Importance of Pericytes in the Pathophysiology of Cerebral Ischemia

Abstract

Various cell types contribute to pathological changes observed in the brain following cerebral ischemia. Pericytes, as a component of neurovascular unit (NVU) and blood brain barrier (BBB), play a key role for cerebral blood flow control and regulation of vessel permeability. It was shown that pericytes can control cerebral blood flow at the level of capillaries, by their contractile property. Their role in BBB development and maintenance are crucial for guidance of brain vessel development, new vessel formation and stabilization of the newly formed vessels. Additionally, they can contribute to inflammation in response to inflammatory stimuli and can differentiate to various cell types by their multipotent differentiation properties. This cell type which is intimately associated with cerebral circulation also plays important roles during cerebral ischemia. Here, we review the properties and physiological functions of pericytes, how these functions change during ischemia to affect the pathophysiology of ischemic stroke and post stroke cognitive impairment. Pericytes are a neglected cell type and they are not unambiguously characterized which in turn led to contradictory findings in the literature. Clear characterization of pericytes by current methods will help better understanding of their role in the pathophysiology of stroke. With the information gained from these efforts it will be possible to develop pericyte specific therapeutic targets and achieve important breakthroughs in clinical recovery in ischemic stroke treatment.

INTRODUCTION

Stroke is the second leading cause of mortality and the third leading cause of disability worldwide and its prevalence is increasing (1). In the pathophysiology of ischemic stroke mechanisms such as cell death, blood-brain barrier (BBB) break down and inflammation play critical roles depending on the intensity and duration of the reduction in the cerebral blood flow; all these mechanisms are orchestrated by neurons, astrocytes, microglia and immune cells originating from blood (2).

The brain has the highest energy requirement relative to its size among organs and its needs are highly dynamic. The neurovascular unit (NVU) is a special structure formed to meet the requirements of the brain (3) (Figure 1). This unit is located between the brain parenchyma and capillary vessels and consists of endothelial cells, astrocytes, neurons, microglia, vessel smooth muscle cells and pericytes (3). Brain and retina have the highest density of pericytes in the body in which they line pre-capillary arterioles, capillaries and venules. They have important roles in the Central Nervous System (CNS) as a part of NVU such as neurovascular coupling and formation of BBB (3).

Figure 1.

Schematic representation of the Neurovascular Unit. The endothelial cells lining the vessels, pericytes and smooth muscle cells surrounding them, astrocytes, microglia and neurons constitute the neurovascular unit. It functions as a structural and functional unit. Pericytes and endothelial cells are in rapid communication with each other via the “peg and socket” connections. Endothelial cells sealed with tight junctions, pericytes, basal lamina and astrocyte end feet form the BBB.

Pericytes can play roles in the pathophysiology of several diseases owing to their position and functions. Dysfunctions of pericytes are shown in stroke, diabetic retinopathy, tumor angiogenesis and dementia (3) (Table 1). In this review, the physiological functions of pericytes in the brain, their roles in the pathophysiology of ischemic stroke, their therapeutic significance and properties that need to be addressed in future studies are discussed.

Table 1.

Functions of pericytes in physiological state and ischemic state

Functions	Physiological state	Ischemic state	References
Regulation of blood flow	Pericytes can contract or relax depending on neuronal activity, energy requirement and vasoactive substances and can regulate cerebral blood flow especially at the level of pre-capillary arterioles and capillaries.	Pericytes contract during ischemia and remain contracted after ischemia. This causes occlusion of microvessels and impairment of tissue perfusion even though recanalization has been achieved in the occluded big artery.	(5,10,11,32)
Maintenance of BBB	They play important roles in BBB formation and maintenance. Through their interactions with endothelial cells, they can regulate tight junctions and can control permeability.	Pericyte death resulting from ischemia causes impaired integrity of BBB and vessels. Additionally, pericytes that are activated by ischemia can release MMP-9 and produce ROS through NOX4 and damage BBB.	(14,16,17,36)
Angiogenesis	Pericytes guide the newly formed vessels during angiogenesis, stabilize them and help their maturation.	Factors important for angiogenesis such as PDGF-β, VEGF, TGF-β increase after ischemia through pericytes, and they proliferate and migrate to newly formed vessels after ischemia. Cells originating from bone marrow can also gain pericyte like phenotype and take role in angiogenesis.	(18–20,42)
Immune function	They can synthesize pro-inflammatory factors in response to inflammatory factors, exhibit phagocytotic activity to clean up toxic substances from brain and regulate leukocyte passage to brain.	In addition to their phagocytotic activity and regulation of leukocyte passage to the brain, pericytes can gain microglia like phenotype and orchestrate inflammation.	(13,22,24,44)
Stem cell properties	They can express same markers as mesenchymal and neural stem cells and can differentiate to astrocytes, microglia, oligodendrocytes, vascular cells and neurons.	Their multipotential differentiation properties increase with ischemia and they can contribute to recovery after ischemia and neurogenesis by differentiating to neural, glial and vascular cell types.	(26,27,45,47)

BBB: Blood Brain Barrier; MMP-9: Matrix Metalloproteinase 9; NOX4: NADPH Oxidase 4; PDGF-β: Platelet-derived growth factor-β; ROS: Reactive Oxygen Species; TGF-β: Transforming Growth Factor; VEGF: Vascular Endothelial Growth Factor.

Highlights

• Pericytes have important functions for cerebral blood flow regulation.

• Effects of pericytes in ischemia have important consequences for tissue survival.

• Pericytes should be better characterized and their roles in ischemia should be elucidated.

• Therapies targeted to pericytes might provide big breakthrough for stroke therapy.

Physiological Functions of Pericytes in the Brain

Similar to vascular smooth muscle cells, pericytes can express contractile proteins such as alpha-smooth muscle actin (α-SMA), myosine and tropomyosine and can contract (4,5). According to their placement on the microvasculature they show heterogeneity in their morphology, function and protein expression. Pericytes that are closer to arterioles show higher α-SMA expression and have ring-like appendages that firmly wrap the vessel in a circular shape.Pericytes that reside on the middle segments of the capillaries show little to no α-SMA expression and have longer fibrillary appendages that can span more vessel area but wrap the vessel less firmly. Pericytes that are closer to venules also show low α-SMA expression and have mesh-like appendages (6,7). Because of this heterogeneity, the distinction between pericytes that are close to arterioles and vascular smooth muscle cells on arterioles was not very well-defined. There was no common terminology in the literature, and this produced controversial results about the characteristics and functions of the pericytes (6). Despite the heterogeneity, CNS pericytes express common markers such as platelet-derived growth factor receptor-β (PDGFR-β), Neural/glial antigen 2 (NG2), regulator of G protein signalling-5 (RGS-5) and they can be identified by these markers (3). However, as these are not specific to pericytes, and can be expressed by other cell types, the identification of pericytes still remains difficult (8). Overall, pericytes play important roles in the regulation of cerebral blood flow, formation and maintenance of BBB, angiogenesis, inflammation and stem cell source in the CNS.

Regulation of Cerebral Blood Flow

Pericytes have the chief role in the regulation of blood flow in the cerebral and retinal microvessels (9,10). In vitro and ex vivo studies, real-time in vivo multiphoton microscopy imaging via cell-specific optogenetic and chemogenetic interventions showed that capillary pericytes can contract.

During in vivo multiphoton microscopy imaging in mice brains, following a sensory stimulus, vessels of different sizes dilate, and these dilatations occurred earlier in the capillaries that are close to arterioles than the penetrating arterioles. Based on the finding that dilatations were at locations that have pericyte soma or processes, it was suggested that activity-dependent dilatations on vessels are carried out by the α-SMA expressing pericytes on capillaries that are close to arterioles (10). In a study, where vascular smooth muscle cells and pericytes were stimulated optogenetically and changes in blood flow were examined with multiphoton imaging, it was seen that arterioles that harbor smooth muscle cells showed significant constriction and dilation, whereas capillaries that only have pericytes showed no significant difference in vessel diameter. Based on these findings, it was suggested that blood flow regulation occurred in arterioles which have smooth muscle cells with high α-SMA expression but not in capillaries (11). These results, which were contradictory to the previous findings, were interpreted to be resulting from the differences in the definition of vessel mural cells and a lack of consensus in the classification of pericytes by other groups (6). In a study conducted to address these conflicting findings, it was determined that when examined using rapid fixation by cold methanol, pericytes on the middle and distal segments of capillaries also expressed α-SMA. It was interpreted that the inability of previous studies to show α-SMA expression in capillary pericytes was because of the slow fixation by transcardial paraformaldehyde perfusion and rapid depolymerization of α-SMA during the fixation process which already has low expression in capillary pericytes (4).

Several vasoactive mediators can affect pericytes to make them constrict or dilate. ATP, noradrenaline, arachidonic acid derivatives such as thromboxane A2 and 20-HETE and endothelin-1 show vasoconstrictive effects on pericytes whereas prostaglandin E2 (and through that glutamate), adenosine, and ATP inactivated potassium channels show a vasodilatory effect on pericytes (9). The finding that these vasoactive substances also affect pericytes supports the notion that pericytes can regulate blood flow in physiological conditions.

Interaction of Pericytes with the Blood-Brain Barrier

Approximately 600 kilometers in length, 85% of cerebral vessels consist of capillaries hosting the BBB, which consists of monolayered endothelial cells sealed with tight junctions to provide low paracellular and transcellular transmission (12). These capillaries are surrounded by a specialized basement membrane, and pericytes in the same basement membrane contribute to the formation of the BBB in the embryonic period, and its maintenance in the adult period. Pericytes are important in terms of forming and maintaining tight junctions in the BBB and affecting endothelial transcytosis. The tight junction proteins that make up the BBB are mainly claudin -1, -3, -5, and -12, and occludin, and they are attached to the intracellular skeleton via the zonula occludens (ZO)-1, 2, 3. There are studies showing that loss of pericyte results in loss of claudin-5, occludin, and ZO-1 (13,14) or a change in its organization (14). In addition to affecting the tight junctions, pericytes have also been shown to increase the permeability of the BBB by controlling the transendothelial transition (15). There are studies showing that the PDGFR-β-PDGFR-BB pathway is effective in the regulation of tight junctions and BBB permeability by pericyte-endothelial interaction (14). PDGFR-β or PDGFBB knockout mice die in the embryonic period due to cerebral hemorrhage and edema. Although the pericyte coverage of capillaries decreased by approximately 50% and the BBB permeability was increased in PDGFR-β −/− mice with bilallelic PDGFR-β knockout, the amount of occludin and claudin-5 was not different from the controls. It has been observed that the proteins are scattered in such a way that they distort the angles of the tight junctions (14). It was observed that microvascular permeability increased in healthy-born mice brains with weakened PDGFR-β alleles (16).

The main problem in transgenic mouse models is that they are created by mating genetically modified parents and they show developmental defects in all processes involving pericytes throughout the embryonic period. Therefore, models created in adulthood are important to examine the role of pericytes in cerebral ischemia, since it would be difficult to say that the increase in permeability in the BBB is unrelated to angiogenetic abnormalities during embryonic period in embryonically created transgenic models. In order to investigate the homeostatic role of pericytes in BBB in adulthood, independent of their role in neurodevelopment, a model was created that provides endothelial-specific deletion of the PDGF-B gene by tamoxifen administration in a 2-month-old adult mouse. In this model, PDGF-B deletion in adulthood did not affect BBB in the short term, but changes were observed in the 3-6 months or longer. A decrease in pericyte coverage of microvessels and permeability increase in BBB was observed. Similar to the embryonic models, tight junction proteins were observed along the microvessels at a similar level to the control, but differences in the expression and distribution of claudin-5 and cadherin proteins were observed at the leakage points of the capillaries (17).

Angiogenesis

Pericytes have important roles in the formation and maintenance of microvessels. Their interaction with endothelial cells especially via PDGF-B/PDGFR-β signal plays an essential role. PDGF-B released from endothelium binds to its receptor PDGFR-β on pericytes, and this causes pericytes to migrate and adhere to budding endothelium for angiogenesis (18). Angiopoietin 1 (Ang-1)/Tie2 also plays a role in angiogenesis through pericyte-endothelium interaction. Ang-1 released from pericytes binds and activates its receptor Tie-2 on endothelial cells and this acts to help the maturation of endothelium (19). In contrast, Ang-2 acts to increase vessel destabilization and new vessel formation (19). Vascular Endothelial Growth Factor (VEGF) is another important factor for angiogenesis. Beyond angiogenesis, VEGF released from pericytes increase endothelial cells’ survival and stabilizes vessels (19). Transforming growth factor-β (TGF-β) helps the stabilization of vessels by making precursors to differentiate into pericytes and reducing the proliferation of endothelial cells (20). Notch-3 signaling in pericytes is also important for the fate of microvessels. This pathway is found to be important for the complete maturation of new vessel networks and helping them become functional mature vessels (21). CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) syndrome is associated with Notch-3 mutations and it is characterized by disruptions in pericyte-vessel interactions (21). The notion that pericytes are also important for the development of brain microvessels is strengthened by observations that when pericytes are ablated in mice reaching adulthood brain capillary perfusion becomes defective, functional hyperemia is disrupted and toxic substances cross to the brain because of BBB breakdown (13).

Inflammation and Immunity

Although it is generally thought that the brain cells which take part in immune response are microglia and astrocytes, it is found that pericytes can also have immune functions. In pericyte cultures, it was observed that after the treatment with pro-inflammatory factors, the genes associated with chemokine and cytokine pathways are expressed more and pro-inflammatory factors are released to the medium (22). In parallel to this response, it was shown that pericytes can express various Pattern Recognition Receptors (PRRs). Pericytes expressing PRRs such as Toll-like receptor 4 (TLR4), TLR2, nucleotide-binding oligomerization domain-containing protein 1 (NOD1) can release pro-inflammatory factors such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-6 in response to pathogen-associated or damage-associated molecular patterns and they can act as antigen presenting cells for immune system cells (22). Because of their position in the BBB, pericytes can often come across toxic and unwanted substances. It was discovered that pericytes have phagocytic activity and plenty of lysosomal granules to clean up these substances (13). Through this function, they can also take part in cleaning up disease-associated proteins such as amyloid β (23). Another contribution of pericytes to the immune response in the brain is controlling the passage of peripheral leukocytes to the brain. It is shown that especially pericytes closer to venules can increase the expression of chemokines and adhesion molecules in response to inflammation and can increase leukocyte passage into the brain (24). Along all these pro-inflammatory effects, pericytes can also synthesize anti-inflammatory factors such as IL-10 and IL-13, hence can protect endothelial cells from the effects of inflammation (25).

Stem Cell Properties

Pericytes show resemblances to mesenchymal stem cells. They can express similar markers, and can both renew their own pool and differentiate into different cell types such as astrocytes, microglia, oligodendrocytes and vascular cells (26). Pericytes can also show neural stem cell properties and transform into neurons and this property of pericytes is believed to play a role in tissue repair after damage (27). It was shown that through neuronal differentiation pericytes can form neurogenic niches and accelerate neurogenesis (27). It is believed that stem cell-like properties of pericytes can be used therapeutically for recovery after a stroke.

Research Methods for Brain Pericytes in Current Literature

Immunohistochemical methods

It is known that pericytes cannot be demonstrated with a single marker immunohistochemically and the expression profile of pericytes differs in different brain regions (8). Although it is common to define them as cells with an ovoid nucleus and a ‘bump on a log’ appearance adjacent to the endothelium considering their location and morphological features; it is now known that there are different types of pericytes (3). The common pericyte markers are PDGFR-β, NG2, α-SMA, desmin and aminopeptidase N (CD13), but none of them are specific to pericytes. Therefore, pericytes are usually detected by colocalization of double markers.

Studying pericytes in vivo

Pericytes were investigated with various transgenic animals with current genetic engineering methods. By attaching various fluorescent proteins to the promoters of genes such as PDGFR-B, NG2, α-SMA, in vivo marking of pericytes with fluorescent proteins has been attained. Also, by interfering with the marker genes in embryological or adult stages, animals with reduced pericyte numbers or different proteins expressed in their pericytes have been obtained as well. NG2-DsRed, PDGFR-β-tdTomato transgenic mice with fluorescently labeled pericytes are used in the literature, and the pericytes of these animals can be imaged fluorescently under confocal or multiphoton microscopy (7). If not genetically labeled, pericytes can be displayed in vivo by applying the fluorescently labeled Nissl dye called Neurotrace 500/525 to the cortical surface (9). By expressing the photosensitive channelorhodopsin-2 protein in NG2-Cre or PDGFRβ-Cre transgenic mice, it is also possible to stimulate pericytes and investigate their contractile properties by optogenetics (12). GCaMP6f transgenic mouse whose neuronal activity was observed with genetically labeled calcium, were crossed with the NG2-Cre animal to yield animals whose pericytic intracellular calcium activity could be observed (28).

Single cell sequencing

Examination of cell-specific transcriptional profiles by single cell sequencing is a very popular technique today. After the brain tissue is enzymatically and mechanically digested into single cell suspensions, the pericytes can be selected individually according to their superficial markers with the Fluorescent Activated Cell Classification (FACS) method (29). Single cell sequencing has become important to understand the multifunctional and heterogeneous nature of pericytes, and to elucidate their behavior in physiological and pathological conditions.

Pericyte Cultures

Pericytes can be isolated from the brain freshly, proliferated and sustained in suitable media for several passages. Signaling pathways, molecular mechanisms, and cell-cell interactions can be studied at the cellular level by culturing them alone or with astrocytes, endothelium or neurons. It is an important advantage that 3D BBB models can be created with cells obtained from human and that pericytic mechanisms can be studied in disease and patient specific-manner (30). However, an important disadvantage of pericyte cultures is that their gene profile and morphology change rapidly in vitro (31).

Roles of Pericytes in Cerebral Ischemia

It is known that pericytes, which are part of NVU, relax in the state of physiological hypoxia and help microvessels dilate to increase blood flow (25). However, in the case of prolonged hypoxia and ischemia, the opposite happens and pericytes contract. First in vivo proofs of pericyte contraction during ischemia were presented by Yemişci et al. (32). They determined that pericyte contractions prevented erythrocyte passage but a fair amount of serum passage continued. And these contractions didn’t revert back after the recanalization of the occluded big artery. It was seen that the factor that causes pericytes to contract during ischemia is reactive oxygen and nitrogen species, especially peroxynitrite, and the agents that inhibit oxidative and nitrative stress suppressed constrictions in the microvessels significantly, reduced the infarct volume and were neuroprotective (32). In addition to pericyte contractions, difficulty of activated leukocytes to pass through capillaries and being trapped in the capillaries also contribute to occlusion of capillaries in the penumbra region after ischemia (33). When it is considered that capillary diameter hardly allows the passage in normal conditions, one may think that a slight contraction or increase in the tone of pericytes might be enough to halt the passage of these elements through capillaries (9). The clinical equivalent of occlusions in microvessels because of pericyte contractions during ischemia is believed to be the “no reflow” phenomenon. “No reflow” phenomenon is defined as incomplete recovery of tissue perfusion and inability to achieve desired functional recovery after the recanalization of the occluded big artery. Its pathophysiology and clinical significance is well known (34).

Pericytes and Blood Brain Barrier During Ischemia

The damage that occurs in pericytes because of ischemia disrupts the function of pericytes to maintain the BBB. After ischemia, disruptions in the pericyte-endothelium interactions cause transcellular passage through endothelial cells to increase in early stages, and degradation of the structure of tight junction proteins in late stages. These factors increase paracellular passage and cause BBB dysfunction (35). Activated pericytes in response to ischemia detach from the basal membrane and migrate to injured tissue, and this migration also contributes to the breakdown of BBB structure (36). They also release matrix metalloproteinase-9 (MMP-9) during this process and contribute further to BBB breakdown (22). It is also shown that when pericytes encounter pro-inflammatory mediators released during ischemia, they generate reactive oxygen and nitrogen species and cause necrosis and/or apoptosis in BBB cells, contributing to BBB breakdown this way (22). Despite these destructive effects, pericytes can also take a role in BBB repair after ischemia. With the help of factors such as VEGF, pericytes can alleviate BBB breakdown and help to reinstate the barrier function (20).

Angiogenesis

To alleviate the stress caused by the occlusion of vessels that support the tissue during ischemia and to ensure tissue survival, the formation of new vessels to support the tissue might be a significant mechanism. It was shown that neuroprotection can be increased by triggering angiogenesis after ischemia (37). The first evidence of the involvement of pericytes in angiogenesis after ischemia came from the observations that factors important for angiogenesis such as PDGFR-β, VEGF, TGF-β had increased expression in pericytes after ischemia (38). In the infarct region, PDGF-β synthesis in endothelial cells increase, and increased PDGF-β/PDGFR-β signaling in pericytes both make pericytes migrate to budding endothelium for angiogenesis to stabilize the newly formed vessels and through Akt phosphorylation trigger pericyte proliferation and save them from apoptosis (39). VEGF, one of the most important factors for angiogenesis, is also shown to be involved in angiogenesis after cerebral ischemia. It was seen that VEGFR-2 receptor, which plays a major role in the angiogenic effect of VEGF, was increased in neurons, glia and endothelial cells after ischemia and was effective in increasing angiogenesis (40). This increase continued for days after ischemia (40). Additionally, VEGFR-2 inhibition after ischemia caused an increase in apoptosis in endothelial cells and enlargement of infarct area (2). VEGFR-1 receptor, previously thought to only regulate VEGF levels and is not involved in angiogenesis directly, was seen to have high expression in pericytes after ischemia. It was shown that VEGFR-1 signaling in pericytes and its interaction with endothelial cells acts to help the formation of endothelium buds during angiogenesis and migration of pericytes. Pericyte specific knockout of VEGFR-1 interrupted vessel formation after ischemia and enlarged infarct area (41). It was seen that after ischemia, cells originating from bone marrow started to settle in the brain. Some of these cells showed microglia phenotype and some of them showed pericyte phenotype and it was seen that they interacted with newly formed vessels. Cells exhibiting pericyte phenotype expressed angiogenic factors such as VEGF and TGF-β and they were involved in the strong angiogenic response after ischemia (42). One of the positive effects of increased angiogenesis to recovery after ischemia might be increased neurogenesis (37). To increase angiogenesis, when combined endothelial and vascular smooth muscle cells progenitors are transplanted to ischemic brains it was seen that more mature vessel networks formed and this caused neurogenesis to increase and helped neuroblast survival (43).

Neuroinflammation

Inflammation also has a role in the pathophysiology of ischemia. It is known that pericytes have phagocytotic activity in a physiologic state (13). It was shown that after a stroke, “granular” pericytes which are rich in lysosomes and have high phagocytotic activity get activated, increase in number and clean up the debris (25). Additionally, in an inflammatory state, pericytes can create pores in the BBB and allow leukocyte entrance to the brain, enabling systemic immunity to affect inflammation in the brain after a stroke (24). Regulator of G protein signaling 5 (RGS 5) expressing pericytes leave vessels as a result of ischemia and migrate to brain parenchyma, change their morphologies and start to express microglia markers such as Iba1, CD11b, GAL-3 (44). It was shown that microglia originating from pericytes in this way express PDGFR-β but not α-SMA, and they exhibited microglial functions like phagocytosis and inflammatory cytokine synthesis (45). It was understood by these findings that pericytes activated by ischemia can exhibit microglia like phenotype and affect inflammatory response after ischemia.

Stem Cell Properties

After ischemia, pericytes can show stem cell like properties and show multipotential differentiation. When both cultured pericytes that had OGD treatment and harvested pericytes from mice who underwent in vivo Middle Cerebral Artery Occlusion (MCAo) model were examined, it was seen that these pericytes show a reprogramming similar to mesenchymal-epithelial transition, start expressing stem cell markers such as nestin, c-myc, Sox2 and can differentiate to different cell types such as neural cells and vascular cells (26, 45). It was also shown that pericytes which gained stem cell properties after ischemia can be acquired from human brains after a stroke (46). The observations that pericytes can differentiate to different cell types such as neurons suggest that they might have important contributions to recovery after stroke and they can be therapeutic targets (47).

Pericytes in Post-Stroke Cognitive Dysfunction

Post-Stroke Dementia (PSD), refers to deterioration in cognitive functions and behavioral changes in a period following stroke (48). Pericyte death after ischemia, breakdown of BBB, accumulation of toxic substances, loss in blood flow regulation and decrease in trophic factors all cause neuronal death and white matter damage (49). Additionally, pericytes can show pro-inflammatory properties after ischemia and contribute to neuronal death by promoting apoptosis (22). Supporting these findings, it was seen that when brains of individuals who had PSD, vascular dementia (VaD), Alzheimer’s Disease (AD) or mixed dementia were examined, they had significantly fewer pericytes in their frontal lobe white matter, compared to age-matched controls or stroke patients who didn’t have dementia. This finding was interpreted as pericyte loss being associated with BBB disruption and cognitive deterioration (50). When the same brains were examined using a similar method, it was seen that pericyte number in the frontal cortex didn’t differ significantly between individuals who have PSD, VaD, AD and mixed dementia and controls. It was also determined that the pericyte number decreased with age (51). These findings suggest that pericytes in the cortex and white matter are affected differently and pericyte loss in white matter is more associated with cognitive dysfunction. In CADASIL, which is a genetic cause of stroke and cerebral small vessel disease, white matter lesions are typical and are associated with cognitive dysfunction (21). Findings from mice experiments suggested that pericyte loss and BBB breakdown are main factors associated with white matter lesions (52). There should be new studies to confirm that pericytes and BBB are important factors in cognitive and psychiatric disorders seen after stroke.

Therapeutic Interventions for Pericytes in Ischemic Stroke

The therapeutic interventions in ischemic stroke for pericytes can be targeted to either their natural properties inspired by the multifunctionality or to modify the pericyte behavior with drug systems rendering biological and functional recovery.

Utilizing the stem cell properties of pericytes to provide therapeutic benefits in ischemic stroke is an attractive target as cellular therapies are extremely popular nowadays. It was claimed that pericyte-derived pluripotent cells that can transform into neurons and microglia arose in the ischemic brain (26). However, a study showed that brain pericytes preserve their identity and do not transform into other cell types in physiological aging or pathological conditions in vivo. It was supported by tracking the transplanted pericytes with lineage-tracing method in an animal model with genetically marked pericytes (53). Therefore, although it is a questionable issue that pericytes can naturally transform into stem cells and differentiate into other cell types in vivo, it is uncontroversial that pericytes can be converted into different cell types in vitro. Hence, the idea that cell loss in the ischemic core can be compensated by utilizing the stem cell potential of pericytes is exciting due to the ease of obtaining pericytes from peripheral tissues, and due to its convenience for the personalized treatment with autologous transplantation. In a recent study, pericytes obtained from human pluripotent stem cells, and transplanted into animals with ischemic stroke resulted in functional recovery (54).

Treatment approaches to protect the BBB by strengthening the pericyte-endothelium interaction and preventing ischemic damage have also been studied. The intracerebroventricular administration of TGF-β before cerebral ischemia resulted in a decrease in edema formation and behavioral disorders, and an increase in occludin and claudin-5 expression, which are tight junction proteins that stabilize BBB, compared to control (55). Human recombinant PDGF-BB molecule, which enhances the pericyte-endothelial interaction, was produced and administered intracerebroventricularly to Parkinson’s Disease patients. Although this clinical phase1/2 study did not emphasize functional improvement since it was planned as a clinical tolerance and safety study, it may be important to show that similar approaches can be tested in cerebral ischemia (56). It was also suggested that exogenous application of PDGF-B releases microvesicles containing various growth factors from pericytes, and may contribute to neuronal recovery (39). Intranasal VEGF-B administration in mice with cerebral ischemia increased pericyte survival in the ischemic region by interacting with VEGFR-1 on the pericyte and contributed to the stabilization of microvessels (57).

Another pericyte-targeted treatment approach is to prevent the separation of pericytes from the microvessel wall in ischemic stroke. The phosphodiesterase type 3 inhibitor cilostazol and its prostacyclin analog ilioprost prevented the separation of pericytes from the vessel wall in stroke-susceptible animals and increased the amount of tight junction proteins through TGF-B1 in vitro (58,59).

In addition, since pericytes are known to play a role in glial scar formation peri-infarction, it was thought that blocking pericyte proliferation might be an intervention to reduce glial scar formation (60).

Voltage-gated Ca²⁺ channel blockers, superoxide radical scavengers such as L-NIO, PBN or L-NA, and nanoparticles loaded with adenosine have been used as treatment approaches to prevent the narrowing of microvessel diameter that causes hypoperfusion with pericyte contraction during ischemia (32,61).

Nanodrugs that contain pericyte-specific peptides or antibodies, and chemicals were tried to create an anti-tumor response by breaking the vascular resistance in the vessels surrounding the tumors where pericytes were intense. They provided controlled drug release in the relevant region. These studies are important as they show that a similar perspective can be used in cerebral ischemia as well. For example, a cyclic peptide that binds to PDGFR-β was conjugated to serum albumin with doxorubucin, and this conjugate taken up by peripheral vascular pericytes resulted in a reduction in tumor volume. Valproic acid, imatinib and sorefenib are small molecules that target pericytes (62).

Future Studies

Although the heterogeneous profiles of pericytes have been studied over years and the transcriptional profiles of the cells have been largely elucidated with current technologies, studies in this field are still ongoing (63). It is important to elucidate the transcriptional signatures of these cells, to characterize them using current proteomics and metabolomics approaches that contribute to understanding their roles in cerebral ischemia and develop novel therapeutic strategies.

Cre-dependent transgenic models play a crucial role in providing temporal-specific control of pericyte gene expression patterns. Although this approach is useful in understanding the pathophysiology and candidate drug mechanisms, it is impractical as transgenic approaches cannot be implemented to humans. For this reason, it is important to use rapidly developing biotechnologies in cerebral ischemia by making them pericyte specific like pericyte-specific peptides, nanodrugs containing DNA fragments, pericyte-specific antibodies and various viral vectors in the future. It has been observed that pericytes undertake different roles at different time points in ischemic stroke. This condition should be taken into account in developing treatments regarding the appropriateness of treatments at acute or chronic time frames. For example, pericyte-mediated microvascular contraction is prominent within 1 hour after ischemia (10), and for this early time point, α-SMA that is responsible for contraction, could be a suitable therapeutic target. Since pericytes contribute to vascular regeneration 3 days after cerebral ischemia, PDGFR-β and VEGF-R can be used as potential therapeutic targets after this time point.

In summary, brain pericyte biology is an integral part of the pathophysiology of cerebral ischemia. Understanding the roles of pericytes in ischemic stroke will be important both in understanding the pathophysiology of ischemic stroke and in the development of new treatments for ischemic stroke.