Plasminogen Activator Inhibitor-1 Expression by Brain Microvessel Endothelial Cells Is Inhibited by Elevated Glucose

Abstract: Patients with diabetes are predisposed to microvascular disease. In the retina and brain, this is characterized by neovascularization and new capillary formation. Because of the potential importance of plasmin generation in these processes, we evaluated the effect of elevated glucose concentrations on expression of plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA), and urokinase (uPA) in cultured bovine brain endothelial cells (BBEC) versus cultured bovine aortic endothelial cells (BAEC). We observed that BBEC PAI-1 mRNA levels were decreased fivefold in cells cultured in media containing 20 m M glucose compared with BBEC cultured in media with 5.5 m M glucose, whereas expression of PAI-1 mRNA in BAEC, bovine mesenteric endothelial cells, and human umbilical vein endothelial cells was not modulated under these conditions. Expression of PAI-1 protein was also inhibited by growth of BBEC in elevated glucose, but the effect was less marked than at the mRNA level. Elevated glucose did not decrease expression of PAI-1 protein by BAEC. Withdrawal of acidic fibroblast growth factor enhanced expression of PAI-1 mRNA and protein in BBEC. Expression of tPA mRNA was not affected by the glucose concentration of the medium, and uPA mRNA was not detected in our BBEC cultures. A decrease in the local tissue activity of PAI-1 by elevated glucose concentrations, with no effect on tPA or uPA expression, would lead to an increase in the plasmin activity and thereby predispose neural tissues, such as the cerebrum and retina, of diabetic patients to neovascularization.     

Stem Cells Downregulate the Elevated Levels of Tissue Plasminogen Activator in Rats After Spinal Cord Injury

We investigated the involvement of tPA after SCI in rats and effect of treatment with human umbilical cord blood derived stem cells. tPA expression and activity were determined in vivo after SCI in rats and in vitro in rat embryonic spinal neurons in response to injury with staurosporine, hydrogen peroxide and glutamate. The activity and/or expression of tPA increased after SCI and reached peak levels on day 21 post-SCI. Notably, the tPA mRNA activity was upregulated by 310-fold compared to controls on day 21 post-SCI. As expected, MBP expression is minimal at the time of peak tPA activity and vice versa. Implantation of hUCB after SCI resulted in the downregulation of elevated tPA activity/expression in vivo in rats as well as in vitro in spinal neurons. Our results demonstrated the involvement of tPA in the secondary pathogenesis after SCI as well as the therapeutic potential of hUCB.     

Cleavage of the NR2B subunit amino terminus of N-methyl-D-aspartate (NMDA) receptor by tissue plasminogen activator: identification of the cleavage site and characterization of ifenprodil and glycine affinities on truncated NMDA receptor

Thrombolysis using tissue plasminogen activator (tPA) has been the key treatment for patients with acute ischemic stroke for the past decade. Recent studies, however, suggest that this clot-busting protease also plays various roles in brain physiological and pathophysiological glutamatergic-dependent processes, such as synaptic plasticity and neurodegeneration. In addition, increasing evidence implicates tPA as an important neuromodulator of the N-methyl-d-aspartate (NMDA) receptors. Here, we demonstrate that recombinant human tPA cleaves the NR2B subunit of NMDA receptor. Analysis of NR2B in rat brain lysates and cortical neurons treated with tPA revealed concentration- and time-dependent degradation of NR2B proteins. Peptide sequencing studies performed on the cleaved-off products obtained from the tPA treatment on a recombinant fusion protein of the amino-terminal domain of NR2B revealed that tPA-mediated cleavage occurred at arginine 67 (Arg(67)). This cleavage is tPA-specific, plasmin-independent, and removes a predicted ~4-kDa fragment (Arg(27)-Arg(67)) from the amino-terminal domain of the NR2B protein. Site-directed mutagenesis of putative cleavage site Arg(67) to Ala(67) impeded tPA-mediated degradation of recombinant protein. This analysis revealed that NR2B is a novel substrate of tPA and suggested that an Arg(27)-Arg(67)-truncated NR2B-containing NMDA receptor could be formed. Heterologous expression of NR2B with Gln(29)-Arg(67) deleted is functional but exhibits reduced ifenprodil inhibition and increased glycine EC(50) with no change in glutamate EC(50). Our results confirmed NR2B as a novel proteolytic substrate of tPA, where tPA may directly interact with NR2B subunits leading to a change in pharmacological properties of NR2B-containing NMDA receptors.     

tPA Regulates Neurite Outgrowth by Phosphorylation of LRP5/6 in Neural Progenitor Cells

Despite the important role of tissue plasminogen activator (tPA) as a neuromodulator in neurons, microglia, and astrocytes, its role in neural progenitor cell (NPC) development is not clear yet. We identified that tPA is highly expressed in NPCs compared with neurons. Inhibition of tPA activity or expression using tPA stop, PAI-1, or tPA siRNA inhibited neurite outgrowth from NPCs, while overexpression or addition of exogenous tPA increased neurite outgrowth. The expression of Wnt and β-catenin as well as phosphorylation of LRP5 and LRP6, which has been implicated in Wnt–β-catenin signaling, was rapidly increased after tPA treatment and was decreased by tPA siRNA transfection. Knockdown of β-catenin or LRP5/6 expression by siRNA prevented tPA-induced neurite extension. NPCs obtained from tPA KO mice showed impaired neurite outgrowth compared with WT NPCs. In ischemic rat brains, axon density was higher in the brains transplanted with WT NPCs than in those with tPA KO NPCs, suggesting increased axonal sprouting by NPC-derived tPA. tPA-mediated regulation of neuronal maturation in NPCs may play an important role during development and in regenerative conditions.     

Activity deprivation induces neuronal cell death: mediation by tissue-type plasminogen activator

Spontaneous activity is an essential attribute of neuronal networks and plays a critical role in their development and maintenance. Upon blockade of activity with tetrodotoxin (TTX), neurons degenerate slowly and die in a manner resembling neurodegenerative diseases-induced neuronal cell death. The molecular cascade leading to this type of slow cell death is not entirely clear. Primary post-natal cortical neurons were exposed to TTX for up to two weeks, followed by molecular, biochemical and immunefluorescence analysis. The expression of the neuronal marker, neuron specific enolase (NSE), was down-regulated, as expected, but surprisingly, there was a concomitant and striking elevation in expression of tissue-type plasminogen activator (tPA). Immunofluorescence analysis indicated that tPA was highly elevated inside affected neurons. Transfection of an endogenous tPA inhibitor, plasminogen activator inhibitor-1 (PAI-1), protected the TTX-exposed neurons from dying. These results indicate that tPA is a pivotal player in slowly progressing activity deprivation-induced neurodegeneration.     

Biphasic regulation of tissue plasminogen activator activity in ischemic rat brain and in cultured neural cells: essential role of astrocyte-derived plasminogen activator inhibitor-1

In brain, the serine protease tissue plasminogen activator (tPA) and its endogenous inhibitor plasminogen activator inhibitor-1 (PAI-1) have been implicated in the regulation of various neurophysiological and pathological responses. In this study, we investigated the differential role of neurons and astrocytes in the regulation of tPA/PAI-1 activity in ischemic brain. The activity of tPA peaked transiently and then decreased in cortex and striatum along with delayed induction of PAI-1 in the inflammatory stage after MCAO/reperfusion injury. In cultured primary cells, glutamate stimulation increased tPA activity in neurons but not in other cells such as microglia and astrocytes. With LPS stimulation, a model of neuroinflammatory insults, robust PAI-1 induction was observed in astrocytes but not in neurons and microglia. The upregulation of PAI-1 by LPS in astrocytes was also verified by RT-PCR analysis as well as PAI-1 promoter reporter assay. Lastly, we checked the effects of hypoxia on tPA/PAI-1 activity. Hypoxia increased tPA release from neurons without effects on microglia, while the activity of tPA in astrocyte was decreased consistent with increased PAI-1 activity in astrocyte. Taken together, the results from the present study suggest that neurons are the major source of tPA and that the glutamate-induced stimulated release is mainly governed by neurons in the acute phase. In contrast, the massive up-regulation of PAI-1 in astrocytes during subchronic and chronic inflammatory conditions, leads to decreased tPA activity in the later stages of MCAO. Differential regulation of tPA and PAI-1 in neurons, astrocytes and microglia suggest more attention is required to understand the role of local tPA activity in the vicinity of individual cell types.     

Tissue plasminogen activator neurovascular toxicity is controlled by activated protein C

Although thrombolytic effects of tissue plasminogen activator (tPA) are beneficial, its neurotoxicity is problematic. Here, we report that tPA potentiates apoptosis in ischemic human brain endothelium and in mouse cortical neurons treated with N-methyl-D-aspartate (NMDA) by shifting the apoptotic pathways from caspase-9 to caspase-8, which directly activates caspase-3 without amplification through the Bid-mediated mitochondrial pathway. In vivo, tPA-induced cerebral ischemic injury in mice was reduced by intracerebroventricular administration of caspase-8 inhibitor, but not by caspase-9 inhibitor, in contrast to controls in which caspase-9 inhibitor, but not caspase-8 inhibitor, was protective. Activated protein C (APC), a serine protease with anticoagulant, anti-inflammatory and antiapoptotic activities, which is neuroprotective during transient ischemia and promotes activation of antiapoptotic mechanisms in brain cells by acting directly on endothelium and neurons, blocked tPA vascular and neuronal toxicities in vitro and in vivo. APC inhibited tPA-induced caspase-8 activation of caspase-3 in endothelium and caspase-3-dependent nuclear translocation of apoptosis-inducing factor in NMDA-treated neurons and reduced tPA-mediated cerebral ischemic injury in mice. Data suggest that tPA shifts the apoptotic signal in stressed brain cells from the intrinsic to the extrinsic pathway which requires caspase-8. APC blocks tPA's neurovascular toxicity and may add substantially to the effectiveness of tPA therapy for stroke.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates the expression and the release of tissue plasminogen activator (tPA) in neuronal cells: involvement of tPA in the neuroprotective effect of PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) and tissue plasminogen activator (tPA) play important roles in neuronal migration and survival. However, a direct link between the neurotrophic effects of PACAP and tPA has never been investigated. In this study, we show that, in PC12 cells, PACAP induced a 9.85-fold increase in tPA gene expression through activation of the protein kinase A- and protein kinase C-dependent signaling pathways. In immature cerebellar granule neurons (CGN), PACAP stimulated tPA mRNA expression and release of proteolytically active tPA. Immunocytochemical labeling revealed the presence of tPA in the cytoplasm and processes of cultured CGN. The inhibitory effect of PACAP on CGN motility was not affected by the tPA substrate plasminogen or the tPA inhibitor plasminogen activator inhibitor-1. In contrast, plasminogen activator inhibitor-1 significantly reduced the stimulatory effect of PACAP on CGN survival. Altogether, these data indicate that tPA gene expression is activated by PACAP in both tumoral and normal neuronal cells. The present study also demonstrates that PACAP stimulates the release of tPA which promotes CGN survival by a mechanism dependent of its proteolytic activity.     

Developmental patterns of doublecortin expression and white matter neuron density in the postnatal primate prefrontal cortex and schizophrenia

Postnatal neurogenesis occurs in the subventricular zone and dentate gyrus, and evidence suggests that new neurons may be present in additional regions of the mature primate brain, including the prefrontal cortex (PFC). Addition of new neurons to the PFC implies local generation of neurons or migration from areas such as the subventricular zone. We examined the putative contribution of new, migrating neurons to postnatal cortical development by determining the density of neurons in white matter subjacent to the cortex and measuring expression of doublecortin (DCX), a microtubule-associated protein involved in neuronal migration, in humans and rhesus macaques. We found a striking decline in DCX expression (human and macaque) and density of white matter neurons (humans) during infancy, consistent with the arrival of new neurons in the early postnatal cortex. Considering the expansion of the brain during this time, the decline in white matter neuron density does not necessarily indicate reduced total numbers of white matter neurons in early postnatal life. Furthermore, numerous cells in the white matter and deep grey matter were positive for the migration-associated glycoprotein polysialiated-neuronal cell adhesion molecule and GAD65/67, suggesting that immature migrating neurons in the adult may be GABAergic. We also examined DCX mRNA in the PFC of adult schizophrenia patients (n?=?37) and matched controls (n?=?37) and did not find any difference in DCX mRNA expression. However, we report a negative correlation between DCX mRNA expression and white matter neuron density in adult schizophrenia patients, in contrast to a positive correlation in human development where DCX mRNA and white matter neuron density are higher earlier in life. Accumulation of neurons in the white matter in schizophrenia would be congruent with a negative correlation between DCX mRNA and white matter neuron density and support the hypothesis of a migration deficit in schizophrenia.     

Reciprocal actions of NCAM and tPA via a Ras-dependent MAPK activation in rat hippocampal neurons

In an attempt to identify the functions of neural cell adhesion molecule (NCAM) and tissue plasminogen activator (tPA) in hippocampal synaptic plasticity, we investigated the relationship between the two molecules by focusing on mitogen-activated protein kinase (MAPK), an essential enzyme in this process. NCAM clustering in cultured hippocampal neurons transiently induced MAPK within 10min. Moreover, soluble NCAM also induced a Ras-dependent MAPK activation. Conversely, MAPK activation led to an increase in the expressions of all three isoforms of NCAM. Treatment of neurons with tPA and plasminogen induced a Ras-dependent MAPK activation and tPA-plasmin degradation of NCAM was mediated in a MAPK-dependent manner. Soluble NCAM transiently inhibited tPA mRNA expression levels in a MAPK-dependent manner, while stimulation of MAPK alone induced tPA reduction in cells. These results collectively indicate that NCAM and tPA reciprocally act as important regulators in the modulation of synaptic plasticity via a Ras-MAPK-involved signaling pathway. In turn, MAPK activation may cause tPA degradation or a decrease in expression to promote synaptic plasticity.     

Tissue plasminogen activator and its receptor in the human amnion, chorion, and decidua at preterm and term

The plasminogen activator system consists of two proteins: tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which act upon their specific receptors to generate plasmin from plasminogen located on the cell surface. Plasmin then acts directly and indirectly to degrade the components of the extracellular matrix (ECM). This process is likely to be important in the normal turnover of the ECM of fetal membranes and in its premature weakening in preterm premature rupture of the fetal membranes. Quantitative Northern analysis and in situ hybridization have shown that the decidua expresses mRNA for tPA. However, the immunolocalized tPA protein was most strongly associated with the amnion and chorion, as was its receptor annexin II, suggesting that the amnion and chorion are the targets for decidual tPA. At term, decidual tPA expression was unaffected by labor, and the tPA receptor was elevated both before and after labor. At preterm, the converse was found: decidual tPA expression was significantly (p < 0. 05) up-regulated by labor, but the tPA receptor was not. The results suggest that the generation of plasmin at term would be controlled by an increased concentration of the tPA receptor in the amnion and chorion, whereas at preterm a pathological increase in plasmin would be generated by an overexpression of tPA, initiated by labor.     

Neuroprotective effect of neuroserpin in rat primary cortical cultures after oxygen and glucose deprivation and tPA

Besides its role as a thrombolytic agent, tissue plasminogen activator (tPA) triggers harmful effects in the brain parenchyma after stroke, such as inflammation, excitotoxicity and basal lamina degradation. Neuroserpin, a natural inhibitor of tPA, has shown neuroprotective effects in animal models of brain infarct. However, the molecular mechanisms of neuroserpin-mediated neuroprotection after brain ischemia remain to be well characterized. Then, our aim was to investigate such mechanisms in primary mixed cortical cell cultures after oxygen and glucose deprivation (OGD). Primary rat mixed cortical cultures containing both astrocytes and neurons were subjected to OGD for 150min and subsequently treated with either tPA (5μg/mL), neuroserpin (0.125, 0.25, 0.5 or 1μM), and tPA together with neuroserpin at the mentioned doses. Twenty-four hours after treatment, LDH release, caspase-3 activity, MCP-1, MIP-2, active MMP-9, GRO/KC and COX-2 were measured. Statistical differences were analyzed using Student's t-test or one-way ANOVA as appropriate. Treatment with tPA after OGD increased LDH release, active MMP-9, MCP-1 and MIP-2 (all p≤0.05), but not caspase-3, GRO/KC or COX-2 compared to control. Treatment with neuroserpin after OGD decreased LDH release and active MMP-9 (all p≤0.05). It had no effect on caspase-3 activity, or on MCP-1, MIP-2, GRO/KC or COX-2 expression compared to control. Administration of tPA together with neuroserpin decreased LDH release, active MMP-9 and MIP-2 (all p≤0.05) and showed no effect on MCP-1, GRO/KC or COX-2 compared to control. Our results suggest that neuroprotective activity of neuroserpin involves attenuation on tPA-mediated mechanisms of inflammation and BBB disruption after brain ischemia.     

Activators and inhibitors of the plasminogen system in Alzheimer's disease

Accumulation and deposition of Aβ is one of the main neuropathological hallmarks of Alzheimer's disease (AD) and impaired Aβ degradation may be one mechanism of accumulation. Plasmin is the key protease of the plasminogen system and can cleave Aβ. Plasmin is activated from plasminogen by tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). The activators are regulated by inhibitors which include plasminogen activator inhibitor-1 (PAI-1) and neuroserpin. Plasmin is also regulated by inhibitors including α2-antiplasmin and α2-macroglobulin. Here, we investigate the mRNA levels of the activators and inhibitors of the plasminogen system and the protein levels of tPA, neuroserpin and α2-antiplasmin in post-mortem AD and control brain tissue. Distribution of the activators and inhibitors in human brain sections was assessed by immunoperoxidase staining. mRNA measurements were made in 20 AD and 20 control brains by real-time PCR. In an expanded cohort of 38 AD and 38 control brains tPA, neuroserpin and α2-antiplasmin protein levels were measured by ELISA. The activators and inhibitors were present mainly in neurons and α2-antiplasmin was also associated with Aβ plaques in AD brain tissue. tPA, uPA, PAI-1 and α2-antiplasmin mRNA were all significantly increased in AD compared to controls, as were tPA and α2-antiplasmin protein, whereas neuroserpin mRNA and protein were significantly reduced. α2-macroglobulin mRNA was not significantly altered in AD. The increases in tPA, uPA, PAI-1 and α2-antiplasmin may counteract each other so that plasmin activity is not significantly altered in AD, but increased tPA may also affect synaptic plasticity, excitotoxic neuronal death and apoptosis.     

Proteomic analysis of neural differentiation of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) can differentiate into different types of cells, and serve as a good model system to study human embryonic stem cells (hESCs). We showed that mESCs differentiated into two types of neurons with different time courses. To determine the global protein expression changes after neural differentiation, we employed a proteomic strategy to analyze the differences between the proteomes of ES cells (E14) and neurons. Using 2-DE plus LC/MS/MS, we have generated proteome reference maps of E14 cells and derived dopaminergic neurons. Around 23 proteins with an increase or decrease in expression or phosphorylation after differentiation have been identified. We confirmed the downregulation of translationally controlled tumor protein (TCTP) and upregulation of alpha-tubulin by Western blotting. We also showed that TCTP was further downregulated in derived motor neurons than in dopaminergic neurons, and its expression level was independent of extracellular Ca(2+) concentration during neural differentiation. Potential roles of TCTP in modulating neural differentiation through binding to Ca(2+), tubulin and Na,K-ATPase, as well as the functional significance of regulation of other proteins such as actin-related protein 3 (Arp3) and Ran GTPase are discussed. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.     

Nectin-like molecule 1 is a high abundance protein in cerebellar neurons

Summary. Nectins and Nectin-like molecules belong to the Ca-independent immunoglobulin superfamily of cell adhesion molecules and are mandatory for various cellular functions such as morphogenesis, differentiation and proliferation. Among them, Nectin-like molecule 1 (Necl-1) is unique for its exclusive expression in the brain where it is localized at the contact sites among axon terminals and glia cell processes, cooperatively forming synapses. We hereby aimed to unambiguously characterize Necl-1 at the protein level in rat brain. Rat cerebellar neurons were lysed, proteins extracted and run on two-dimensional gel electrophoresis with subsequent in-gel digestion and mass spectrometrical (MS/MS) analysis of protein spots. One spot at pI 5.96 with an observed molecular weight of 26 kDa was identified as Nectin-like molecule 1. MS/MS analyses of three matching peptides warranted unambiguous identification for the first time. Additionally, we verified the result by immunoblotting and detected two bands at about 48 kDa and 60 kDa. The proposed roles of Necl-1 in cerebellar morphogenesis as well as plasticity of synapses challenge further research on its function in more detail and we hereby provide a fair analytical tool for the unequivocal determination of Necl-1, independent of antibody availability and specificity.     

Tissue plasminogen activator mediates amyloid-induced neurotoxicity via Erk1/2 activation

Tissue plasminogen activator (tPA) is the main activator of plasminogen into plasmin in the brain where it may have beneficial roles but also neurotoxic effects that could be plasmin dependent or not. Little is known about the substrates and pathways that mediate plasmin-independent tPA neurotoxicity. Here we show in primary hippocampal neurons that tPA promotes a catalytic-independent activation of the extracellular regulated kinase (Erk)1/2 signal transduction pathway through the N-methyl-D-aspartate receptor, G-proteins and protein kinase C. This results in GSK3 activation in a process that requires de novo synthesis of proteins, and leads to tau aberrant phosphorylation, microtubule destabilization and apoptosis. Similar effects are produced by amyloid aggregates in a tPA-dependent manner, as demonstrated by pharmacological treatments and in wt and tPA-/- mice neurons. Consistently, in Alzheimer's disease (AD) patients' brains, high levels of tPA colocalize with amyloid-rich areas, activated Erk1/2 and phosphorylated tau. This is the first demonstration of an intracellular pathway by which tPA triggers kinase activation, tau phosphorylation and neurotoxicity, suggesting a key role for this molecule in AD pathology.