Transl. Stroke Res. DOI 10.1007/s12975-014-0331-4

ORIGINAL ARTICLE

Plasticity of Cerebrovascular Smooth Muscle Cells After Subarachnoid Hemorrhage Lars Edvinsson & Stine Schmidt Larsen & Aida Maddahi & Janne Nielsen

Received: 16 October 2013 / Revised: 19 December 2013 / Accepted: 6 January 2014 # Springer Science+Business Media New York 2014

Abstract Subarachnoid hemorrhage (SAH) is most often followed by a delayed phase of cerebral ischemia which is associated with high morbidity and mortality rates. The causes underlying this delayed phase are still unsettled, but are believed to include cerebral vasospasm, cortical spreading depression, inflammatory reactions, and microthrombosis. Additionally, a large body of evidence indicates that vascular plasticity plays an important role in SAH pathophysiology, and this review aims to summarize our current knowledge on the phenotypic changes of vascular smooth muscle cells of the cerebral vasculature following SAH. In light of the emerging view that the whole cerebral vasculature and the cells of the brain parenchyma should be viewed as one integrated neurovascular network, phenotypical changes are discussed both for the cerebral arteries and the microvasculature. Furthermore, the intracellular signaling involved in the vascular plasticity is discussed with a focus on the Raf–MEK1/2–ERK1/2 pathway which seems to play a crucial role in SAH pathology. Keywords Subarachnoid hemorrhage . Vascular plasticity . Cerebral ischemia . Cerebral arteries . Microvasculature . Vasoconstrictor receptors . Pro-inflammatory cytokines

Introduction Without a properly regulated blood circulation in the brain in the days following subarachnoid hemorrhage (SAH), any L. Edvinsson : S. S. Larsen : J. Nielsen Department of Clinical Experimental Research, Glostrup Research Institute, Glostrup University Hospital, Glostrup, Denmark L. Edvinsson (*) : A. Maddahi Experimental Vascular Research, Department of Medicine, Institute of Clinical Sciences, Lund University, 221 85 Lund, Sweden e-mail: [email protected]

attempt of neuroprotection will have low chances of success. To develop better therapies it is necessary to understand how the brain vasculature and its interactions with neurons and glial cells are altered after SAH. A combined effort may be useful in order to save neurons, glial, and vascular cells (sometimes referred to as the neurovascular unit) [1], which are strongly dependent on each other for survival. Neurovascular coupling, the process by which neuronal activity is ‘sensed’ by the local vasculature that responds by increasing the blood flow to active brain areas, occurs at the level of smaller arterioles and capillaries inside the brain (the microvasculature) [1–3]. Current work centers much on understanding the basic facts of the neurovascular unit and little work has until now examined pathological changes in cerebral microvessels after SAH.1 The larger arteries on the brain surface (the pial arteries) and the parenchymal arterioles are, however, equally important for regulation of brain blood flow [4]. In recent years a growing awareness of the importance of investigating the vascular pathologic changes that occur following cerebral ischemia of both subsets of vasculature with their unique properties is recognized in the field of cerebral ischemic research. We share the view of Zhang and co-workers that the focus for studies of cerebral ischemia should shift to a more integrated view on the interplay between the whole cerebral vasculature and the cells of the brain parenchyma, a concept which has been termed the neurovascular network [5], but in our view the pial arteries should also more clearly be included in the neurovascular network.

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Litterature search strategy and selection criteria: Medline and PubMed were searched for papers in English with the following search terms: cerebral arterioles AND SAH, cerebral arterioles AND SAH AND vasoconstrictor, microvessel AND SAH, microvessel AND SAH AND vasoconstrictor, SAH AND SMC AND vasoconstrictor, SAH AND inflammation AND SMC. The last search was performed on October 14, 2013.

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The importance of the concept of this extended neurovascular network is illustrated by a long series of studies from our laboratory which has revealed that the large cerebral arteries undergo phenotypic changes after experimental focal and global cerebral ischemia and SAH which results in decreased blood supply to ischemic areas and worsens the outcome [6, 7]. In the first part of this review an overview of the phenotypic changes of the pial arteries, also referred to as vascular plasticity as previously defined [6], observed after SAH is presented. Furthermore, the underlying mechanisms for these phenotypic changes are presented, as well as an account of our current view on how these changes are triggered. Interestingly, despite the massive focus on the neurovascular unit in previous studies on cerebral ischemia, very few studies have investigated the pathological changes occurring in the microvasculature itself, but it is our belief that the phenotypic changes in the cerebrovascular smooth muscle cells (SMC) of the large cerebral/pial arteries is a phenomenon inherent to the entire cerebral vasculature including the parenchymal arterioles and that these changes are an important unexplored part of cerebral ischemic research. As mentioned, research has primarily focused on vascular plasticity of the SMC of the pial arteries while work is now in progress on pathological changes of the SMC of the microvasculature. Recent reports have shown similar vascular phenotypic changes of the microvessels in the tissue surrounding the pial arteries following experimental SAH in addition to the phenotypic changes observed in the pial arteries. This includes increased expression of contractile G protein coupled receptors ETB and 5-HT1B in vascular SMC (VSMC) in conjunction with an increased expression of proinflammatory cytokines. The second part of this review will focus on changes of the microvasculature following SAH and their importance in the pathophysiology emphasizing our working hypothesis that important pathological microvascular changes occurs following cerebral ischemia. SAH is most often caused by the rupture of a cerebral arterial aneurysm, which results in a bleeding into the subarachnoid space between the arachnoid mater and the pia mater, and is associated with high mortality and morbidity. Clinically, SAH often shows a biphasic course, with an acute drop in cerebral blood flow (CBF) during, and immediately after, the bleeding and a later phase which results in delayed cerebral ischemia (DCI) and begins between day 2 and 4 postSAH and lasts for up to 14 days in man. This delayed phase is associated with pathological constriction of cerebral arteries (known as cerebral vasospasm, CVS) and is thought to include cortical spreading depression and microthrombosis. A considerable number of molecular mechanisms have been forwarded and these include enhanced superoxide radical generation, inflammation in the brain and in the cerebral vasculature, reduced relaxation by endothelial factors, and enhanced production of contractile factors such as endothelin-1 (ET-1) and 5-hydroxytryptamine (5-HT) [8]. One key factor that clearly

correlates with CVS and DCI is the amount of extravasated blood in the subarachnoid space [9]. The constriction of the cerebral arteries is controlled by the VSMC in the vessel wall. VSMC exhibit a remarkable degree of plasticity and are highly responsive to changes in their environment such as the SAH-related drop in CBF [10], and during the last decade a series of studies have revealed that VSMC plasticity plays a key role in cerebrovascular pathology in SAH.

Plasticity of Cerebral Arteries After SAH Upregulation of Vasoconstrictor Receptors A series of studies from our group have revealed a novel aspect of the cerebrovascular pathology in SAH; it is associated with expressional upregulation of vasoconstrictor receptors in pial arteries (Fig. 1) [thoroughly reviewed in 6, 7]. In a rat model of SAH [11] mRNA and protein levels of contractile endothelin B (ETB) [12], 5-hydroxytryptamine 1B (5-HT1B) [13], angiotensin II type 1 (AT1) [14], and thromboxane A2 (TP) [15] receptors in VSMC is increased in the large pial arteries during the first days post-SAH. This delayed upregulation of vasoconstrictor receptors results in an increased contractile reactivity of pial arteries towards agonists of these receptors [12–15] and the increase in vasoconstrictor receptor expression has been found to correlate with the reduction in regional cerebral blood flow and the level of neurological deficits [10, 16]. Importantly, the above described phenotypic changes in vasoconstrictor receptors in the cerebral vasculature have also been shown to occur in human tissue. To this end organ culture of segments of human cerebral arteries was performed, which was previously shown to be a useful in vitro model for studies of phenotypic changes in the vasculature [6]. Thus, when fresh human cortical arteries removed after neurosurgery are cultured for 48 h, there is an upregulated expression of contractile ETB, 5-HT1B, and AT1 receptors in the VSMC and an altered contractile response to agonists of these receptors [17]. Furthermore, although altered expression of vasoconstrictor receptors in cerebral arteries from SAH patients at this point have not been investigated, gene expression profiling studies and immunohistochemistry studies of cerebral arteries from patients suffering a thromboembolic stroke have shown a similar upregulation of ETB, 5-HT1B, and AT1 receptors in VSMC [18]. The Raf–MEK1/2–ERK1/2 Signal Transduction Pathway in Plasticity of Cerebral Arteries The upregulation of vasoconstrictor receptors occurring after SAH as described above has been shown to be mediated by

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Fig. 1 ETB, 5-HT1B, AT1, and pERK1/2 are upregulated in cerebral arteries after SAH. Representative micrographs which illustrate immunohistochemistry stainings for ETB, 5-HT1B and AT1 receptors, and pERK1/ 2 in the wall of basilar arteries in sham and SAH groups 48 h after induction of experimental SAH. There was a significant increase of all

the investigated proteins in the wall of basilar arteries from animals subjected to SAH as compared to arteries from animals from the sham group. The expression was mostly located in the VSMCs in the medial layer

intracellular signaling via the Raf–mitogen activated protein kinase kinase (MEK1/2)–extracellular regulated kinase 1/2 (ERK1/2) pathway [thoroughly reviewed in 6, 7]. The trigger(s) of receptor upregulation is described in a separate section of this review. The Raf–MEK1/2–ERK1/2 pathway is one of six mammalian mitogen-activated protein kinase (MAPK) signal transduction pathways which are extensively involved in cell regulation and of which the best studied is the ERK, JNK, and p38 MAPK pathways. Each MAPK pathway have a central three-tiered part consisting of a MAPK kinase kinase (MAPKKK) that phosphorylates and activates a MAPK kinase (MAPKK) which then phosphorylates and activates a MAPK (Fig. 2). Downstream targets of the MAPK include transcription factors, other kinases and cytoskeletal components [19]. ERK1/2 is activated by phosphorylation by MEK1/2 and the level of phosphorylated ERK1/2 is increased

in VSMC of pial arteries shortly after the induction of experimental SAH in rats (Fig. 1) [14]. In contrast, p38 and JNK MAPK kinases are only activated 48 h after induction of SAH [14]. In organ cultured human cerebral arteries, phosphorylation of ERK1/2 is also increased [17]. Furthermore, inhibitors of Raf, ERK1/2, or MEK1/2 normalizes vasoconstrictor receptor expression in rats subjected to SAH [14, 20, 21] as do MEK inhibition in human cerebral vessels in organ culture [17]. Moreover, when rats subjected to SAH are treated with inhibitors of the Raf–MEK1/2–ERK1/2 pathway the delayed phase of cerebral hypoperfusion is abrogated and neurological outcome is improved [14, 20–22]. These data indicates that the Raf–MEK1/2–ERK1/2 pathway may be a target for clinical intervention in SAH. In this relation it is noteworthy that the beneficial effect of inhibition of the Raf–MEK1/2–ERK1/ 2 pathway is present even when initiation of treatment is postponed to 6 h post insult, which is a time frame that is relevant in a clinical setting [21–23]. The data presented above underline that signaling through the Raf–MEK1/2–ERK1/2 pathway plays a central role in the pathological changes in constrictive activity of cerebral arteries observed in SAH. However, as described below, the pathological changes in VSMC post-SAH are not restricted to the vasoconstrictor receptors. Induction of Immune Response and Blood–Brain Barrier Breakdown

Fig. 2 Mitogen-activated protein kinase (MAPK) signaling cascades. The figure illustrates the three-tiered core which is common for MAPK signaling pathways, along with the specific kinases involved in the ERK, JNK, and p38 MAPK pathways. Typical stimuli and outcome are also indicated

The pathological changes observed after a cerebral ischemic insult have a strong inflammatory aspect, but its nature differs according to the origin of the cerebral ischemia [24]. The inflammatory processes involved in cerebral damage following SAH are primarily elicited by the spillage of blood cells and blood components into the subarachnoid space, which triggers complement and platelet activation and leads to clot formation. Subsequently, hemolysis of blood cells and

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degradation of blood lead to generation of reactive oxygen species (ROS) which are strong initiators of inflammation [25, 26]. In the early phases of cerebral ischemia the inflammatory responses in the affected cerebral tissue are characterized by production of pro-inflammatory cytokines and chemokines, proteolytic enzymes, and adhesion molecules facilitating leukocyte infiltration into the subarachnoid space and brain parenchyma [25, 27]. The migration of the leukocytes is mediated by adherence of the leukocyte to the adhesion molecules expressed in the endothelial cells and breakdown of the blood–brain barrier (BBB). The increased expression of adhesion molecules, enabling leukocyte infiltration and exacerbation of tissue inflammation in the affected brain regions, is induced by the extravasated blood and the pro-inflammatory cytokines tumor necrosis factor (TNF) α and interleukin (IL)1β [27, 28]. Leukocytes further promote and propagate the inflammatory response by releasing ROS and cytokines [25]. Several studies have shown that the pro-inflammatory cytokines TNFα, IL-1β, and IL-6 are increased in cerebrospinal fluid (CSF) or blood from patients suffering from SAH or in animals subjected to experimental SAH [reviewed in 25]. These cytokines are involved in the induction and regulation of the inflammatory events occurring after SAH. The cytokine TNFα exists both in a membrane-bound form and a soluble form, the latter of which is generated by the proteolytic action of TNFα-converting enzyme. Membrane-bound TNFα interacts with the p75 TNF receptor (TNFR), and p75TNFR can then interact with TNFR associated factors (TRAFs) to induce phosphorylation of inhibitory-κB (IκB), MAPKs, and JNK [reviewed in 28]. Phosphorylation of IκB leads to activation of NFκB, a transcription factor known to be involved in transcription of many inflammatory genes [29]. The p55TNFR interacts with both membrane-bound and soluble TNFα and p55TNFR can induce apoptosis through the adaptor protein TNFR-associated death domain, or activation of MAPK and NFκB through the adaptor protein MAPKactivating death domain [28]. TNFα can be expressed on/ released from endothelial cells, perivascular macrophages, mast cells, microglia, and astrocytes [24, 28], but TNFα is also upregulated at mRNA [30, 31] and protein levels [23] (Fig. 3) in cerebral arteries after experimental SAH. In accordance with our findings, TNFα has been found to be expressed in the vascular wall of the basilar artery after induction of SAH [32]. The expression of TNFα protein was found in VSMC and it increased from 24 h post-SAH and stayed elevated until 96 h post-SAH where the study was terminated [23]. Interestingly, an initial upregulation of TNFα in astrocytes within the brain parenchyma that preceded the upregulation in VSMC was also found, which is in accordance with a previous study showing TNFα expression in rat microglia and astrocytes after transient forebrain ischemia [33]. However, the expression of TNFα in astrocytes started to decrease again at the time when TNFα was upregulated in VSMC, which

suggest that at approximately 24 h post-SAH the expression of TNFα shifts from occurring mainly in the brain parenchyma to occurring mainly in the vasculature [23]. IL-1β is produced as an immature pro-peptide that is cleaved to mature IL-1β by IL-1β-converting enzyme/ caspase 1 in secretory lysozymes, but the exact mechanisms by which IL-1β is then secreted are still discussed [28, 34]. IL1β exerts its effects through the IL-1R1 that associates with the IL-1R accessory protein and recruits the adaptor protein MyD88 which facilitates the interaction with TRAFs and thus activates the same signaling as described above for TNFα [28]. IL-1β and TNFα can be released from endothelial cells, perivascular macrophages, mast cells, microglia, and astrocytes [24, 28]. IL-1β mRNA is upregulated in rat cerebral arteries 24 and 48 h after induction of experimental SAH [30, 31], and Western blotting demonstrated that IL-1β protein levels were also elevated 48 h post-SAH [35]. The finding of upregulation of IL-1β protein was confirmed using immunohistochemistry (Fig. 3), which further showed that the upregulation of IL-1β was found in the VSMC as staining for IL-1β co-localized with staining for smooth muscle actin [35]. Furthermore, in a time–course study the upregulation of IL-1β was evident from 48 to 72 h post-SAH [23]. In accordance with our findings, IL-1β was found to be expressed in VSMC post-SAH in a mouse model of cerebral aneurysms [36]. In contrast to earlier studies [30, 31], a newer study did not detect an upregulation of IL-1β mRNA in the cerebral arteries post-SAH [35], suggesting that the upregulation of IL1β could also be due to a translational upregulation. Alternatively, the observed staining for IL-1β in VSMC may reflect binding of the cytokine to receptors expressed on the VSMC. IL-6 exerts its effects through a receptor complex consisting of IL-6 Rα and gp130 [37]. TNFα and IL-1β can induce the expression of IL-6, and IL-6 is therefore often expressed together with these two cytokines [38]. Interestingly, the level of IL-6 in CSF from SAH patients is higher in patients with a poor clinical outcome [39] and in patients experiencing CVS after SAH [40], indicating that IL-6 plays an important role in SAH pathophysiology. In a gene expression profiling study on cerebral arteries from rats subjected to experimental SAH for 24 h it was found that there was an upregulation of IL-6 mRNA, which was confirmed by quantitative RT-PCR [30, 31, 41]. Upregulation of IL-6 mRNA 48 h post-SAH was not detected, but Western blotting showed that protein levels of IL-6 were elevated in cerebral arteries 48 h post-SAH [35]. From these data it appears that upregulation of IL-6 might be due to both transcriptional and translational regulation, but further studies are required to clarify this. Immunohistochemistry of cerebral arteries showed that IL-6 co-localized with smooth muscle actin, demonstrating that the upregulated IL-6 was found in the VSMC [35] (Fig. 3). A subsequent time–course study showed that IL-6 upregulation had a biphasic profile as IL-6 protein levels were

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Fig. 3 TNF-α, IL-1β, IL-6, iNOS, and MMP-9 are upregulated in cerebral arteries after SAH. Representative micrographs which illustrate immunohistochemistry stainings for TNF-α, IL-1β, IL-6, iNOS, and MMP-9 in the wall of cerebral arteries in sham and SAH groups 48 h after induction of experimental SAH. There was a significant increase of

all the investigated proteins in the wall of cerebral arteries from animals subjected to SAH as compared to arteries from animals from the sham group. The expression of TNF-α, IL-1β, iNOS, and MMP-9 proteins was located in the VSMCs in the medial layer, whereas IL-6 was located in both VSMC and endothelial cells

upregulated at 6 h, but not at 12 h, and then again from 24 h to 72 h post-SAH [23]. Inducible nitric oxide synthase (iNOS) is not expressed in the CNS under normal conditions [42], but under inflammatory conditions iNOS expression can be induced by cytokines such as IL-1β and TNFα, most likely through activation of NFκB, which leads to the expression of iNOS in astrocytes, microglia, and macrophages [43]. NO is a potent vasodilator, and it thus seems counterintuitive that iNOS expression contributes to stroke pathology as treatment with an iNOS inhibitor reduced infarct size and improved neurological outcome after experimental ischemic stroke [44]. However, NO can react with the superoxide anion, a ROS produced during inflammatory processes, to form the potent oxidant peroxynitrite, and both NO and peroxynitrite are highly toxic as they promote protein breakdown, lipid peroxidation, and damage to DNA and mitochondria [43]. In a gene expression profiling study on cerebral arteries from rats subjected to experimental SAH for 24 h the level of iNOS mRNA was also found to be elevated. This finding has been confirmed by quantitative RT-PCR [30, 31, 41]. As for IL-6, upregulation of iNOS mRNA 48 h post-SAH could in a later study not be detected, whereas, Western blotting clearly showed that protein levels of iNOS were increased in cerebral arteries 48 h post-SAH [35]. Thus further studies are required to clarify if upregulation of iNOS in cerebral arteries post-SAH are due to transcriptional or translational regulation or both. Using immunohistochemistry it was demonstrated by co-localization with smooth muscle actin that iNOS is upregulated in VSMC of cerebral arteries [35] (Fig. 3). Signaling through the MAPK signal transduction pathway has been suggested to be involved in the inflammatory response following SAH [45], and in line with this suggestion pERK1/2 was seen to be upregulated in VSMC of cerebral arteries along with the SAH-induced upregulation of TNFα,

IL-1β, IL-6, and iNOS [23, 35]. The upregulation of pERK1/2 was evident from 1 to 72 h post-SAH [23]; further supporting the suggestion that MAPK signaling is involved in the SAHinduced inflammatory response. Noteworthy, inhibition of signaling through the Raf–MEK1/2–ERK1/2 pathway using inhibitors of Raf [35] or MEK1/2 [23] almost completely prevented the upregulation of TNFα, IL-1β, IL-6, and iNOS protein in VSMC of cerebral arteries. This occurred even when the inhibitors were not administered until 6 h postSAH, again underlining that inhibition of the Raf–MEK1/2– ERK1/2 pathway could be applicable within a relevant clinical time-frame. The cells of the brain parenchyma are protected against adverse molecules or cells in the bloodstream by the BBB which restricts the passage of solutes and cells between the blood and the brain parenchyma. The BBB is formed by the endothelial cells lining the cerebral vasculature throughout the brain, and the tightness of the barrier builds mainly on the existence of tight junctions between the endothelial cells [46]. The BBB is disrupted early on in SAH patients [47] and this contributes to formation of brain edema [48]. Additionally, BBB breakdown is as mentioned above thought to contribute to SAH pathophysiology by facilitating the infiltration of vascular walls of cerebral arteries and microvessels by activated leukocytes extravasated from the bloodstream [49]. It is thought that one of the mechanisms behind the BBB breakdown observed in SAH is the proteolysis of tight junction proteins by matrix metalloproteinases (MMPs), especially MMP-9, which would increase BBB permeability and cause edemas. MMP-9 can degrade the tight junction protein zonula occludens-1 [50], and it has been shown that levels of MMP-9 are increased in brains of patients suffering from ischemic or hemorrhagic stroke [51] and in the hippocampus of rats subjected to experimental SAH [52]. MMP-9 can be released from activated platelets, leukocytes, endothelium, and

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astrocytes [24, 26], but our studies show that MMP-9 also is expressed in the vascular wall. Using immunohistochemistry (Fig. 3) and Western blotting an upregulation of MMP-9 in the wall of large cerebral (pial) arteries was demonstrated from rats subjected to SAH [35]. The upregulation is present from 48 h after induction of SAH and disappears again 4 days postSAH [23]. The co-staining for smooth muscle actin showed that the MMP-9 expression localizes to the VSMC [35]. Interestingly, the expression of MMP-9 has also been observed to be increased in the walls of human cerebral aneurysms [53]. The mRNA levels for MMP-9 were found to be upregulated, indicating that there is an increased transcription of the MMP-9 gene in the VSMC [30, 31, 35]. The transcription of MMP-9 is possibly activated downstream of the Raf– MEK1/2–ERK1/2 pathway as inhibitors of Raf and MEK1/2 prevented the upregulation of MMP-9 [23, 35], thereby suggesting that the adverse effects of MMP-9 activation in SAH may be prevented by inhibition of this pathway. Furthermore, the endogenous MMP-9 inhibitor, tissue inhibitor of metalloproteinase (TIMP)-1, was found to be upregulated at mRNA and protein levels in VSMC of pial arteries from rats subjected to SAH, although to a lesser degree than MMP-9 [35], suggesting that there is a compensatory reaction of the vasculature to the MMP-9 activation. Currently, the pathophysiological importance of cytokines and MMPs produced in the cerebrovascular SMC is relatively limited, except that organ culturing cerebral vessel segments in the presence of TNFα or IL-1β results in increased ETA and ETB receptor expression and/or functionality [54].

What Triggers the Plasticity of Cerebral Arteries After SAH? As described above, the Raf–MEK1/2–ERK1/2 pathway is seen to play an essential role in the signal transduction induced by SAH, and the natural next question is then, what initiates the activation of this pathway? The present hypothesis (Fig. 4) suggests that it is the drop in brain blood flow and the decrease in wall tension (reduced shear stress) experienced by cerebral arteries in acute SAH that is the key triggering event which through early activation of the Raf–MEK1/2–ERK1/2 pathway in cerebral arteries initiates the process of delayed cerebrovascular vasoconstrictor receptor upregulation, thereby contributing to the development of DCI. This hypothesis is built on data obtained using three different approaches. First, the stretch-dependence of increased contractility to ETB specific agonists has been investigated in organ cultured cerebral arteries [55]. Here it was found that arterial segments incubated without stretch showed an increased contractility in response to ETB receptor agonist compared to arterial segments incubated with stretch. These in vitro data thus supports the hypothesis that it is the absence of vessel wall stretch that

is the initial trigger for increase in VSMC contractile receptor reactivity. Furthermore, the findings from the organ culture have been confirmed using arterial segments from rats subjected to an MCA occlusion model of thromboembolic stroke. Here, only arterial segments obtained downstream of the occlusion, where there is a drop in blood flow and vascular wall tension, showed increased contractile responses to agonists of vasoconstrictor receptors, whereas this was not seen for arterial segments obtained upstream of the occlusion, although both upstream and downstream segments were subjected to the same low levels of ischemia [56]. Second, the importance of the acute drop in CBF and its duration in experimental SAH has been examined. In the model of experimental SAH employed in our studies [11] blood is injected prechiasmatically, which produces an acute increase in intracranial pressure (ICP) that results in an acute drop in CBF. This is followed by a delayed phase of changes in CBF, neurological deficits, and mortality, which resembles the signs of clinical DCI. The delayed changes are linked more to the increase in ICP, and thus the drop in CBF, rather than to the way it is induced; with saline or blood [57]. Furthermore, when the importance of the duration of the acute drop in CBF during experimental SAH in rats was examined, it was found that the degree of early ERK1/2 activation was determined by the duration of acute drop in CBF, as was the level of delayed upregulation of vasoconstrictor receptors [10]. Cerebrovascular expression of ETB and 5-HT1B receptors was much more clearly upregulated in animals subjected to a prolonged acute drop in CBF, and neurological deficits and mortality at 3 days post-SAH were significantly stronger in animals with a prolonged CBF drop during and acutely after SAH when compared to animals subjected to a short acute CBF drop [10]. In accordance with our findings, the duration of the drop in CBF have also been found to have a major influence on mortality and delayed neuronal cell death in another experimental model of SAH, the cerebral puncture model [58, 59]. As in previous studies [21, 23], inhibition of the Raf–MEK1/2–ERK1/2 pathway by treatment with a MEK inhibitor prevented the SAHinduced delayed vasoconstrictor receptor upregulation, and decreased neurological deficits and delayed mortality [10]. Noteworthy, it was only necessary to treat the animals during the first 24 h post-SAH to achieve these effects [10], and to prevent the upregulation of cytokines and MMP-9 [23] described earlier, which indicates that the signaling through ERK1/2 is no longer critically involved after 24 h, and suggests that ERK1/2 activation acts as a switch-on mechanism for induction of SAH pathology, both with regard to alterations in vasoconstrictor receptors and induction of inflammatory response. Third, the early signal transduction in cerebral arteries after experimental SAH has been studied using a quantitative phosphoproteomic approach [60]. Rat cerebral arteries obtained 0.5 and 1 h after induction of experimental SAH were used for this study and major SAH-induced phosphorylations were

Transl. Stroke Res. Fig. 4 Plasticity of cerebrovascular SMC of pial arteries after SAH. The figure illustrates our current hypothesis as presented in the text. Noteworthy, recent data show that this plasticity also occurs in the cerebrovascular SMC of parenchymal microvessels. Figure updated from [6]

seen on focal adhesion complexes, ERK1/2, calcium calmodulin-dependent kinase II (CaMKII), signal transducer and activator of transcription (STAT3), and c-Jun. Focal adhesion complexes are positioned at the plasma membrane where they serve as a link between the actin cytoskeleton of the cell and the extracellular matrix. Focal adhesion complexes are thus ideally positioned to sense mechanical stimuli such as shear stress, cyclic stretch, and contractile state of the vascular wall and convert these to intracellular signal transduction, and the focal adhesion kinase (FAK) plays a key role in activation of downstream signaling [61]. An upregulated phosphorylation of numerous components of focal adhesion complexes and activation of FAK occurred in response to SAH, which indicates a profound SAH-induced regulation of focal adhesion complexes and actin cytoskeleton dynamics [60]. Furthermore, these findings suggest that FAK may be an early key factor in SAH-induced signaling that, in turn, may activate the Raf–MEK1/2–ERK1/2 pathway which leads to transcription of not only some vasoconstrictor receptor genes but also results in upregulation of cytokines and metalloproteinases in cerebral arteries. In support of this suggestion, lack of wall tension induces activation of FAK in VSMC and downstream activation of ERK1/2 in organcultured cerebral arteries [55]. Additionally, inhibition of the Src kinase which is activated downstream of FAK has been found to reduce ERK1/2 activation and cerebral vasospasm in dogs subjected to experimental SAH [62]. ERK1/2 was as in previous studies found to be activated early on post-SAH induction and the central role of ERK1/2 in

SAH pathophysiology was underlined by the finding that inhibition of ERK1/2 activation using a MEK1/2 inhibitor decreased the phosphorylation of more downstream signaling molecules such as STAT3 and c-Jun. Moreover, treatment with the MEK1/2 inhibitor 6 h post-SAH induction prevented the delayed upregulation of vasoconstrictor receptors and MMP-9, reduced vascular wall thickening, and improved neurological outcome [60]. As already mentioned, phosphoproteomic analysis showed that the transcription factor STAT3 is activated in response to SAH induction, most probably downstream of ERK1/2 as STAT3 phosphorylation was inhibited by MEK1/2 inhibition. This suggests that STAT3 mediates some of the expressional changes induced downstream of ERK1/2 activation. In line with this suggestion, inhibition of STAT3 was found to prevent upregulation of ETB receptor expression and decrease MMP-9 expression in VSMC after SAH, whereas upregulation of 5-HT1B receptors was unaffected [60]. Furthermore, an analysis of genes that were differentially expressed in response to experimental SAH showed that there was enrichment of genes with promoter binding sites for STAT3 within animals subjected to SAH (unpublished). Another transcription factor that may be acting downstream of ERK1/2 is specificity protein 1 (Sp1). Sp1 is found to be upregulated in organ cultures of cerebral arteries, a response that is prevented by ERK1/2 inhibition, and inhibition of Sp1 prevents upregulation of ETB receptors in the VSMC of organ cultured cerebral arteries (unpublished).

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Does Vascular Plasticity Occur in the Cerebral Microvasculature? The Cerebral Pial Vasculature Versus the Parenchymal Vasculature The cerebral vasculature comprises a network of arteries, arterioles, capillaries, venules, and veins which ensures an adequate blood supply to the brain at all times. Thereby a constant delivery of glucose and oxygen to the neurons is obtained and CO2 and metabolic waste products removed. Even so, the function of the pial (large arteries on the surface of the brain) and parenchymal vasculature differs due to the cellular components they come into contact with. Oxygenated blood is continuously led into the brain by the pial arteries as a consequence of flow-metabolism coupling. In addition, the autoregulation mechanism ensures a constant CBF over the physiological range of perfusion pressures. Furthermore, the vascular tone of the cerebral arteries can be modulated by input from perivascular nerves [4]. As mentioned in the introduction, the regional blood supply is regulated by the neurovascular coupling processes in arterioles and capillaries which ensures increased blood supply to regions with high neuronal activity [2, 3]. Accordingly, the regional blood supply is the product of the upstream blood supply and the local microvascular tone. The tone of parenchymal arterioles and capillaries are affected by both intracerebral nerves and astrocytes [1]. The entire parenchymal vasculature is ensheathed by astrocytic processes [63] and the close proximity of these astrocytic endfeet to the arterioles enables release of vasoactive substances acting directly on the local vasculature. Likewise, specific centrally originating interneurons have been shown to be involved in the regional neurally evoked increases in CBF in vivo [64]. The cellular mechanisms governing vasodilatation/vasoconstriction at the neuronal–vascular interface have been thoroughly studied in recent years. The increased neuronal activity leads to release of the neurotransmitter glutamate, which also acts on nearby astrocytic metabotropic receptors initiating a Ca2+ wave in the astrocyte. When the wave of increased Ca2+ reaches the astrocytic endfoot it results in generation of arachidonic acid metabolites and local increases in potassium concentration, which causes VSMC hyperpolarization, leading to vasodilatation in the healthy brain [65]. Changes in the Microvasculature and Neurovascular Coupling After SAH Spreading depolarizations are a pathological phenomenon following SAH that affects both the pial vessels believed to be involved in CVS and the microvasculature where it causes microvascular constriction [66, 67]. Spreading depolarizations are waves of excessive depolarizations propagating through

the cortical grey matter and they are seen in migraine, traumatic brain injury, and SAH patients [67, 68]. Propagation of these depolarizing waves causes neuronal activation with excessive release of excitatory neurotransmitters, and therefore vascular dilations due to the neurovascular coupling response in the normal brain [67]. In contrast, spreading depolarizations occurring in SAH patients results in regional microvascular constrictions and thereby worsens ischemic damage due to transient or prolonged regional cerebral hypoperfusion [66, 69]. Recently, it was shown that neurovascular coupling after SAH is inverted, meaning that electrical neuronal activation in brain slices from SAH rats resulted in regional parenchymal vascular constrictions instead of the dilations seen in healthy brains [70]. These constrictions was found to be transient or prolonged [71] resulting in hypoperfusion of affected brain regions and thereby potentially worsening of ischemic damage. This inverted response was found to be due to elevated local potassium concentrations resulting in vasoconstriction, whereas signaling by arachidonic acid metabolites remained unchanged after SAH [68, 70]. Collectively, these studies point to important pathological changes in the microvascular reactivity following SAH that should be investigated further, both in vivo and in vitro using isolated vascular preparations. Vasoconstrictor Receptor Upregulation and Immune Response Induction in Microvessels As described above, novel insights regarding plastic changes in the vessel wall receptors, proteins associated with the blood–brain barrier, and cytokines has occurred within recent years. The ETB, 5-HT1B, AT1, and TP receptors are upregulated via transcription and translation, and this process might contribute to the development of DCI after SAH. However, there is limited information regarding this process in the cerebral microcirculation. We hypothesize that the plasticity observed in VSMC of large cerebral (pial) arteries also takes place in the cerebral microcirculation and contributes to the increases in microvascular tone following cerebral ischemia. Recent data show that vasoconstrictor receptors are upregulated within the walls of parenchymal microvessels located in close proximity of the large cerebral arteries from rats subjected to experimental SAH. Using immunohistochemistry (Fig. 5), it was found that ETB, 5-HT1B, and AT1 receptors were upregulated in the wall of microvessels [22]. Whether the upregulation of vasoconstrictor receptors following SAH also leads to an increased constriction of microvessels in response to agonists of the upregulated receptors is currently unknown, but should clearly be investigated in future studies to establish the functionality of the observed changes. Furthermore, we are currently undertaking investigations of the cerebral microvasculature in a broader sense, including also

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Fig. 5 ETB, 5-HT1B, AT1, and pERK1/2 are upregulated in microvessels after SAH. Representative micrographs which illustrate immunohistochemistry stainings for ETB, 5-HT1B, and AT1 receptors, and pERK1/2 in the wall of microvessels and in the brain parenchyma in sham and SAH groups 48 h after induction of experimental SAH. There was a significant

increase of all the investigated proteins in the wall of microvessels (arrows) from animals subjected to SAH as compared to microvessels from animals from the sham group. There was also a modest expression of ETB and 5-HT1B proteins in the brain parenchyma in animals from the SAH group

the microvasculature that is located more distantly from the cerebral arteries, with the aim to clarify if the below findings are generally representative for the microvasculature. In microvessels from rats subjected to experimental SAH immunohistochemistry (Fig. 6) showed an upregulation of the cytokines TNFα, IL-1β, and IL-6 in the walls of microvessels 48 and 72 h post-SAH [23, 35]. Protein levels of iNOS appears to be elevated in the walls of microvessels 48 h after induction of SAH (Fig. 6) [35], indicating that there is increased production of NO within and around the microvasculature and consequently a higher risk for cellular damage induced by NO and peroxynitrite. Our data indicates that there could be an increased MMP-9mediated breakdown of the BBB in microvessels after SAH, as immunohistochemistry (Fig. 6) showed an upregulation of MMP-9 in the microvascular wall 48 and 72 h after induction of experimental SAH [23, 35]. The upregulation of MMP-9 was in the microvessels accompanied by upregulation of the endogenous MMP-9 inhibitor TIMP-1 [35].

Role of the Raf–MEK1/2–ERK1/2 Pathway in Microvessels

Fig. 6 TNF-α, IL-1β, IL-6, iNOS, and MMP-9 are upregulated in microvessels after SAH. Representative micrographs which illustrate immunohistochemistry stainings for TNF-α, IL-1β, IL-6, iNOS, and MMP-9 in the wall of microvessels and in the brain parenchyma in sham and SAH groups 48 h after induction of experimental SAH. There was a

significant increase of all the investigated proteins in the wall of microvessels (arrows) from animals subjected to SAH as compared to microvessels from animals from the sham group. Additionally, there was a slight increase of IL-1β and iNOS brain parenchyma of animals subjected to SAH

Signaling through the Raf–MEK1/2–ERK1/2 pathway plays a major role in the vascular changes seen in cerebral arteries after experimental SAH, and our current data points toward an equally important role of the Raf–MEK1/2–ERK1/2 pathway in the microvasculature. Western blotting results show that already 1 h after induction of SAH the levels of pERK1/2 are elevated in microvessels isolated from rat brains, whereas the levels of activated JNK and p38 were not elevated until 48 h post-SAH [14]. The levels of pERK1/2 remain upregulated at 48 h postSAH [14] and localization to the VSMCs was illustrated using immunohistochemistry (Fig. 5). Additionally, an upregulation of pERK1/2 in the walls of microvessels at 48 and 72 h after SAH was found [23, 35]. In line with these findings, it has in humans been shown that there is an activation of ERK1/2 in microvessels within the penumbra after acute ischemic stroke [72].

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The importance of the Raf–MEK1/2–ERK1/2 pathway for the changes observed in the microvasculature in response to SAH has further been supported by results obtained from studies where rats were treated with inhibitors of this pathway after induction of SAH. In these studies inhibition of Raf or MEK1/2 prevented activation of ERK1/2 [14, 22, 23] and inhibited upregulation of the ETB, 5-HT1B, and AT1 vasoconstrictor receptors [22]. The upregulated expression of the cytokines TNFα, IL-1β, and IL-6 was also prevented by inhibition of the Raf–MEK1/2–ERK1/2 pathway as was the upregulation of iNOS and MMP-9 [23, 35]. Notably, upregulation was prevented even when treatment was not initiated until 6 h post-SAH [22, 23, 35], supporting that inhibition of the Raf–MEK1/2–ERK1/2 pathway to prevent pathological changes post-SAH is possible within a clinical relevant timewindow also for changes in the microvasculature.

Concluding Remarks Despite decades of intense research, the mechanisms leading to DCI after SAH are still not fully understood, and at present no effective and specific clinical treatment exists. We have above summarized data obtained which show that SAH is followed by extensive vascular plasticity and that pathological changes in the VSMC of the vasculature includes upregulation of vasoconstrictor receptors and resulting increased contractility, expression of pro-inflammatory cytokines and iNOS as part of an inflammatory response, and increased MMP-9 expression associated with BBB breakdown. Furthermore, these changes appear not only to affect the large cerebral arteries, but probably also occurs in the microvasculature, thereby pointing to a general pathophysiological mechanism affecting the entire cerebral vasculature post-SAH (Fig. 4). The described pathological changes may act in concert with yet other pathological events such as spreading depression, inverted neurovascular coupling and microthrombosis, and this multimodality of the delayed phase of SAH pathophysiology indicates that a future effective treatment of SAH relies on simultaneous targeting multiple mechanisms. Importantly, the data summarized above point out a central role for the Raf–MEK1/2–ERK1/2 signal transduction pathway in mediating the intracellular signaling leading to the described pathological changes, and inhibition of this pathway has beneficial effects on multiple aspects of SAH pathophysiology including upregulation of vasoconstrictor receptors and induction of inflammatory response. Moreover, it seems, on the basis of the reviewed data, plausible that ERK1/2 activation functions as a switch-on mechanism for SAH pathology as prevention of ERK1/2 activation during the first 24 h after a subarachnoid hemorrhage is crucial for improving the outcome. Even more importantly, the data indicate that initiation of inhibition of ERK1/2 activation can be postponed to 6 h

after the bleeding, which means that it would be possible to initiate treatment of patients within a clinically relevant timeframe. Thus, it appears that inhibitors of the Raf–MEK1/2– ERK1/2 pathway act to target several aspects of the pathophysiological mechanisms underlying SAH-related morbidity and mortality, and that such inhibitors are promising candidates for development for clinical use. Acknowledgments The financial support to L.E. from the Swedish Research Council (grant no 5958), the Swedish Heart and Lung Foundation, and the Lundbeck Foundation is gratefully acknowledged. Conflict of Interest The authors, Lars Edvinsson, Stine Schmidt Larsen, Aida Maddahi, and Janne Nielsen declare no conflicts of interest.

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