3.2.3. Astrocytes

tPA is abundantly found in astrocytes, and several stimuli including hypoxia [21] and mechanical injury [22] trigger its release, both in vivo and in vitro. In line with these observations, tPA activates the NF-κB pathway in astrocytes [23], and its release into the basement membrane increases the permeability of the NVU [21]. The mechanism whereby tPA activates the NF-κB pathway is not completely understood. However, work with cerebral cortical astrocytes and rat kidney interstitial fibroblasts (NRK-49F) [24] revealed that the interaction between tPA and LRP1 triggers the phosphorylation of IKK α, which then allows p65/p50 to translocate to the nucleus [25]. Further work with kidney cells has shown that another mechanism whereby tPA activates the NF-κB is by triggering annexin 2-mediated aggregation of the integrin CD11B, which in turn prompts the phosphorylation of IKβ with the resultant nuclear translocation of p65/p50 [26] (Figure 3). In addition to its proinflammatory effect, an increase in the expression and activity of astrocytic tPA induced by multipotent mesenchymal stromal cells has been associated with neurite growth [27]. Furthermore, it has been proposed that astrocytes recycle tPA released in the synaptic cleft in response to glutamatergic signals [28], and that tPA released from astrocytes modulates the microglial response to endotoxins [29]. Together, these data indicate that the release of astrocytic tPA plays a pivotal role in the NVU as a regulator of the permeability of the astrocyte–basement membrane–endothelial cell interface, synaptic transmission, neuroinflammation and microglial function.

**Figure 3.** Mechanisms of tPA-induced NF-κB activation. Representative diagram of the proposed mechanisms whereby tPA activates the NF-κB pathway in the kidney and cerebral cortical astrocytes. In both cases, IKBα phosphorylation is followed by the nuclear translocation of p65/p50.

#### 3.2.4. tPA in Microglia

Inasmuch as a functional link between tPA and microglial activation has been experimentally demonstrated, it is not clear if microglia release tPA. However, it has been proposed that injured neurons release tPA, and that this tPA triggers the release of microglial tPA, which in turn causes neurodegeneration [30]. Independently of these considerations, experimental evidence indicates that tPA mediates endotoxin- and kainic acid-induced microglial activation via an annexin II-mediated mechanism [31] that does not require plasmin generation [32] and triggers neuronal apoptosis [33].

#### 3.2.5. Neuronal tPA

Neurons are a major reservoir of tPA in the central nervous system (CNS), and the release of neuronal tPA in the developing and mature brain plays a central role in the regulation of synaptic function and the response of the CNS to a variety of injuries. Indeed, the release of tPA by neuronal growth cones in the developing brain [34] induces neuronal migration [35] and neurite outgrowth and remodeling [36]. Noticeably, a similar sequence of events in the mature brain promotes neuronal recovery following an ischemic injury [37,38]. In contrast with the developing CNS, in situ zymography studies have revealed that only well-defined areas of the mature brain exhibit tPA-catalyzed proteolytic activity, namely the hippocampus, hypothalamus, thalamus, amygdala, cerebellum and meningeal blood vessels [39]. Furthermore, the interaction of tPA with *N*-methyl-D-aspartate (NMDA) receptors in these structures [40] regulates glutamatergic neurotransmission [41] and promotes the development of synaptic plasticity, as demonstrated in in vitro and in vivo models of long-term potentiation [42], learning [43,44], stress-induced anxiety [45] and visual cortex plasticity [37].

#### *3.3. Urokinase-Type Plasminogen Activator*

Urokinase-type plasminogen activator (uPA) is a 53 kDa serine proteinase secreted as a single-chain uPA (scuPA) with 411 amino acids assembled into three domains: an N-terminal domain homologous to the epidermal growth factor (responsible for its binding to uPAR), a kringle domain that interacts with plasminogen activator inhibitor-1 (PAI-1) and a C-terminal catalytic domain that harbors the active site with the His204, Asp255 and Ser356 amino acids triad [46]. The binding of scuPA to its receptor (uPAR) triggers its cleavage at K158-I159, thus prompting its conversion into an active two-chain form (tcuPA), with an A chain with the epidermal growth factor and kringle domains, and a B chain with the proteolytic domain [47]. In turn, tcuPA catalyzes the conversion of plasminogen into plasmin on the cell surface [48].

The receptor for uPA (uPAR) has 270 amino acids assembled into three cysteinerich Cd59-like sequence domains (D1, D2 and D3) connected by short linker regions and bound to the surface of the plasma membrane by a glycosyl phosphatidylinositol (GPI) tail. Regulation of uPAR is accomplished by either an inactivating uPA-induced cleavage of the receptor between D1 and D2, or by endocytosis of a PAI-1–uPA–uPAR low-density lipoprotein receptor-related protein-1 (LRP1) complex assembled on the cell surface, which then recycles free uPAR back to the membrane to bind to more uPA [49].

#### *3.4. Urokinase-Type Plasminogen Activator in the Neurovascular Unit (NVU)* 3.4.1.CerebralEndothelialCells

A substantial body of experimental evidence indicates that uPA and uPAR are found in endothelial cells [50], and that the release of this uPA and the increase in the expression of uPAR in endothelial cells triggered by a variety of stimuli [51,52] induces cell migration, angiogenesis [53] and capillary branching. However, it is important to take into account that most of these studies have been performed with cell lines, and that very few studies have assessed the in vivo expression of uPA in cerebral endothelial cells. With that in mind, it has been reported that *Cryptococcus neoformans* increases the expression of uPA in cerebral endothelial cells [54], and that microvascular endothelial cells upregulate uPA following an ischemic injury to the spinal cord in vivo [55].

#### 3.4.2. Astrocytes and Microglia

The abundance of uPA and uPAR is increased in glial cell tumors, particularly glioblastoma multiforme, where they have been reported to play a role in tumor growth [56]. In contrast, the role of astrocytic uPA and uPAR under physiological conditions is less well understood. However, recent studies indicate that the release of uPA under physiological conditions triggers astrocytic activation [57] and induces the formation of peripheral astrocytic processes [58]. The expression and role of microglial uPA and uPAR under non-pathological conditions is largely unknown, although in vitro studies have shown that endotoxins, kainic acid and neurogeneration increase their abundance in microglia [59].

## 3.4.3. Neurons

Despite the fact that uPA and uPAR are abundantly found in developing neurons [60–62], their expression changes dramatically over a few days. Hence, while day in vitro (DIV) 3 neurons harbor uPAR in their cell body and neurites, at DIV 7 the expression of this receptor shifts to the axon shaft and growth cones, and at DIV 16 is restricted to the distal segmen<sup>t</sup> of some axons and very few growth cones [60]. Significantly, uPA/uPAR binding during the early stages of development induces neuritogenesis and neuronal migration via a plethora of mechanisms that do not always require plasmin generation [63,64]. More specifically, by promoting activation of integrins and the focal kinase adhesion (FAK) pathway, uPAR regulates the reorganization of the cytoskeleton [63], thus triggering axonal growth, neuronal migration [65] and dendritic branching [66]. In line with these observations, uPAR seems to be pivotal for the formation of neuronal circuits that underlie the development of language and cognition, and dysregulation of the uPA/uPAR system has been linked to epilepsy and cognitive and developmental disorders [67].

In contrast, the expression and role of uPA/uPAR in the mature brain have been less studied. However, recent studies with human, murine and non-human primate brains indicate that uPA is abundantly found in synapses of the second and fifth cortical layers of the cerebral cortex, and that uPA/uPAR binding modulates excitatory neurotransmission by regulating the synaptic expression of neuronal cadherin (NCAD) [68]. Additionally, these studies showed that uPA induces the expression of NCAD in the synapse, and that the resultant generation of NCAD-dimers leads to the formation of synaptic contacts in neurons maintained under physiological conditions [68].

#### **4. Plasminogen Activators in the Neurovascular Unit (NVU) under Ischemic Conditions**

Ischemic stroke is a leading cause of mortality and disability in the world [69]. Significantly, plasminogen activators are pivotal for the protection and repair of the NVU that has suffered an ischemic injury. Indeed, while acute cerebral ischemia causes the rapid release of tPA from each cellular compartment of the NVU [70], the abundance and activity of uPA increase only during the recovery stages from the ischemic insult [61]. These data have led to the proposal that while the early release of tPA restores the patency of the occluded blood vessel and has a neuroprotective effect, the delayed release of uPA promotes the repair of the damaged NVU. Below we will review data on the role of tPA and uPA in each component of the NVU under hypoxic/ischemic conditions.

#### *4.1. Endothelial Cells*

#### 4.1.1. Tissue-Type Plasminogen Activator

The effect of hypoxia on the release of tPA from human cerebrovascular endothelial cells has been poorly characterized. However, studies with human saphenous and umbilical veins [71,72] have shown that hypoxia decreases the abundance and activity of tPA in endothelial cells, and that this effect is accompanied by an increase in the expression and activity of PAI-1. Furthermore, in vitro studies with rat brain microvascular endothelial cells indicate that tPA has a harmful effect on endothelial cells exposed to oxygen and glucose deprivation conditions [4,73]. In contrast with these in vitro studies, in vivo observations have revealed an increase in the concentrations of tPA and PAI-1 in the intravascular space of patients suffering an acute ischemic stroke [74], suggesting that endothelial cells release tPA into the intravascular space as an attempt to restore the patency of the occluded blood vessel. This is supported by the observation of complete or nearly complete recovery of neurological function in acute ischemic stroke patients intravascularly treated with recombinant tPA within 3–4.5 h of the onset of symptoms [75,76].

A growing body of experimental evidence indicates a link between plasminogen activation and the immune system. Indeed, while some studies have revealed that plasminogen activators play a role in bradykinin-mediated endothelial cell activation [77], others show that an interaction between plasmin and factor XII increases the permeability of the neurovascular unit in neurodegenerative diseases [78,79]. Importantly, in apparent discrepancy with a proinflammatory role of plasmin, in vivo studies with an animal model of cerebral ischemia sugges<sup>t</sup> that tPA attenuates the activation of the immune response in the neurovascular unit that has suffered an ischemic injury [80].

#### 4.1.2. Urokinase-Type Plasminogen Activator

The role of uPA in hypoxic/ischemic cerebral endothelial cells is even less well studied. Indeed, although studies with human umbilical endothelial cells (HUVEC) [81] and human microvascular endothelial cells [82] have revealed that a hypoxia-induced, hypoxiainducible factor (HIF)-mediated increase in uPAR expression in endothelial cells triggers angiogenesis and cell migration [83], the effect of hypoxia on uPA and uPAR expression and function in cerebral endothelial cells has not been characterized. Independently of these considerations, clinical studies indicate that the intravascular administration of recombinant uPA effectively restores the patency of the occluded blood vessel and improves neurological outcome in acute ischemic stroke patients [84,85].

## *4.2. Astrocytes*

#### 4.2.1. Tissue-Type Plasminogen Activator

As discussed above, tPA is abundantly found in astrocytes [21], and the release of astrocytic tPA has a direct effect on the permeability of the NVU. Indeed, the interaction between tPA, released from perivascular astrocytes in response to the ischemic injury, and the low-density lipoprotein receptor-related protein-1 (LRP-1) found in astrocytic end-feet processes, activates an NF-κ-regulated inflammatory response [86] and triggers the detachment of perivascular astrocytes from the basement membrane, which in turn increases the permeability of the blood–brain barrier, thus causing ischemic cerebral edema [21]. In line with these observations, the intracerebroventricular administration of recombinant tPA induces an LRP-1-mediated increase in the permeability of the NVU [87]. The translational relevance of these observations is underscored by neuroradiological studies showing that the intravenous administration of recombinant tPA to acute ischemic stroke patients also increases the permeability of the blood–brain barrier [88] which is in line with a reported 10-fold increase in the risk of hemorrhagic complications in recombinant tPA (rtPA)-treated stroke patients [75]. Interestingly, besides its effect on the permeability of the NVU, experimental evidence indicates that multipotent mesenchymal stromal cell-induced tPA activity in astrocytes promotes neurorepair after stroke by facilitating neurite outgrowth in the ischemic area [27,89].

#### 4.2.2. Urokinase-Type Plasminogen Activator

The roles of astrocytic uPA and uPAR in the ischemic brain have only recently been studied. Accordingly, it was reported that the binding of uPA released from neurons to uPAR recruited to the astrocytic plasma membrane in response to the ischemic injury, induces astrocytic activation [57]. Ezrin is a protein that regulates the reorganization of the actin cytoskeleton [90] and the formation of microvilli, filopodia and lamellipodia [91]. In the cytosol, ezrin exists in an inactive conformation. However, following its recruitment to the plasma membrane, ezrin is activated by phosphorylation at a conserved Thr567 residue [92]. Ezrin is abundantly found in astrocytic filopodia [93], and its activation is required for the formation of peripheral astrocytic processes [94]. Significantly, uPA induces the expression of ezrin in astrocytes, thus triggering the formation of peripheral astrocytic processes that, upon embracing the pre- and post-synaptic compartments, preserve the integrity of the tripartite synapse that has suffered an ischemic insult [58].
