We hypothesized that urokinase plasminogen activator (uPA) contributes to age-dependent early hyperemia after fluid percussion brain injury (FPI) by activating extracellular signal-related kinase (ERK) mitogen-activated protein kinase (MAPK), leading to histopathologic changes in the underlying cortex. Both cerebrospinal fluid (CSF) uPA and phosphorylation of CSF ERK MAPK was increased at 1 min after FPI in newborn pigs, but was unchanged in juvenile pigs. uPA and phosphorylated ERK MAPK, detectable in sham piglet brain by immunohistochemistry, was markedly elevated and associated with histopathology 4 h after FPI in the newborn but there was minimal staining and histopathology in the juvenile. EEIIMD, a peptide derived from PA inhibitor-1 that does not affect proteolysis, blunted FPI-induced phosphorylation of ERK MAPK. FPI produced pial artery dilation and increased cerebral blood flow at 1 min after insult in the newborn, but not in the juvenile. Antilipoprotein-related protein (LRP) antibody, EEIIMD, a soluble uPA antagonist, and the ERK MAPK antagonist U 0126 inhibited FPI-associated hyperemia. These data indicate that uPA is upregulated after FPI and produces an age-dependent early hyperemia followed by histopathology through an LRP- and ERK MAPK-dependent pathway.

Objective

NMDA induced pial artery dilation (PAD) is reversed to vasoconstriction after fluid percussion brain injury (FPI). Tissue type plasminogen activator (tPA) is upregulated and the tPA antagonist, EEIIMD, prevents impaired NMDA PAD after FPI. Mitogen activated protein kinase (MAPK), a family of at least 3 kinases, ERK, p38 and JNK, is also upregulated after TBI. We hypothesize that tPA impairs NMDA induced cerebrovasodilation after FPI in a MAPK isoform dependent mechanism.

Methods

Lateral FPI was induced in newborn pigs. The closed cranial window technique was used to measure pial artery diameter and to collect CSF. ERK, p38, and JNK MAPK concentrations in CSF were quantified by ELISA.

Results

CSF JNK MAPK was increased by FPI, increased further by tPA, but blocked by JNK antagonists SP600125 and D-JNKI1. FPI modestly increased p38 and ERK isoforms of MAPK. NMDA induced PAD was reversed to vasoconstriction after FPI, whereas dilator responses to papaverine were unchanged. tPA, in post FPI CSF concentration, potentiated NMDA induced vasoconstriction while papaverine dilation was unchanged. SP 600125 and D-JNKI1, blocked NMDA induced vasoconstriction and fully restored PAD. The ERK antagonist U 0126 partially restored NMDA-induced PAD, while the p38 inhibitor SB203580 aggravated NMDA-induced vasoconstriction observed in the presence of tPA after FPI.

Discussion

These data indicate that tPA contributes to impairment of NMDA mediated cerebrovasodilation after FPI through JNK, while p38 may be protective. These data suggest that inhibition of the endogenous plasminogen activator system and JNK may improve cerebral hemodynamic outcome post TBI.

The concentration of urokinase plasminogen activator (uPA) is elevated in pathological settings such as acute lung injury, where pulmonary arterial contractility and permeability are disrupted. uPA limits the accretion of fibrin after injury. Here we investigated whether uPA also regulates pulmonary arterial contractility and permeability. Contractility was measured using isolated pulmonary arterial rings. Pulmonary blood flow was measured in vivo by Doppler and pulmonary vascular permeability, according to the extravasation of Evans blue. Our data show that uPA regulates the in vitro pulmonary arterial contractility induced by phenylephrine in a dose-dependent manner through two receptor-dependent pathways, and regulates vascular contractility and permeability in vivo. Physiological concentrations of uPA (≤1 nM) stimulate the contractility of pulmonary arterial rings induced by phenylephrine through the low-density lipoprotein receptor–related protein receptor. The procontractile effect of uPA is independent of its catalytic activity. At pathophysiological concentrations, uPA (20 nM) inhibits contractility and increases vascular permeability. The inhibition of vascular contractility and increase of vascular permeability is mediated through a two-step process that involves docking to N-methyl-d-aspartate receptor–1 (NMDA-R1) on pulmonary vascular smooth muscle cells, and requires catalytic activity. Peptides that specifically inhibit the docking of uPA to NMDA-R, or the uPA variant with a mutated receptor docking site, abolished both the effects of uPA on vascular contractility and permeability, without affecting its catalytic activity. These data show that uPA, at concentrations found under pathological conditions, reduces pulmonary arterial contractility and increases permeability though the activation of NMDA-R1. The selective inhibition of NMDAR-1 activation by uPA can be accomplished without a loss of fibrinolytic activity.

Abstract

Outcome of traumatic brain injury (TBI) is impaired by hyperglycemia, hypotension, and glutamate, and improved by insulin. Insulin reduces glutamate concentration, making it uncertain whether its beneficial effect accrues from euglycemia. Glucagon decreases CNS glutamate, lessens neuronal cell injury, and improves neurological scores in mice after TBI. In vitro, glucagon limits NMDA-mediated excitotoxicity by increasing cAMP and protein kinase A (PKA). NMDA receptor activation couples cerebral blood flow (CBF) to metabolism. Dilation induced by NMDA is impaired after fluid percussion brain injury (FPI) due to upregulation of endogenous tPA, which further disturbs cerebral autoregulation during hypotension after fluid percussion injury (FPI). We hypothesized that glucagon prevents impaired NMDA receptor-mediated dilation after FPI by upregulating cAMP, which decreases release of tPA. NMDA-induced pial artery dilation (PAD) was reversed to vasoconstriction after FPI. Glucagon 30 min before or 30 min after FPI blocked NMDA-mediated vasoconstriction and restored the response to vasodilation. PAD during hypotension was blunted after FPI, but protected by glucagon. Glucagon prevented FPI-induced reductions in CSF cAMP, yielding a net increase in cAMP, and blocked FPI-induced elevation of CSF tPA. Co-administration of the PKA antagonist Rp 8Br cAMPs prevented glucagon-mediated preservation of NMDA-mediated dilation after FPI. The pKA agonist Sp 8Br cAMPs prevented impairment of NMDA-induced dilation. These data indicate that glucagon protects against impaired cerebrovasodilation by upregulating cAMP, which decreases release of tPA, suggesting that it may provide neuroprotection when given after TBI, or prior to certain neurosurgical or cardiac interventions in which the incidence of perioperative ischemia is high.

The sole FDA approved treatment for acute stroke is tissue type plasminogen activator (tPA). However, tPA potentiates impairment of pial artery dilation in response to hypotension after hypoxia/ischemia (H/I) in pigs. ATP and Ca sensitive K channels (Katp and Kca) are important regulators of cerebrovascular tone and mediate cerebrovasodilation in response to hypotension. Mitogen activated protein kinase (MAPK), a family of at least 3 kinases, ERK, p38 and JNK, is upregulated after H/I, with the ERK isoform contributing to vasodilator impairment. This study examined the effect of H/I on Katp and Kca induced pial artery dilation and the roles of tPA and ERK during/after injury in piglets equipped with a closed cranial window. H/I blunted vasodilation induced by the Katp agonists cromakalim, calcitonin gene related peptide (CGRP) and the Kca agonist NS 1619; the effect of each was exacerbated by tPA. Pre- or post-injury treatment with EEIIMD, a hexapeptide derived from plasminogen activator-1, and ERK antagonist U 0126 prevented Katp and Kca channel agonist induced vasodilator impairment while the inactive analogue EEIIMR had no effect. ERK was upregulated after H/I, which was potentiated by tPA. These data indicate that H/I impairs K channel mediated cerebrovasodilation. tPA augments loss of K channel function after injury by upregulating ERK. These data suggest that thrombolytic therapy for treatment of CNS ischemic disorders can dysregulate cerebrohemodynamics by impairing cation-mediated control of cerebrovascular tone.

Reactive airway disease is mediated by smooth muscle contraction initiated through several agonist-dependent pathways. Activation of type 1 N-methyl-D-aspartate receptors (NMDA-R1s) by plasminogen activators (PAs) has been linked to control of vascular tone, but their effect on airway smooth muscle contractility has not previously been studied to our knowledge. We observed that NMDA-R1s are expressed by human airway smooth muscle cells and constitutively inhibit the contraction of isolated rat tracheal rings in response to acetylcholine (Ach). Both tissue-type PA (tPA) and urokinase-type PA (uPA) bind to NMDA-R1 and reverse this effect, thereby enhancing Ach-induced tracheal contractility. Tracheal contractility initiated by Ach is reduced in rings isolated from tPA−/− and uPA−/− mice compared with their wild-type counterparts. The procontractile effect of uPA or tPA was mimicked and augmented by the nitric oxide synthase inhibitor, l-NAME. uPA and tPA further enhanced the contractility of rings denuded of epithelium, an effect that was inhibited by the NMDA-R antagonist, MK-801. Binding of PAs to NMDA-R1 and the subsequent activation of the receptor were inhibited by PA inhibitor type 1, by a PA inhibitor type 1–derived hexapeptide that recognizes the tPA and uPA docking domains, as well as by specific mutations within the docking site of tPA. These studies identify involvement of PAs and NMDA-R1 in airway contractility, and define new loci that could lead to the development of novel interventions for reactive airway disease.

Rationale: The involvement of neutrophil activation in the sentinel, potentially reversible, events in the pathogenesis of acute lung injury (ALI) is only partially understood. α-Defensins are the most abundant proteins secreted by activated human neutrophils, but their contribution to ALI in mouse models is hindered by their absence from murine neutrophils and the inability to study their effects in isolation in other species.

Objectives: To study the role of α-defensins in the pathogenesis of ALI in a clinically relevant setting using mice transgenic for polymorphonuclear leukocyte expression of α-defensins.

Methods: Transgenic mice expressing polymorphonuclear leukocyte α-defensins were generated. ALI was induced by acid aspiration. Pulmonary vascular permeability was studied in vivo using labeled dextran and fibrin deposition. The role of the low-density lipoprotein–related receptor (LRP) in permeability was examined.

Measurements and Main Results: Acid aspiration induced neutrophil migration and release of α-defensins into lung parenchyma and airways. ALI was more severe in α-defensin–expressing mice than in wild-type mice, as determined by inspection, influx of neutrophils into the interstitial space and airways, histological evidence of epithelial injury, interstitial edema, extravascular fibrin deposition, impaired oxygenation, and reduced survival. Within 4 hours of insult, α-defensin–expressing mice showed greater disruption of capillary–epithelial barrier function and ALI that was attenuated by systemic or intratracheal administration of specific inhibitors of the LRP.

Conclusions: α-Defensins mediate ALI through LRP-mediated loss of capillary–epithelial barrier function, suggesting a potential new approach to intervention.

Coupling plasminogen activators to carrier red blood cells (RBC) prolongs their life-time in the circulation and restricts extravascular side effects, thereby allowing their utility for short-term thromboprophylaxis. Unlike constitutively active plasminogen activators, single chain urokinase plasminogen activator (scuPA) is activated by plasmin proteolysis or binding to its receptor, uPAR. In this study we conjugated recombinant soluble uPAR (suPAR) to rat RBC, forming RBC/suPAR complex. RBC carrying suPAR circulated in rats similarly to naïve RBC and markedly prolonged the circulation time of suPAR. RBC/suPAR carrying ~3×104 suPAR molecules per RBC specifically bound up to 2×104 molecules of scuPA, retained ~75% of scuPA binding capacity after circulation in rats and markedly altered the functional profile of bound scuPA. RBC carrying directly conjugated scuPA adhered to endothelial cells, while showing no appreciable fibrinolytic activity. In contrast, RBC/suPAR loaded with scuPA did not exhibit increased adhesion to endothelium, while effectively dissolving fibrin clots. This molecular design, capitalizing on unique biological features of the interaction of scuPA with its receptor, provides a promising modality to deliver a pro-drug for prevention of thrombosis.

Abstract

Pial artery dilation in response to prostaglandin (PG)E2 and the nitric oxide (NO) releaser sodium nitroprus-side (SNP) are blunted after fluid percussion brain injury (FPI), whereas responses to papaverine are unchanged. Urokinase plasminogen activator (uPA) and ERK mitogen-activated protein kinase (MAPK) are upregulated and contribute to the impairment of cerebrohemodynamics seen after FPI. PA vascular activity is mediated through the low-density lipoprotein receptor (LRP). Therefore, we investigated the role of uPA, LRP, and ERK MAPK in the impaired cerebrovasodilation response to PGE2 and SNP after FPI. Lateral FPI (2 atm) was induced in anesthetized piglets equipped with a closed cranial window. Cerebrospinal fluid (CSF) ERK MAPK was quantified by enzyme-linked immunosorbent assay (ELISA). Pretreatment with soluble uPA receptor (su-PAR), which antagonizes the vascular action of uPA, blunted the impairment of SNP and PGE2-mediated dilation seen after FPI. Pretreatment with the LRP antagonist RAP, a monoclonal antibody against LRP (Mab ag LRP) and the ERK MAPK antagonist, U 0126, all provided similar protection, whereas control immunoglobulin G (IgG) had no effect. Responses to papaverine were unchanged after FPI. Upregulation of ERK MAPK phosphorylation in CSF after FPI was blunted in animals pretreated with suPAR, RAP, MAb ag LRP, or U 0126, whereas control IgG had no effect. These data indicate that uPA contributes to the impairment of SNP and PGE2-mediated cerebrovasodilation seen after brain injury through activation of LRP and ERK MAPK.

We have previously observed that soluble urokinase plasminogn activator receptor (suPAR) prevents impairment of cerebrovasodilation induced by hypercapnia and hypotension after hypoxia/ischemia (H/I) in the newborn pig. In this study, we investigated the role of low-density lipoprotein related protein (LRP) receptor and the ERK isoform of mitogen activated protein kinase (MAPK) in uPA-mediated impairment of vasodilation after H/I in piglets equipped with a closed cranial window. CSF uPA increased from 9 ± 2 to 52 ± 8 and 140 ± 21 ng/ml at 1 and 4h after H/I, respectively. The LRP antagonist receptor associated protein (RAP) and anti-LRP antibody blunted the increase in CSF uPA at 1h (17 ± 2 ng/ml) but not 4h post insult. uPA detectable in sham-treated cortex by immunhistochemistry was markedly elevated 4h after H/I. Phosphorylation (activation) of CSF ERK MAPK was detected at 1 and 4h post H/I and blocked by RAP. Exogenous uPA administered at 4h post H/I further stimulated ERK MAPK phosphorylation, which was blocked by RAP. Pre-treatment of piglets with RAP, anti-LRP, and suPAR completely prevented, and the ERK MAPK antagonist U 0126 partially prevented, impaired responses to hypotension and hypercapnia post H/I, but none of these antagonists affected the response to isoproterenol. These data indicate that uPA is upregulated after H/I through an LRP-dependent process and that the released uPA impairs hypercapnic and hypotensive dilation through an LRP- and ERK MAPK dependent pathway. These data suggest that modulation of uPA upregulation and/or uPA-mediated signal transduction may preserve cerebrohemodynamic control after hypoxia/ischemia.