The molecular mechanisms underlying cerebral angiogenesis have not been fully investigated. Using primary mouse brain endothelial cells (MBECs) and a capillary-like tube formation assay, we investigated whether the sonic hedgehog (Shh) signaling pathway is coupled with the plasminogen/plasmin system in mediating cerebral angiogenesis. We found that incubation of MBECs with recombinant human Shh (rhShh) substantially increased the tube formation in naïve MBECs. This was associated with increases in tissue plasminogen activator (tPA) activation and reduction of plasminogen activator inhibitor 1 (PAI-1). Blockage of the Shh pathway with cyclopamine abolished the induction of tube formation and the effect of rhShh on tPA and PAI-1. Addition of PAI-1 reduced rhShh-augmented tube formation. Genetic ablation of tPA in MBECs impaired tube formation and downregulated of vascular endothelial growth factor (VEGF) and angiopoietin 1 (Ang1). Addition of rhShh to tPA-/- MBECs only partially restored the tube formation and upregulated Ang1, but not VEGF, although rhShh increased VEGF and Ang1 expression on wild-type MBECs. Complete restoration of tube formation in tPA-/- MBECs was observed only when both exogenous Shh and tPA were added. The present study provides evidence that tPA and PAI-1 contribute to Shh-induced in vitro cerebral angiogenesis.

Tissue plasminogen activator (tPA) has been implicated in neurite outgrowth and neurological recovery post stroke. tPA converts the zymogen plasminogen (Plg) into plasmin. In this study, using plasminogen knockout (Plg-/-) mice and their Plg-native littermates (Plg+/+), we investigated the role of Plg in axonal remodeling and neurological recovery after stroke. Plg+/+ and Plg-/- mice (n = 10/group) were subjected to permanent intraluminal monofilament middle cerebral artery occlusion (MCAo). A foot-fault test and a single pellet reaching test were performed prior to and on day 3 after stroke, and weekly thereafter to monitor functional deficit and recovery. Biotinylated dextran amine (BDA) was injected into the left motor cortex to anterogradely label the corticospinal tract (CST). Animals were euthanized 4 weeks after stroke. Neurite outgrowth was also measured in primary cultured cortical neurons harvested from Plg+/+ and Plg-/- embryos. In Plg+/+ mice, the motor functional deficiency after stroke progressively recovered with time. In contrast, recovery in Plg-/- mice was significantly impaired compared to Plg+/+ mice (p<0.01). BDA-positive axonal density of the CST originating from the contralesional cortex in the denervated side of the cervical gray matter was significantly reduced in Plg-/- mice compared with Plg+/+ mice (p<0.05). The behavioral outcome was highly correlated with the midline-crossing CST axonal density (R2>0.82, p<0.01). Plg-/- neurons exhibited significantly reduced neurite outgrowth. Our data suggest that plasminogen-dependent proteolysis has a beneficial effect during neurological recovery after stroke, at least in part, by promoting axonal remodeling in the denervated spinal cord.

Traumatic brain injury (TBI) is a major cause of death and long-term disability worldwide. To date, there are no effective pharmacological treatments for TBI. Recombinant human tissue plasminogen activator (tPA) is the effective drug for the treatment of acute ischemic stroke. In addition to its thrombolytic effect, tPA is also involved in neuroplasticity in the central nervous system. However, tPA has potential adverse side effects when administered intravenously including brain edema and hemorrhage. Here we report that tPA, administered by intranasal delivery during the subacute phase after TBI, provides therapeutic benefit. Animals with TBI were treated intranasally with saline or tPA initiated 7 days after TBI. Compared with saline treatment, subacute intranasal tPA treatment significantly 1) improved cognitive (Morris water maze test) and sensorimotor (footfault and modified neurological severity score) functional recovery in rats after TBI, 2) reduced the cortical stimulation threshold evoking ipsilateral forelimb movement, 3) enhanced neurogenesis in the dentate gyrus and axonal sprouting of the corticospinal tract originating from the contralesional cortex into the denervated side of the cervical gray matter, and 4) increased the level of mature brain-derived neurotrophic factor. Our data suggest that subacute intranasal tPA treatment improves functional recovery and promotes brain neurogenesis and spinal cord axonal sprouting after TBI, which may be mediated, at least in part, by tPA/plasmin-dependent maturation of brain-derived neurotrophic factor.

Multipotent mesenchymal stromal cells (MSCs) decrease the expression of transforming growth factor β1 (TGFβ1) in astrocytes and subsequently decrease astrocytic plasminogen activator inhibitor 1 (PAI-1) level in an autocrine manner. Since activated microglia/macrophages are also a source of TGFβ1 after stroke, we therefore tested whether MSCs regulate TGFβ1 expression in microglia/macrophages and subsequently alters PAI-1 expression after ischemia. TGFβ1 and its downstream effector phosphorylated SMAD 2/3 (p-SMAD 2/3) were measured in mice subjected to middle cerebral artery occlusion (MCAo). MSC treatment significantly decreased TGFβ1 protein expression in both astrocytes and microglia/macrophages in the ischemic boundary zone (IBZ) at day 14 after stroke. However, the p-SMAD 2/3 was only detected in astrocytes and decreased after MSC treatment. In vitro, RT-PCR results showed that the TGFβ1 mRNA level was increased in both astrocytes and microglia/macrophages in an astrocyte-microglia/macrophage co-culture system after oxygen-glucose deprived (OGD) treatment. MSCs treatment significantly decreased the above TGFβ1 mRNA level under OGD conditions, respectively. OGD increased the PAI-1 mRNA in astrocytes in the astrocyte-microglia/macrophage co-culture system, and MSC administration significantly decreased this level. PAI-1 mRNA was very low in microglia/macrophages compared with that in astrocytes under different conditions. Western blot results also verified that MSC administration significantly decreased p-SMAD 2/3 and PAI-1 level in astrocytes in astrocyte-microglia/macrophage co-culture system under OGD conditions. Our in vivo and in vitro data, in concert, suggest that MSCs decrease TGFβ1 expression in microglia/macrophages in the IBZ which contribute to the down-regulation of PAI-1 level in astrocytes.

We demonstrate that tissue plasminogen activator (tPA) and its inhibitors contribute to neurite outgrowth in the central nervous system (CNS) after treatment of stroke with multipotent mesenchymal stromal cells (MSCs). In vivo, administration of MSCs to mice subjected to middle cerebral artery occlusion (MCAo) significantly increased activation of tPA and downregulated PAI-1 levels in the ischemic boundary zone (IBZ) compared with control PBS treated mice, concurrently with increases of myelinated axons and synaptophysin. In vitro, MSCs significantly increased tPA levels and concomitantly reduced plasminogen activator inhibitor 1 (PAI-1) expression in astrocytes under normal and oxygen and glucose deprivation (OGD) conditions. ELISA analysis of conditioned medium revealed that MSCs stimulated astrocytes to secrete tPA. When primary cortical neurons were cultured in the conditioned medium from MSC co-cultured astrocytes, these neurons exhibited a significant increase in neurite outgrowth compared to conditioned medium from astrocytes alone. Blockage of tPA with a neutralizing antibody or knock-down of tPA with siRNA significantly attenuated the effect of the conditioned medium on neurite outgrowth. Addition of recombinant human tPA into cortical neuronal cultures also substantially enhanced neurite outgrowth. Collectively, these in vivo and in vitro data suggest that the MSC mediated increased activation of tPA in astrocytes promotes neurite outgrowth after stroke.

Stroke is a leading cause of death and disability worldwide, mainly affecting the elderly. Unfortunately, current treatments for acute ischemic stroke warrant improvement. To date, tissue plasminogen activator (tPA) is of limited use in stroke patients mainly due to its narrow therapeutic window and potential for hemorrhagic complication. The adjuvant treatment with Vepoloxamer, a purified amphipathic polymer has been shown to enhance the thrombolytic efficacy of tPA treatment in young adult male rats after embolic stroke. However, most stroke patients are aged; therefore, the current study investigated the therapeutic effect of the combined tPA and Vepoloxamer treatment in aged male and female rats subjected to embolic stroke.

Bone marrow stromal cells (MSCs) improve neurologic recovery after middle cerebral artery occlusion (MCAo). To examine whether in vivo blockage of the endogenous sonic hedgehog (Shh) pathway affects grafted MSC-induced neurologic benefits, MCAo mice were administered: vehicle (control); cyclopamine (CP)- a specific Shh pathway inhibitor; MSC; and MSC and cyclopamine (MSC-CP). Neurologic function was evaluated after MCAo. Electron microscopy and immunofluorescence staining were employed to measure synapse density, protein expression of tissue plasminogen activator (tPA), and Shh in parenchymal cells in the ischemic boundary zone (IBZ), respectively. Marrow stromal cell treatment significantly enhanced functional recovery after ischemia, concurrent with increases of synaptophysin, synapse density, and myelinated axons along the IBZ, and significantly increased tPA and Shh expression in astrocytes and neurons compared with control. After treatment with MSC-CP or CP, the above effects were reversed. Co-culture of MSCs with cortical neurons confirmed the effect of Shh on MSC-mediated neurite outgrowth. Our data support the hypothesis that the Shh pathway mediates brain plasticity via tPA and thereby functional recovery after treatment of stroke with MSCs.

Tissue plasminogen activator (tPA) is a serine protease primarily involved in mediating thrombus breakdown and regulating catabolism of amyloid-beta (Aβ). The aim of this study is to investigate age-dependent decline of endogenous tPA and the effects of tPA decline on glymphatic function and cognitive outcome in mice. Male, young (3m), adult (6m) and middle-aged (12m) C57/BL6 (wild type) and tPA knockout (tPA-/-) mice were subject to a battery of cognitive tests and white matter (WM) integrity, neuroinflammation, and glymphatic function were evaluated. Adult WT mice exhibit significantly decreased brain tPA level compared to young WT mice and middle-aged WT mice have significantly lower brain tPA levels than young and adult WT mice. Middle-aged WT mice exhibit significant neuroinflammation, reduced WM integrity and increased thrombin deposition compared to young and adult mice, and increased blood brain barrier (BBB) permeability and reduced cognitive ability compared to young WT mice. In comparison to adult WT mice, adult tPA-/- mice exhibit significant BBB leakage, decreased dendritic spine density, increased thrombin deposition, neuroinflammation, and impaired functioning of the glymphatic system. Compared to age-matched WT mice, adult and middle-aged tPA-/- mice exhibit significantly increased D-Dimer expression and decreased perivascular Aquaporin-4 expression. Compared to age-matched WT mice, young, adult and middle-aged tPA-/- mice exhibit significant cognitive impairment, axonal damage, and increased deposition of amyloid precursor protein (APP), Aβ, and fibrin. Endogenous tPA may play an important role in contributing to aging induced cognitive decline, axonal/WM damage, BBB disruption and glymphatic dysfunction in the brain.

Diabetes leads to an elevated risk of stroke and worse functional outcome compared to the general population. We investigate whether L-4F, an economical ApoA-I mimetic peptide, reduces neurovascular and white-matter damage in db/db type-2 diabetic (T2DM) stroke mice. L-4F (16 mg/kg, subcutaneously administered initially 2 h after stroke and subsequently daily for 4 days) reduced hemorrhagic transformation, decreased infarct-volume and mortality, and treated mice exhibited increased cerebral arteriole diameter and smooth muscle cell number, decreased blood-brain barrier leakage and white-matter damage in the ischemic brain as well as improved neurological functional outcome after stroke compared with vehicle-control T2DM mice (p < 0.05, n = 11/group). Moreover, administration of L-4F mitigated macrophage infiltration, and reduced the level of proinflammatory mediators tumor necrosis factor alpha (TNFα), high-mobility group box-1 (HMGB-1)/advanced glycation end-product receptor (RAGE) and plasminogen activator inhibitor-1 (PAI-1) in the ischemic brain in T2DM mice (p < 0.05, n = 6/group). In vitro, L-4F treatment did not increase capillary-like tube formation in mouse-brain endothelial cells, but increased primary artery explant cell migration derived from C57BL/6-aorta 1 day after middle cerebral artery occlusion (MCAo), and enhanced neurite-outgrowth after 2 h of oxygen-glucose deprivation and axonal-outgrowth in primary cortical neurons derived from the C57BL/6-embryos subjected to high-glucose condition. This study suggests that early treatment with L-4F provides a potential strategy to reduce neuroinflammation and vascular and white-matter damage in the T2DM stroke population.

We reported that mesenchymal stromal cells (MSCs) enhance neurological recovery from experimental stroke and increase tissue plasminogen activator (tPA) expression in astrocytes. Here, we investigate mechanisms by which tPA mediates MSC enhanced axonal outgrowth. Primary murine neurons and astrocytes were isolated from wild-type (WT) and tPA-knockout (KO) cortices of embryos. Mouse MSCs (WT) were purchased from Cognate Inc. Neurons (WT or KO) were seeded in soma side of Xona microfluidic chambers, and astrocytes (WT or KO) and/or MSCs in axon side. The chambers were cultured as usual (normoxia) or subjected to oxygen deprivation. Primary neurons (seeded in plates) were co-cultured with astrocytes and/or MSCs (in inserts) for Western blot. In chambers, WT axons grew significantly longer than KO axons and exogenous tPA enhanced axonal outgrowth. MSCs increased WT axonal outgrowth alone and synergistically with WT astrocytes at both normoxia and oxygen deprivation conditions. The synergistic effect was inhibited by U0126, an ERK inhibitor, and receptor associated protein (RAP), a low density lipoprotein receptor related protein 1 (LRP1) ligand antagonist. However, MSCs exerted neither individual nor synergistic effects on KO axonal outgrowth. Western blot showed that MSCs promoted astrocytic tPA expression and increased neuronal tPA alone and synergistically with astrocytes. Also, MSCs activated neuronal ERK alone and synergistically with astrocytes, which was inhibited by RAP. We conclude: (1) MSCs promote axonal outgrowth via neuronal tPA and synergistically with astrocytic tPA; (2) neuronal tPA is critical to observe the synergistic effect of MSC and astrocytes on axonal outgrowth; and (3) tPA mediates MSC treatment-induced axonal outgrowth through the LRP1 receptor and ERK.

APX3330 is a selective inhibitor of APE1/Ref-1 redox activity. In this study, we investigate the therapeutic effects and underlying mechanisms of APX3330 treatment in type one diabetes mellitus (T1DM) stroke rats. Adult male Wistar rats were induced with T1DM and subjected to transient middle cerebral artery occlusion (MCAo) and treated with either PBS or APX3330 (10mg/kg, oral gavage) starting at 24h after MCAo, and daily for 14 days. Rats were sacrificed at 14 days after MCAo and, blood brain barrier (BBB) permeability, ischemic lesion volume, immunohistochemistry, cell death assay, Western blot, real time PCR, and angiogenic ELISA array were performed. Compared to PBS treatment, APX3330 treatment of stroke in T1DM rats significantly improves neurological functional outcome, decreases lesion volume, and improves BBB integrity as well as decreases total vessel density and VEGF expression, while significantly increases arterial density in the ischemic border zone (IBZ). APX3330 significantly increases myelin density, oligodendrocyte number, oligodendrocyte progenitor cell number, synaptic protein expression, and induces M2 macrophage polarization in the IBZ of T1DM stroke rats. Compared to PBS treatment, APX3330 treatment significantly decreases plasminogen activator inhibitor type-1 (PAI-1), monocyte chemotactic protein-1 and matrix metalloproteinase 9 (MMP9) and receptor for advanced glycation endproducts expression in the ischemic brain of T1DM stroke rats. APX3330 treatment significantly decreases cell death and MMP9 and PAI-1 gene expression in cultured primary cortical neurons subjected to high glucose and oxygen glucose deprivation, compared to untreated control cells. APX3330 treatment increases M2 macrophage polarization and decreases inflammatory factor expression in the ischemic brain as well as promotes neuroprotective and neurorestorative effects after stroke in T1DM rats.