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Cerebral microvascular pericytes and Neurogliovascular signaling in health and disease Turgay Dalkara, Luis Alarcon-Martinez

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Received date:6 January 2015 Revised date: 10 March 2015 Accepted date: 27 March 2015 Cite this article as: Turgay Dalkara, Luis Alarcon-Martinez, Cerebral microvascular pericytes and Neurogliovascular signaling in health and disease, Brain Research, http://dx.doi.org/10.1016/j.brainres.2015.03.047 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Cerebral Microvascular Pericytes and Neurogliovascular Signaling in Health and Disease

Turgay Dalkara MD, PhD1,2,3 and Luis Alarcon-Martinez PhD2 1

; Department of Neurology, Faculty of Medicine, 2Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey 3 Departments of Neurology and Radiology, Massachusetts General Hospital, Harvard University, Boston, USA

Corresponding author: Turgay Dalkara e-mail: [email protected] Phone number: +90 0312 305 21 30 Postal address: Institute of Neurological Sciences and Psychiatry, Hacettepe University, 06100 Sıhhıye, Ankara, Turkey Abstract Increases in neuronal activity cause an enhanced blood flow to the active brain area. This neurovascular coupling is regulated by multiple mechanisms: Adenosine and lactate produced as metabolic end-products couple activity with flow by inducing vasodilation. As a specific mechanism to the brain, synaptic activity-induced Ca2+ increases in astrocytes, interneurons and neurons translate neuronal activity to vasoactive signals such as arachidonic acid metabolites and NO. K+ released onto smooth muscle cells through Ca2+-activated K+ channels on end-feet can also induce vasodilation during neuronal activity. An intense communication between the endothelia, pericytes and astrocytes is required for development and functioning of the neurovascular unit as well as the BBB. The ratio of pericytes to endothelial cells is higher in the cerebral microcirculation than other tissues. Pericytes play a role in distribution of microvascular blood flow in response to the local demand as a final regulatory step after arterioles, which feed a larger cohort of cells. Pericyteendothelial communication is essential for vasculogenesis. Pericyte also take part in leukocyte infiltration and immune responses. The microvascular injury induced by ischemia/reperfusion plays a critical role in tissue survival after recanalization by inducing sustained pericyte contraction and microcirculatory clogging (no-reflow) and by disrupting BBB integrity. Suppression of oxidative/nitrative stress or sustained adenosine delivery during re-opening of an occluded artery improves the outcome of recanalization by promoting microcirculatory reflow. Pericyte dysfunction in retinal microvessels is the main cause of diabetic retinopathy. Recent findings suggest that the age-related microvascular dysfunction may initiate the neurodegenerative changes seen Alzheimer’s dementia.

Index 1. Cerebral circulation 2. Neurovascular unit and neurovascular coupling 3. Pericytes 3.1. Regulation of microcirculation by pericytes 3.2. Angioneurogenesis and pericytes 3.3. Inflammation and pericytes 3.4. Pericytes are vulnerable to injury 4. Microvascular injury after recanalization therapies for stroke 5. Incomplete microcirculatory reflow after recanalization 6. Pericyte dysfunction causes diabetic retinopathy 7. Microvascular dysfunction as a cause of neurodegenerative diseases 8. Conclusion 9. Bibliography

1.- Cerebral circulation The brain surface is covered by a network of pial arteries and veins (Duvernoy et al., 1981). Arteries branching off the pial network dive in the brain, while intracortical veins surface and join to pial veins (Duvernoy et al., 1981; Lauwers et al., 2008) (Fig. 1). The honeycomb-like structure of pial arterial/arteriolar network allows redistribution of blood during activation of cortical columns to match the increased focal demand of the activated brain area via penetrating arteries (Blinder et al., 2010; Schaffer et al., 2006). Penetrating arteries branch into arterioles and terminate in an extensive network of capillaries (Fig. 2). There is a circular capillary free space surrounding intracortical arteries (Fig. 1). Capillaries are not required around large vessels possibly because they can supply the surrounding tissue by passive diffusion of O2 and glucose (Duvernoy et al., 1981; Kasischke et al., 2011; Sakadžiü et al., 2014) (Fig. 2B). Away from these perivascular areas, there is a microvessel located within 15 µm of every neuron soma (Mabuchi et al., 2005; Tsai et al., 2009). Unlike other organs where temporary closing or recruitment of capillaries matches the varying tissue O2 demand, in the brain, this is likely regulated by varying red blood cell (RBC) transit times across capillaries (Jespersen and Østergaard, 2012; Pawlik et al., 1981; Villringer et al., 1994). Heterogeneous RBC transit times among capillary branches decrease O2 extraction during resting conditions. On neuronal activation, RBC transit times are homogenized and the mean RBC flux increases, allowing more O2 to be extracted (Jespersen and Østergaard, 2012; Ostergaard et al., 2013) (Fig. 2C, D). Altogether these observations suggest that, whereas regulation of the blood flow in intraparenchymal arterioles may be sufficient for the perivascular tissue, a control at arteriolar level is solely not adequate to match the local tissue O2 demand at capillary level. Recent evidence suggests that pericyte-mediated capillary dilatation in response to neurotransmitters released from nearby active neurons demanding oxygen may yield higher oxygen extraction (Peppiatt et al., 2006). The capillary dilation is followed by dilation of pre-capillary arteriole in one second to meet the increased downstream blood volume need (Hall et al., 2014). The capillary endothelia, pericytes and the surrounding astrocyte end-feet are thought to transmit the dilatory signals upstream to the pre-capillary arterioles (Itoh and Suzuki, 2012; Peppiatt et al., 2006; Puro, 2007).

2.- Neurovascular unit and neurovascular coupling The neurovascular unit (NVU), which is composed of the endothelia, pericytes, neurons and astrocyte end-feet, plays an integrating role in matching the metabolic demand with the blood flow in addition to the vasodilation induced by adenosine and lactate produced as end products of the metabolic activity and by NO of endothelial origin (Fig. 3)
(Abbott et al., 2006; Attwell et al., 2010; Iadecola, 2004; Ko et al., 1990; Li and Iadecola, 1994). Recent studies suggest that astrocytes play an important part to translate neuronal activity to vasoactive signals (Attwell et al., 2010; CornellBell et al., 1990; Filosa et al., 2004; Harder et al., 1998; Haydon and Carmignoto, 2006; Koehler et al., 2009; Metea and Newman, 2006; Mulligan and MacVicar, 2004; Petzold and Murthy, 2011; Takano et al., 2006; Zonta et al., 2003). Briefly, synaptic activity initiates Ca2+ rises in peri-synaptic endfeet, which propagate through the astrocyte processes and, by activating phospholipase A2, trigger synthesis of vasoactive arachidonic acid metabolites in the end-feet surrounding arterioles and

capillaries by cyclooxygenases and epoxygenases (Bosetti, 2007; Harder et al., 1998; Kroetz and Xu, 2005; Shi et al., 2008) (Fig. 3). The arachidonic acid formed also diffuses to the adjacent vascular smooth muscle cells or pericytes on the vessel wall and is metabolized to 20-hydroxyeicosatetraenoic (20-HETE) by Ȧ–hydroxylase. Depending on the end-feet Ca2+ concentration reached and local O2 concentration, PGE2/EET or 20-HETE syntheses dominates, which lead to vascular dilation or constriction, respectively (Attwell et al., 2010; Gordon et al., 2008). Of note, the role of synaptically released glutamate in astrocytic Ca2+ signaling in adult animals has recently been questioned because the expression of mGluR5 was found to be confined to the young rodent pups commonly used in in vitro studies, suggesting that other mediators released during synaptic activity such as purines as well as changes in extracellular Ca2+ may be the trigger for astrocytic Ca2+ signaling (Calcinaghi et al., 2011; Sun et al., 2013). As another astrocytic Ca2+ wave-mediated mechanism, it has been shown that Ca2+ increase in astrocytes can release K+ onto smooth muscle cells in vessel wall by opening the large-conductance Ca2+-activated K+ channels abundantly expressed on perivascular end-feet plasmalemma (Filosa et al., 2006; Price et al., 2002). Depending on the amount of K+ released through Ca2+-activated K+ channels, modest increases in end-feet Ca2+ induce dilation, whereas larger increases cause constriction (Girouard et al., 2010). However, these in vitro findings need to be confirmated in vivo. Recent studies have also questioned the role of IP3mediated astrocytic Ca2+ signaling at least in the early functional hyperemia response (Nizar et al., 2013), however, these controversies appear to be largely related to the techniques and models used (Lind et al., 2013).
In addition to the astrocyte Ca2+ signaling, activity-induced Ca2+ increases in interneurons and neurons also contribute to the regulation the local blood flow (Fig. 3). For example, NO synthesis starts in neuronal NO synthase (nNOS) expressing GABAergic interneurons on intracellular Ca2+ rise induced by glutamatergic collateral activity originating from the neighboring principal neurons (Steinert et al., 2010; Tricoire and Vitalis, 2012). NO readily spreads across the brain tissue and relaxes smooth muscle cells and pericytes on vasculature (Atochin and Huang, 2011; Haefliger et al., 1994; Sakagami et al., 2001; Toda et al., 2009). It has also been demonstrated that a group of pyramidal neurons, whose activity is regulated by parvalbumin or VIP/ChAT expressing GABAergic interneurons, are the primary source of dilatory COX-2-derived prostanoids in the hyperemic response to whisker stimulation in addition to the direct activation of astrocytic Ca2+ signaling by pyramidal and VIP/ChAT expressing GABAergic interneurons (Lecrux et al., 2011). In summary, the hyperemic response to neuronal activity is a complex signal involving several vasoactive mediators as well as all components of the NVU (vascular, astrocytic and neuronal). Relative contributions to the each pathway briefly outlined above vary in different brain regions; for example, the NO pathway is more dominant in the cerebellum (Yang et al., 1999), whereas COX2 is in the sensory cortex (Lecrux et al., 2011). Several regional differences such as the type of local interneurons activated by glutamatergic collaterals and complex topographical relationship between the cellular compartments of NVU may account for these disparities (Attwell et al., 2010; Lecrux et al., 2011). Quick, short lasting vs. sustained vasodilatory responses may also differ with regard to mediators and pathways involved (Duchemin et al., 2012).

3.- Pericytes Pericytes are uniquely positioned within the NVU; they communicate with other cells of the NVU and regulate several microcirculatory functions such as maintenance of the blood-brain barrier (BBB) and basal lamina, regulation of the angiogenesis, immune responses and scar formation in addition to their role in control of the microvascular flow (Attwell et al., 2010; Göritz et al., 2011; Hamilton et al., 2010; Krueger and Bechmann, 2010; Thomas, 1999) (Fig. 4). They may also function as pluripotent stem cells (Dore-Duffy et al., 2006). Pericytes may change phenotype along the course vasculature and under pathological conditions (Dore-Duffy et al., 2011). Pericytes were first described by Rouget in 1873 (Rouget, 1873) and later named as “pericytes” based on their prominent location at the periphery of microvessels (Zimmermann, 1923). However, their importance in vascular physiology and cell-to-cell communications on microvasculature has recently been recognized. Pericytes cover 30-90% of the microvessel wall with their processes (Mathiisen et al., 2010; Winkler et al., 2011). Pericytes are present on pre-capillary arterioles, capillaries and post-capillary venules (Dore-Duffy et al., 2011). Although pericytes are also present on peripheral microvessels, the density of pericytes on microvessels is highest in the CNS and retina (Armulik et al., 2011; Frank et al., 1987). 3.1.- Regulation of microcirculation by pericytes In vitro studies on cerebellar, cerebral and retinal slices or on isolated microvessels and recent in vivo studies have clearly disclosed that pericytes contract or dilate in response to vasoactive mediators applied (Fernandez-Klett et al., 2010; Peppiatt et al., 2006; Puro, 2007). As briefly noted before, a recent study from David Attwell’s laboratory showed that cortical capillaries dilated before arterioles during sensory stimulation, supporting the view that microvascular blood flow in the CNS is regulated by pericytes in response to the very focal demand originating from a small group of nearby cells as a final step of flow regulation after the arterioles, which serve a larger cohort of cells (Hall et al., 2014). This flow regulation with fine spatial resolution may be essential for tissues with high functional specialization such as the brain. However, it should be noted that all microvascular pericytes may not be contractile and proportion of the contractile ones may vary with the tissue, species and developmental stage as well as along the arteriovenous axis (Krueger and Bechmann, 2010). It has also been shown that a close interaction between the endothelia and pericytes as well as astrocytes is required for development and functioning of the neurovascular unit and BBB (Armulik et al., 2010; Daneman et al., 2010). Pericytes promote formation of tight junctions and inhibit transendothelial vesicular transport (Armulik et al., 2010; Daneman et al., 2010; Kim et al., 2009). The number of pericytes per endothelial cell and the surface area of the vascular wall covered by pericytes determine the relative permeability of capillaries. Accordingly, pericyte dysfunction or deficiency causes increased BBB permeability (Armulik et al., 2011, 2010; Daneman et al., 2010).

3.2.- Angioneurogenesis and pericytes Angiogenesis, the formation of new vessels from pre-existing ones is mainly triggered by hypoxia under physiological (e.g. in response to an increased metabolic demand), and pathological conditions (e.g. cancer, stroke) (Acker and Plate, 2003; Beck and Plate, 2009; Plate et al., 2012). Pericytes are essential especially at the early phase of neovascularization (angiogenic sprouting) (Gerhardt et al., 2000; Gerhardt and Betsholtz, 2003; Ozerdem and Stallcup, 2003)(Fig. 5). Normally, pericytes and endothelial cells, which are in close proximity and directly connected by way of junction proteins, intensely communicate with each other for regulation of angiogenesis (Díaz-Flores et al., 2009; Gerhardt et al., 2000; Li et al., 2011; Stratman et al., 2009) (Fig. 3 and 5). It has been shown that pericytes i) travel to the vascular sprouts to regulate endothelial cell proliferation, ii) attach to newly formed vessels for vascular maturation, iii) contribute to the endothelial-pericyte common basement membrane formation and stabilization, and, finally, iv) assist in forming the blood brain barrier. These actions are mediated by a number of signaling pathways such as the platelet-derived growth factor-ȕ (PDGFȕ) and its receptor (PDGFRȕ), transforming growth factor-b (TGFβ) and its receptor (TGFβR2), Notch ligands and their receptors, angiopoietin and its receptors (Ang-1 and 2/Tie2) and sphingosine-1phosphate (S1P) (Armulik et al., 2011; Gaengel et al., 2009). Increasing evidence suggests that pericytes also play an important role in angiogenesis after stroke (Beck and Plate, 2009; Ergul et al., 2012; Kokovay et al., 2006; Zechariah et al., 2013a, 2013b), Kokovay et al. showed that, following brain ischemia, bone marrow-derived cells with a pericytic phenotype and expressing angiogenic factors were recruited to cerebral capillaries (Kokovay et al., 2006). Several other laboratories have shown that pericytes migrate and populate vessels in peri-ischemic areas, probably driven by an upregulated PDGF/PDGFR signaling (Kamouchi et al., 2012; Renner et al., 2003; Shen et al., 2012). Accordingly, adult postnatally-induced PDGF/PDGFR conditional knockout mice, which have normallydeveloped vasculature, had larger infarcts than controls when subjected to cerebral ischemia (Shen et al., 2012). Moreover, Ang-1, -2 and their receptors Tie-1, -2, which are essential for the migration and maturation of endothelial cells from mature vessels to form new vessels, are reportedly upregulated after stroke in a rat model (Lin et al., 2000). Recently, Zechariah et al. showed that pericytes did not appropriately cover the brain capillaries in hyperlipidemic mice exposed to ischemia and, this was associated with attenuation of post-stroke angiogenesis (Zechariah et al., 2013a, 2013b). In vitro studies have clearly shown that the brain-derived pericytes have a potential to differentiate into neurons (Dore-Duffy et al., 2006; Karow, 2013; Karow et al., 2012; Paul et al., 2012), raising the possibility that they may play a role in post-stroke neural plasticity in addition to angiogenesis. Pericytes differentiate into cells of neural lineage in response to basic fibroblast growth factor (Dore-Duffy et al., 2006) or to other trophic factors such as Sox2 and Mash1 (Ascl1). Differentiated neurons exhibit neuronal electrophysiological properties and NeuN expression (Karow, 2013; Karow et al., 2012). In line with these findings, an in vitro study showed that perivascular mesenchymal stem cells could also differentiate to neural cells (Paul et al., 2012). Further supporting the possibility that pericytes may play a role in post-stroke neural plasticity, pericytes obtained from ischemic MCA tissue of adult animals or pericytes cultured under ischemic conditions showed capability to differentiate to cells of neural as well as vascular lineage (Nakagomi et al., 2015). Pericytes probably also

play a role in the formation of the vascular niche, which is considered to be essential also for neurogenesis (Kamouchi et al., 2012). Indeed, when diabetics, whose microvascular pericytes are dysfunctional, suffer from stroke, pericyte number and coverage on the newly formed vessels as well as functional recovery is reduced compared to non-diabetic patients (Ergul et al., n.d.). 3.3.- Inflammation and pericytes Owing to their position between parenchyma and vessels, and their role in maintenance of the BBB, pericytes play important roles in leukocyte infiltration to the CNS (Daneman et al., 2010; Dohgu and Banks, 2013; Kim et al., 2006). Pericytes also present inflammatory cues and release inflammatory mediators including IL-1 , IL-6, TNF , ROS, NO and matrix metalloproteinases when activated with immune stimuli (Jansson et al., 2014; Kovac et al., 2011; Proebstl et al., 2012; Stark et al., 2013; Wang et al., 2012). They can even assume a macrophage phenotype and migrate to perivascular space, becoming indistinguishable from resident macrophages and activated microglia (Balabanov et al., 1996; Hurtado-Alvarado et al., 2014; Ozen et al., 2012; Pieper et al., 2014; Thomas, 1999). Pericytes initially respond to an inflammatory stimulus by increased expression of pro-inflammatory cytokines (Hurtado-Alvarado et al., 2014), which open tight junctions and facilitate entry of inflammatory cells into the vessel wall (Alvarez and Teale, 2007). Neutrophils crossing the endothelial cells are guided by intercellular adhesion molecule-1 (ICAM1) and its integrin ligands (Mac-1 and LFA-1) expressed on pericytes and leukocytes, respectively. Then, neutrophils travel along pericyte processes and arrive to the junctions between neighboring pericytes, which are widened secondary to inflammation-induced change in pericyte shape (Proebstl et al., 2012; Voisin et al., 2010; Wang et al., 2012, 2006). Some sites, where NG2- pericytes are located, are more preferentially used as exit points by immune cells (Leick et al., 2014; Proebstl et al., 2012; Stark et al., 2013). Once immune cells are in the parenchyma, they are guided via chemokines produced by NG2+ pericytes to the sites of inflammation (Leick et al., 2014; Stark et al., 2013).

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3.4.- Pericytes are vulnerable to injury The pericyte contractility is regulated by intracellular Ca2+ concentration as in vascular smooth muscle cells on the upstream vessels (Hamilton et al., 2010; Kamouchi et al., 2004; Puro, 2007). Accordingly, these highly dynamic cells bear the risk of Ca2+ overload when they unable to maintain intracellular Ca2+ balance, as observed in other vulnerable cells such as neurons. In addition to energy insufficiency, several other factors may cause an uncontrolled rise in intracellular Ca2+ such as reactive oxygen species (ROS), which are produced in excess amounts on the vascular wall (Gursoy-Ozdemir et al., 2012, 2004, 2000; Yemisci et al., 2009) (Fig. 6). Pericytes have large elongated mitochondria, which follow the central core of the pericyte longitudinally, and may be a significant source of ROS under pathological conditions (Mathiisen et al., 2010). NADPH oxidase, a major superoxide-producing enzyme is highly expressed in brain pericytes (Kuroda et al., n.d.; Manea et al., 2005). Indeed, ROS has been shown to cause a sustained increase in Ca2+ in cultured human brain microvascular pericytes (Kamouchi et al., 2007; Nakamura et al., 2009). Reactive oxygen and nitrogen species, especially peroxynitrite, have been reported to induce pericyte contraction during focal ischemia / reperfusion in the intact mouse brain (Yemisci et al., 2009).

4.- Microvascular injury after recanalization therapies for stroke Thrombolysis trials have unambiguously demonstrated the presence of a salvageable brain tissue after ischemic stroke (Donnan et al., 2011; Heiss, 2011). However, a short therapeutic time window limits the use of recanalization therapies for majority of stroke patients (Donnan et al., 2009; Lees et al., 2010). This brief therapeutic window is attributed to rapid loss of neuronal viability in the ischemic penumbra supported by collaterals (del Zoppo et al., 2011; Donnan et al., 2011). However, recent experimental and clinical evidence suggest that the microvascular injury induced by ischemia / reperfusion also plays a critical role in determining tissue survival after recanalization by inducing microcirculatory clogging (no-reflow) (del Zoppo and Hamann, 2011; De Silva et al., 2009; Soares et al., 2010; Yemisci et al., 2009). Several recent imaging studies serially analyzing recanalization and reperfusion in ischemic stroke patients report that, on average, 26% of successfully recanalized patients with thrombolytics do not show reperfusion (Dalkara and Arsava, 2012). Clinical trials have repeatedly demonstrated that a good outcome was better correlated with reperfusion than recanalization in stroke patients treated with tPA or interventional methods (Al-Ali et al., 2012; De Silva et al., 2009; Soares et al., 2010). The reperfusion injury also enhances the BBB permeability, predisposing to intraparenchymal hemorrhage and brain swelling in about 6% of patients receiving i.v. tPA (Donnan et al., 2011). Among many complex mechanisms, overproduction of oxygen and nitrogen radicals on the microvascular wall appear to contribute to both of these pathological processes (Gursoy-Ozdemir et al., 2012; Yemisci et al., 2009) (Fig 6). These developments bring about the exciting possibility that effective suppression of oxidative/nitrative stress during re-opening of the occluded artery may significantly improve the outcome of recanalization therapies by promoting microcirculatory reperfusion as well as by preventing hemorrhagic conversion and vasogenic edema (Gursoy-Ozdemir et al., 2012).

5.- Incomplete microcirculatory reflow after recanalization An impaired tissue reperfusion due to loss of microvascular patency (no-reflow phenomenon) was first noted after global and focal cerebral ischemia more than 50 years ago (Ames et al., 1968; Crowell and Olsson, 1972). Starting one hour after MCA occlusion, some of the capillaries show constrictions whereas precapillary arterioles generally remain open (Belayev et al., 2002; Little et al., 1976). Narrowed capillary lumina are filled with entrapped erythrocytes, leukocytes and fibrin-platelet deposits (Belayev et al., 2002; Garcia et al., 1994; Little et al., 1976; Morris et al., 2000; Zhang et al., 1999). RBCs are the predominant cell type in aggregates as there are 1000 times more RBCs than leukocytes in circulation. To infiltrate the parenchyma leukocytes adhere to postcapillary venules and may also form luminal aggregates together with fibrin and platelets (Belayev et al., 2002; del Zoppo et al., 1991; Ritter et al., 2000; Zhang et al., 1999). Incomplete restoration of the microcirculatory blood flow is expected to negatively impact tissue recovery even if re-opening of the occluded artery is achieved when there is still salvageable penumbral tissue. Therefore, improving microcirculatory reperfusion may be a promising approach for recanalization therapies. In fact,

pharmacological agents and genetic manipulations reducing microvascular clogging by inhibiting leukocyte adherence, platelet activation or fibrin-platelet interactions have been shown to restore microcirculation and improve stroke outcome in animal models (Belayev et al., 2002; Choudhri et al., 1998; Hallenbeck et al., 1986; Ishikawa et al., 2005; Mori et al., 1992). A BBB-impermeable NO synthase inhibitor (L-N-5(1-iminoethyl)-ornithine) also decreases microvascular clogging and reduces the infarct volume, in line with the idea that restoring microvascular patency can improve stroke outcome without direct parenchymal neuroprotection (Yemisci et al., 2009). In the past, microvessel constrictions were generally attributed to compression by swollen astrocyte end-feet encircling microvessels and, the resultant luminal narrowing aggravated by endothelial cell edema was thought to further hinder flow (Little et al., 1976; Zhang et al., 1999). Recently, pericytes on microvessels were reported to play an important role in incomplete microcirculatory reperfusion as they contracted during ischemia and remained contracted despite re-opening of the occluded artery 2 hours after ischemia (Yemisci et al., 2009) (Fig. 7). Small decreases in capillary radius caused by pericyte contractions lead to erythrocyte entrapments because capillary luminal size hardly allows passage of erythrocytes even under normal physiological conditions (Hamilton et al., 2010; Yemisci et al., 2009) (Fig. 7 middle row). Entrapped erythrocytes may impede passage of other blood cells and, promote platelet and fibrin aggregation (del Zoppo and Hamann, 2011; Zhang et al., 1999). The failure of erythrocyte circulation within some of the microvessels and increased heterogeneity of RBC transit times through patent capillaries (due to varying degrees of capillary resistances) significantly reduce O2 delivery to the tissue trying to recover from ischemia (Fig. 2D). Since the plasma flow in constricted capillaries is relatively less restricted compared to RBCs, glucose supply to some parts of the tissue may exceed O2 supply and promote anaerobic glycolysis, hence, lactic acidosis. Restoration of microcirculatory patency with agents inhibiting radical formation administered during recanalization was correlated with the size of tissue surviving, suggesting that microcirculatory obstructions negatively impact recovery after recanalization (Yemisci et al., 2009). More recently, sustained release of adenosine to circulation from nano-assemblies has been shown to reduce ischemiainduced erythrocyte entrapments and improve microcirculatory reflow by relaxing contracted pericytes after 2 hours of MCA occlusion (Gaudin et al., 2014) (Fig. 8). Since pericytes also play an important role in maintenance of the BBB integrity (Armulik et al., 2010; Daneman et al., 2010; Winkler et al., 2011), the ischemia / reperfusion-induced pericyte dysfunction may therefore contribute to BBB leakiness as well. 6.- Pericyte dysfunction causes diabetic retinopathy Diabetic retinopathy is characterized by occlusion of retinal microvessels, acellular capillaries, microaneurysms, breakdown of the blood–retinal barrier, hemorrhages, macular edema and angiogenesis (Willard and Herman, 2012). Pericyte loss in the retinal microvessels is a hallmark of diabetic retinopathy. Platelet-derived growth factor ȕ (PDGFȕ) knockout mice provided the first insight to the role of pericytes in diabetic retinopathy because their microvessels were devoid of pericytes and their retina displayed similar pathological changes seen in diabetes (Lindahl et al., 1997). Mice deficient of PDGFȕ receptor (PDGFRȕ) also exhibit similar features (Enge et al., 2002). PDGFȕ signaling from endothelial cells to pericytes is important to

maintain their presence on capillaries. Retinal microvessels appear particularly dependent on pericytes for maintaining blood/retina barrier stability, preventing microaneurysm formation and excessive angiogenesis. High glucose levels have been shown to activate protein kinase C-į and downstream signaling cascade in pericytes (Geraldes et al., 2009). This leads to PDGFRȕ dephosphorylation, hence, to decreased activity of PDGFȕ-activated pro-survival pathways. The same signaling system has also been found to increase ROS-mediated activation of NF-țB and pericyte apoptosis (Ejaz et al., 2008). 7.- Microvascular dysfunction as a cause of neurodegenerative diseases Dementia had been attributed to age-related changes in major cerebral arteries until the second half of 20th century when the interest shifted to deficiency in cholinergic innervation of the hippocampus and neocortex as well as to cerebral amyloid metabolism. However, the vascular hypothesis has recently been reawakened but, this time, based on the discoveries at microcirculatory level (Brown and Thore, 2011, 2011; de la Torre and Mussivand, 1993; Iadecola, 2010, 2004; Stanimirovic and Friedman, 2012; Zlokovic, 2008a, 2008b). Reduced microvascular density, atrophic capillaries that do not harbor blood (string vessels), increased irregularity of capillary surfaces, capillary basement membrane thickening are observed in brain specimens of patients with Alzheimer’s disease (AD) (Bailey et al., 2004; Farkas and Luiten, 2001). ȕ-amyloid, which accumulates on capillaries has also been shown within degenerating pericytes on microvessels (Wisniewski et al., 1992). These pathological findings together with several lines of evidence from clinical, imaging and experimental studies suggest that vascular changes may play a role in AD pathophysiology (de la Torre and Mussivand, 1993; Iadecola, 2010, 2004; Zlokovic, 2010). According to the vascular hypothesis of AD, vascular risk factors such as aging, hypertension, and hypercholesterolemia damage the microvasculature, leading to chronic focal hypoperfusion, BBB dysfunction and ȕ-amyloid accumulation (Breteler, 2000; Iadecola, 2010; Zlokovic, 2010). The microvascular dysfunction might secondarily trigger degeneration of nerve endings of the subcortical projections innervating the cortex and, retrograde death of neurons (e.g. basal forebrain cholinergic neurons). This exciting hypothesis, indeed, proposes a novel temporal sequence of events in chronic neurodegeneration, in which microvascular dysfunction initiates secondary neurodegenerative changes (Bell and Zlokovic, 2009; Zlokovic, 2010). Supporting this view, experimental studies suggest that the BBB dysfunction diminishes clearance of the soluble brain ȕ-amyloid, leading to its accumulation as neurotoxic ȕ-amyloid oligomers in parenchyma and vessel walls (Iadecola, 2010; Sagare et al., 2013; Stanimirovic and Friedman, 2012; Zlokovic, 2008a, 2008b; Zlokovic et al., 1996). ȕ-amyloid has been shown to be toxic to pericytes (Baloyannis and Baloyannis, 2012; Verbeek et al., 1997; Wilhelmus et al., 2007, 2007). Indeed, amyloid deposits within degenerating pericytes have been observed in the brains of patients with Alzheimer’s disease (Szpak et al., 2007; Wisniewski et al., 1992). 8.- Conclusion Introduction of the NVU concept has shifted the focus of research on neurovascular coupling and cerebral vascular diseases from neuron-centric views to the complex communication between elements of the NVU. With this new perspective, we now

better appreciate how the activity-flow coupling in CNS works and that a successful neuroprotection is not feasible without microvascular protection. The ischemia/reperfusion-induced NVU injury, incomplete recirculation after recanalization and the role of NVU in post-stroke recovery has emerged as research areas with significant translational potential. Recent studies on pericytes, the longneglected cells of the microvasculature, and on their intense communication with other cells of the NVU have started to provide significant insight into the regulation of microcirculatory flow, BBB maintenance, leukocyte infiltration and and vasculogenesis as well as to the pathological conditions such as stroke, diabetic retinopathy and dementia.

Acknowledgments: Dr. Turgay Dalkara’s research is supported by The Turkish Academy of Sciences. Dr. Luis Alarcon-Martinez’s research is supported by the Co-funded Brain Circulation Scheme of Marie Curie Actions into the 7th Framework Programme of European Union. Dr. Luis Alarcon-Martinez prepared Figs. 3 and 5.

LEGENDS: Figure 1. Vascular cast of the human occipital cerebral cortex. Pial vessels (1) and the cortical arterioles (2), which course through the cerebral cortex give rise to the cortical capillary network (3). Note the capillary-free area around arterioles. Scale bar = 500 ȝm. (Reproduced from Jespersen and Rodriguez-Baeza et al., 1998 with permission) Figure 2. O2 extraction from brain capillary networks is heterogeneous. A) Corrosion cast scanning electron micrograph of the capillary plexus in chinchilla auditory cortex. Several interconnected capillary paths between a pre-capillary arteriole (red) and post-capillary venule (purple) are illustrated in different colors. Scale bar = 50 ȝm. Note smooth muscle cells wrapping around arterioles (Reproduced from Harrison et al., 2002 with permission). B, C) PO2 measurements during rest in cortical microvascular segments and the histogram of measured capillary PO2: arterioles (red), capillaries (green) and venules (blue). The PO2 in diving cortical arterioles is high enough to supply the surrounding capillary-free tissue and is significantly reduced as the blood moves downstream (Reproduced from Sakadzic et al., 2014 with permission). D) Effects of capillary transit time heterogeneity on oxygen extraction. Compare the extraction of oxygen from individual capillaries when the same flow is distributed across the same number of parallel capillary paths with homogenous (during activation, b) capillary flow velocities (arrows) and heterogeneous flow velocities (at rest, a), respectively. Notice that venous outflow oxygen concentration is affected by the heterogeneity of capillary flows, in spite of identical total blood flows and number of open capillaries (Reproduced from Jespersen and Ostergaard, 2012 with permission) Figure 3. Neurovascular unit and coupling. Right) The neurovascular unit is composed of the endothelia and tight junctions between them, pericytes, the basal lamina encircling endothelia and pericytes, and astrocyte end-feet surrounding the microvessel. Note the peg and socket type contacts between endothelia and pericytes. Left) The neuronal activity induces vasodilation by several mechanisms mediated by Ca2+ increases in astrocytes, interneurons and neurons. Synaptic activity initiates inositol(1,4,5)triphosphate (IP3)-induced intracellular Ca2+ increases starting from peri-synaptic astrocyte end-feet, which is propagated throughout the neighboring astrocytes by the release of ATP to the extracellular medium and activation of astrocytic purinergic receptors to promote further intracellular Ca2+ release. The Ca2+ wave reaching to the astrocyte foot processes around vessels activates phospholipase A2 (PLA2) and liberates arachidonic acid (AA), from which prostaglandin E2 (PGE2) (by cyclooxygenase1, COX1) or epoxyeicosatrienoic acids (EETs) (by cytochrome P450 2C11, CYP 2C11) are generated. Cytochrome P450 4A (CYP 4A) synthesizes 20-hydroxyeicosatetraenoic (20-HETE) from AA diffusing to smooth muscle cells/pericytes. PGE2 and EETs induce vasodilation, whereas 20-HETE causes vasoconstriction. The Ca2+ rise in perivascular end-feet also stimulates Ca2+-activated K+ channels and cause vasodilation by releasing K+ onto smooth muscle cells. Synaptic activity also activates NMDA receptors on interneurons expressing neuronal nitric oxide (NO) synthase (nNOS) and, by increasing Ca2+, leads to NO generation. NO diffuses to smooth muscle cells/pericytes and induces vasodilation by stimulating guanylyl cyclase (GC) and inhibiting CYP 4A. The vessel tonus is also reduced by NO generated from the endothelial NOS (eNOS) and by prostaglandin I2 (PGI2) from

endothelial COX-1. Neuronal activity triggers prostaglandin (PG) synthesis in some neurons and vasoactive intestinal peptide (VIP) synthesis in some interneurons, both of which promote vasodilation. The broken arrow depicts inhibition of CYP 4A by NO. Red lines indicate vasodilatory pathways and the blue line indicates vasoconstriction. Figure 4. Pericytes regulate blood flow downstream of arterioles: A) Potential blood flow control sites in cerebral vasculature: smooth muscles on arterioles and pericytes on capillaries (Reproduced from Peppiatt et al., 1998 with permission). B) Scanning micrograph of a vascular cast of a cortical capillary (1) with a pericyte-like structure (2) having primary and secondary processes (3) distributed around the vascular cast and the capillary branching points (4). Scale bar = 11.5 ȝm. (Reproduced from Jespersen and Rodriguez-Baeza et al., 1998 with permission) Figure 5. Role of pericytes in angiogenesis. The interaction between PDGFȕ secreted by the endothelium and its receptor localized on pericytes (PDGFRȕ) is essential for recruitment of undifferentiated mesenchymal cells / pericytes to newly formed vessels. Once pericytes are at the vascular wall, reciprocal Notch signaling between the endothelia and pericytes as well as interactions between TGFβ secreted by endothelial cells and its receptor TGFβR2 located at pericytes differentiate mural cells and attach them to the newly formed vessels. The TGFβ/TGFβR2 interaction also promotes formation of the common basement membrane and stabilizes newly formed vessels by inhibiting endothelial proliferation. Ang-1, which is secreted by pericytes, activates its endothelial receptor Tie2 and promotes blood brain barrier formation. Finally, S1P, whose receptor is abundantly expressed on pericytes down regulates genes related to vascular permeability and promotes both endothelialendothelial (VE-cadherin) and pericyte-endothelial cell (N-cadherin) interconnections. Figure 6. Reactive oxygen and nitrogen species are produced in excess amounts on the vascular wall during ischemia/reperfusion. Superoxide (red dots) and NO (green circular cloud) production lead to formation of the strong oxidant peroxynitrite (yellow dots). The increased oxidative/nitrative stress causes loss of BBB integrity by disrupting tight junctions, basal lamina, and endothelial functions. It also causes pericyte contraction, which hinders erythrocyte circulation by narrowing the lumen and, hence, may impair reflow after recanalization (Reproduced from GursoyOzdemir et al., 2012 with permission) Figure 7. Ischemia causes persistent pericyte contraction, which is not restored after complete recanalization of the occluded artery. Mice were subjected to 2 h of MCA occlusion and intravenously injected with horseradish peroxidase (HRP) before decapitation 6 h after reopening of the MCA. HRP-filled microvessels exhibited sausage-like segmental constrictions in ischemic areas on brain sections (upper row). The differential interference contrast (DIC) microscopy images illustrate frequent interruptions in the erythrocyte column in an ischemic capillary contrary to a continuous row of erythrocytes flowing through an intact capillary (middle row). The constricted segments colocalized with -smooth muscle actin ( -SMA) immunoreactive pericytes (bottom row). IF denotes immunofluorescence. Scale bar for upper and middle row, 20 ȝm; bottom row 10 ȝm (Reproduced from Yemisci et al., 2009 with permission)

Figure 8. Systemic administration of squalenoyl-adenosine (SQAd) nanoassemblies (NAs) provides significant neuroprotection in a mouse model of focal cerebral ischaemia. A) Infarct areas in control and treated mice subjected to transient (2 h MCAo and 22 h reperfusion) and permanent (24 h MCAo) focal cerebral ischemia were identified by reduced Nissl staining under a light microscope (magnification ×10, insets) (data are presented as mean (mm3) ± s.d., N = 6 animals per group; † and * indicate P < 0.05 compared to respective controls). Intravenous administration of 7.5 mg kg−1 or 15 mg kg−1 SQAd nano-assemblies just before ischemia or 2 h post-ischemia significantly decreased the infarct volume compared with control groups that received vehicle (dextrose 5%), adenosine-unconjugated SQ nano-assemblies (9.45 mg kg−1) or free adenosine (5.5 mg kg−1). A significant therapeutic effect was also observed when SQAd nano-assemblies were administered 2 h post-ischemia in the permanent MCAo model. B, C) In untreated mice, capillaries in the ischemic brain were filled with trapped erythrocytes, whose hemoglobin was rendered fluorescent by treating brain sections with NaBH4 (C, red, arrowheads) 6 h after re-opening of the MCA following 2 h of occlusion, whereas the majority of capillaries were not clogged in SQAd nano-assemblies-treated mice (B).

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Highlights

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Communication between the elements of NVU is needed for neurovascular coupling.

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Pericytes contribute to regulation of the microvascular blood flow and BBB integrity.

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Pericyte-endothelia communication controls vasculogenesis and leukocyte infiltration.

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Ischemia induces pericyte contraction, hampering reperfusion after recanalization.

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Pericyte dysfunction may contribute to diabetic retinopathy and dementia.

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Microvascular protection is essential for successful neuroprotection.

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