Urokinase Plasminogen Activator Regulates Pulmonary Arterial Contractility and Vascular Permeability in Mice

Abstract

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-Daspartate 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.

tPA-S481A Prevents Impairment of Cerebrovascular Autoregulation by Endogenous tPA after Traumatic Brain Injury by Upregulating p38 MAPK and Inhibiting ET-1

“…[22][23][24] Previous work by others indicates that tPA cleaves the NR-1 subunit of NMDA-Rs, which contributes to excitoxicity by increasing the influx of calcium, 12,27 by signaling through an interaction with the NR2B subunit. 28 Human TBI is heterogenous, limiting the ability of animal models to mimic the complexity of the human situation.…”

“…[22][23][24] Each protein contains two extra amino acids, RS-, at the NH 2 terminus, resulting from the introduction of the Bgl II cloning site. Proteins were expressed in S2 Drosophila Expression System (Invitrogen) according to the manufacturer's protocol and were purified by affinity chromatography using anti-tPA coupled to CN-Br activated Sepharose.…”

tPA-S481A Prevents Neurotoxicity of Endogenous tPA in Traumatic Brain Injury

“…The variant tPA-S481A is predicted to compete with wild-type tPA for binding to the NMDA receptor, and thus protect it from activation by high levels of endogenous wildtype tPA that occur post-TBI. Indeed, tPA-S481A binds to the R1 subunit of NMDA and prevents its activation in the lung (Nassar et al, 2011a(Nassar et al, ,2011b(Nassar et al, ,2011c. Use of this tPA variant in the present study is proposed as a novel approach to limiting neurotoxicity by excessive NMDA-receptor activation associated with the robust increase of glutamate and tPA that occurs within the brain following TBI Katayama et al, 1990).…”

“…To generate tPA-S481A, a mutation was introduced into wild-type tPA by polymerase chain reaction (PCR) using the QuickChange Mutagenesis kit (Stratagene, La Jolla, CA), and the complete sequence was verified (Nassar et al, 2011a(Nassar et al, , 2011b(Nassar et al, , 2011c. The protein contains two extra amino acids, RS-, at the NH2 terminus, resulting from the introduction of the Bgl II cloning site.…”

“…However, this variant had no effect on pial artery diameter or CBF in the absence of brain injury. This compound is a catalytically-inactive tPA variant with a single mutation in the active site that maintains its ''docking site,'' through which it binds to the NMDA receptor (Nassar et al, 2011a(Nassar et al, ,2011b(Nassar et al, ,2011c, but cannot cleave and thereby activate the receptor. Therefore, tPA-S481A competes with wild-type tPA for binding to the NMDA receptor, and protects it from activation by wild-type tPA (Fig.…”