation, and phagocytosis. They also participate in many aspects of CNS inflammatory responses (Lange et al., 2013; Pieper et al., 2013, 2014; ElAli et al., 2014). In the inflammatory response after IS, pericytes play both pro-inflammatory and immunoregulatory roles. In pericyte-deficient mice (PDGFRβ^–/–), intercellular adhesion molecule-1 (ICAM-1) is significantly upregulated, and leukocytes, which are normally absent from the CNS, are present. This indicates that pericytes can protect the brain from the invasion of leukocytes. Pericytes also show phagocytic and migratory characteristics after IS (Rustenhoven et al., 2018). Studies have shown that brain microvascular pericytes express damage-associated molecular patterns and toll-like receptor 4 (Guijarro-Muñoz et al., 2014), triggering an immune inflammatory response. In addition, several in vitro studies have shown that various inflammatory conditions can induce pericytes to express multiple cytokines, including interleukin (IL)-9, IL-10, and tumor necrosis factor-α (TNF-α) (Lange et al., 2013; Pieper et al., 2013, 2014; Liu et al., 2016). In addition, a variety of inflammatory conditions can also induce pericytes to express pro-inflammatory factors as shown by several in vitro studies. For example, TNF-α and IL-1β stimulate microglia to secrete the chemokine IL-8 (Pieper et al., 2013); consequently, TNF-α and TGFβ1 enhance the expression of IL-6 in human pericytes, which can disrupt BBB functioning (Rustenhoven et al., 2016). Pericytes can also upregulate the expression of ICAM-1 and vascular cell adhesion molecule-1 in response to interferon-γ (Balabanov et al., 1999; Lyck and Enzmann, 2015). Therefore, pericytes may be considered as effectors and amplifiers of the inflammatory process in the brain during IS.

Pericytes can transport substances through phagocytosis, pinocytosis and endocytosis and participate in the first line of immune defense in the brain after IS (Thomas, 1999; Girolamo et al., 2021). Pericytes may transform into other cell types to participate in inflammation and immune processes during the occurrence of IS. Pericytes can differentiate into various cell types such as microglia-like cells with phagocytic capacity, which are involved in the immune inflammatory response in IS (Sakuma et al., 2016). This evidence indicates that pericyte loss may be the cause of aggravation of leukocyte infiltration during IS.

In IS, the microcirculation of the ischemic brain tissue cannot be fully perfused after recanalization. This phenomenon, termed reperfusion no-reflow, is harmful to the recovery of ischemic tissues. However, angiogenesis promotes the accumulation of new neuronal cells in the peri-infarct area and contributes to the repair of brain injury after IS, which is significant for tissue regeneration and provides new entry for blood reflow to the hypoperfusion area (Di et al., 2021). The remodeling of microvascular system after IS includes four stages: proliferation of vascular component cells; recruitment of pericytes; formation, migration, and stabilization of vascular budding; and maturation of blood vessels (Stapor et al., 2014; Yang and Torbey, 2020). Pericytes regulate all these stages through a dynamic balance between pro-angiogenic and anti-angiogenic factors. In recent years, pericytes have received increasing attention as potential targets for pro-angiogenesis therapy after stroke. During angiogenesis, bidirectional EC-pericyte signaling is essential (Winkler et al., 2011; Hall et al., 2014; Iadecola, 2017). First, after exposure to pro-angiogenic stimuli, pericytes upregulate Erk1/2-MMP axis to degrade basal membrane components, leading to the detachment of pericytes and subsequent release of ECs and increased vascular permeability (Carmeliet and Collen, 2000; Potente et al., 2011; Xiao et al., 2020). This promotes the extravasation of plasma proteins and provides a stent for the production of vascular sprouts (Carmeliet and Collen, 2000; Zhu et al., 2021). Second, pericytes secrete vascular endothelial growth factor (VEGF)-A, which stimulates ECs to transform into tip cells. Further, stem cells proliferate to support the elongation of vascular sprouts and form blood vessels. In this process, pericytes attach to the ECs, regulate the formation of the extracellular matrix and tight junctions between endothelial cells, and maintain the integrity of immature blood vessels (Franco et al., 2011). Pericytes interact with ECs through a variety of signaling pathways to regulate angiogenesis, including the PDGF-B/PDGFR-B, angiopoietin (Ang)/Tie2, VEGF/VEGFR, Notch1, and Notch 3 signaling pathways (Berger et al., 2005; Nakamura et al., 2016; Leligdowicz et al., 2018; Özen et al., 2018; Shibahara et al., 2020a; Gong et al., 2021; Nguyen et al., 2021), and these pathways are summarized in Figure 2. Finally, the tip cells anastomose with adjacent vascular buds to form a microvascular ring. After these processes, pericytes inhibit the proliferation of ECs by downregulating the expression of VEGF and establishing a resting state in ECs to stop angiogenesis (Jain, 2003; Huang, 2020). Therefore, further research for the treatment of IS can help in developing new therapies that promote angiogenesis.

Neurogenesis can be observed in the brain during IS (Rüber and Schlaug, 2018; Hatakeyama et al., 2020; Yang and Torbey, 2020; Zhan et al., 2020). A study reported that neural stem cells developed in the injured areas of a mouse model of transient brain ischemia/reperfusion injury. Surprisingly, some PDGFR-B+ pericytes can be a potential source of perivascular neural stem cells, which have the potential to generate neurons and repair the NVU in patients with IS. Bone marrow-derived cells that have a phenotype of pericytes and express angiogenic factors such as VEGF, are recruited to the brain capillaries. Accumulating evidence shows that angiogenesis is closely connected with neurogenesis. Further, newly formed neurons are always located near the remodeled vessels, probably because the vascular cells recruit and form the niche of neural stem cells. Some angiogenic factors, such as VEGF and PDGF, also play a vital role in neurogenesis after stroke (Bai et al., 2015; Eilken et al., 2017; Caporarello et al., 2019; Nguyen et al., 2021). VEGF can protect pericytes and NVUs from IS by enhancing the MEK1/2/ERK1/2 and PI3K/AKT signaling pathways, which triggers a part of the feedforward loop of IS. Wang et al. showed that after IS, the PDGF/PDGFR-B signaling pathway is essential for the recruitment of neuroblasts from the subventricular zone to the infarct area in a mouse model (Wang et al., 2017). They downregulated the PDGFR signaling pathway with crenolanib, a nonselective PDGFR inhibitor, and found that crenolanib increased pericyte apoptosis, decreased pericyte and vascular coverage, increased neuroblast apoptosis, and inhibited neuroblast migration in the subventricular zone. These results indicated that the PDGF/PDGFR-B signaling pathway can promote the migration and survival of neuroblasts after IS and affect stroke-induced neurogenesis. Furthermore, it has also been reported that pericytes can stabilize quiescent microvasculature, enrich local neuronal microcircuits, and promote neurogenesis (Farahani et al., 2019). Pericytes with neuro-angiogenic potential can be considered as potential targets for repairing the damaged CNS after IS.

Several studies show that capillary PCs are rapidly lost after cerebral ischemia in both experimental and human stroke (Carmeliet and Jain, 2011; Fernández-Klett et al., 2013). As previously described, a low count of pericytes greatly increases the permeability of BBB (Bell et al., 2010), which indicates that the number of pericytes is a factor in determining BBB permeability. A study shows that after IS, a ubiquitous healing mechanism, scar formation, repairs damaged tissue by producing ECM. Coinciding with this loss was a massive proliferation of PDGFR-B+ cells, originating from the neurovascular unit and depositing ECM in the mouse brain after IS. And this fibrotic response is distinct from astrocyte scarring, which, despite some regenerative function, has a potentially deleterious function in the brain (Fernández-Klett et al., 2013). In another study of Dias et al., they report that the primary source of scar-forming fibroblasts is pericytes in a mouse model of fibrotic scar tissue formation after IS. Although it is unclear whether scar-forming stromal cells have the same origin throughout the CNS and across different types of lesions, pericyte can also be considered as a therapeutic target to improve recovery after CNS injury and need further researches (Dias et al., 2021). Therefore, promoting the proliferation and maturation of pericytes can be regarded as an potential strategies of IS.

The recanalization of blood vessels and restoration of CBF as early as possible are currently recognized as effective methods for the treatment of acute IS. Therefore, regulating CBF by targeting pericytes is a potential therapeutic strategy for the treatment of IS. As described previously, α-SMA can dilate and contract blood vessels, participate in neurovascular coupling, and regulate CBF (Cheng et al., 2018). Most of the pericytes that differentiated by TGFβ expressed functional α-SMA and higher levels of permeability factors such as VEGF, MMP-2, and MMP-9, indicating that TGFβ-induced differentiation of pericytes had an angiogenic effect (Thanabalasundaram et al., 2011). Nuclear factor of activated T-cells cytoplasmic 3 (NFATc3), a calcium-dependent transcriptional factor, also plays a critical role in regulating vascular cell contractility and CBF (Filosa et al., 2007; Serrano-Pérez et al., 2011). Glutamate released by neurons can activate metabotropic glutamate receptors on astrocytes, thereby increasing the expression of activated NFATc3 in pericytes, which is dependent on the activation of Ca²⁺/calmodulin-dependent phosphatase calcineurin (Filosa et al., 2007). In a study of Filosa et al., they inhibit the activation of calcineurin by using gliotoxin l-α-aminoadipic acid, and found that the NFATc3 nuclear accumulation in pericytes is prevented. This results shows that Ca2+/calcineurin is vital between mGluR and NFATc3 (Filosa et al., 2007). Based on these results, NFATc3 can be regarded as a new therapeutic target for IS.

Studies have shown that pericytes play a key role in the formation and maintenance of BBB (Daneman et al., 2010; Winkler et al., 2012). Pericytes are recruited to new blood vessels during early embryogenesis, which induces the formation of a functional BBB (Daneman et al., 2010). Therefore, regulating pericyte recruitment to promote angiogenesis can be regarded as a strategy to treat IS. Neuron-glial 2 (NG2) is an important component of activated pericyte-like cells (PCs). NG2 is expressed by microvascular pericytes in newly formed blood vessels under both physiological and pathological conditions (Fukushi et al., 2004; Huang et al., 2010; Yang et al., 2013). Research on angiogenesis in cancer has shown that NG2 plays a key role in pericyte recruitment and interaction with ECs during microvascular development, which can affect the maturation of pericytes and ECs (Chekenya et al., 2002; Ozerdem et al., 2002; Ozerdem and Stallcup, 2004; You et al., 2012). Another study demonstrated that CD146, a single-cell receptor, could mediate pericyte recruitment to cerebrovascular ECs and help in angiogenesis by functioning as a co-receptor for PDGFR-β (Chen et al., 2017). These results demonstrate that NG2 and CD124 may be therapeutic targets for IS.

Since neurogenesis is always accompanied by angiogenesis, factors that contribute significantly to angiogenesis such as PDGFRβ, VEGF, and Notch proteins could regulate neurogenesis, remodel the NVU, and treat BBB dysfunction (Farahani et al., 2019). A recent study demonstrated that human pluripotent stem cells (hPSCs) can differentiate into PCs. Further, PCs can express typical pericyte markers including PDGFRβ and CD146 (Sun et al., 2020). In this study, hPSC-induced PCs were transplanted into a transient middle cerebral artery occlusion mouse model, and the PCs promoted neurogenesis by rebuilding BBB integrity and preventing neuronal apoptosis. This result indicated that hPSC-induced PCs may be an ideal cell source for IS treatment.

Epigenetic mechanisms such DNA methylation, histone modification, post-transcriptional regulation of microRNAs (miRNAs), and non-coding RNA interference, are widely involved in pathophysiological processes after the occurrence of IS such as apoptosis, inflammation, BBB regulation, and neuro-angiogenesis. The role of miRNAs in the NVU after IS is being researched extensively. miRNAs can regulate endothelial function and angiogenesis, but their significance in pericyte biology has not been determined (Caporali et al., 2017). Wan et al. (2018) found that the level of miR-149-5p in pericytes decreases during IS. They also showed that in vivo and in vitro, miR-149-5p could significantly upregulate sphingosine-1-phosphate receptor 2 (S1PR2) expression in pericytes after IS. S1PR2 increased the expression of N-cadherin, which decreased pericyte migration and BBB permeability. Another study indicated that miR-532-5p was upregulated early on in murine muscular pericytes after experimentally-induced ischemia. The overexpression of miR-532-5p increased pericyte coverage by inhibiting BACH1 and promoting the expression of Ang-1 and vascular maturation in vivo (Slater et al., 2018). Additionally, Let-7 miRNA is involved in pericyte differentiation in response to hypoxia (Esen et al., 2016). Under conditions that mimic hyperglycemia and ischemia, pericytes can reduce capillary coverage and increase capillary permeability by absorbing endothelial cell-derived miR-503 (Caporali et al., 2015). Therefore, blocking miR-503 can increase the coverage of capillary vessels and reduce BBB permeability. In contrast, stroke can significantly alter the expression profile of long non-coding RNAs (lncRNAs) in the brain (Yan et al., 2015). However, research on the relationship between lncRNAs and pericytes is currently in the exploratory stage. Recent studies have found that hypoxia can upregulate the expression of hypoxia-induced endoplasmic reticulum stress regulating lncRNA in pericytes and regulate the differentiation, proliferation, and recruitment of pericytes to ECs (Bischoff et al., 2017). Since research on miRNAs and lncRNAs in pericytes is still in its infancy, it is necessary to carry out research in this field to explore their use as a target for IS treatment.