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1 Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612
2 The R. W. Johnson Pharmaceutical Research Institute, Spring House, Pennsylvania 19477
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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7- and 1.5-fold, respectively) within 20 min of 
-thrombin (100 nM) challenge. PAR-1 agonist peptide (5 µM) gave similar results, whereas control peptide (5 µM; FTLLRNPNDK-NH2) was ineffective. At relatively high concentrations, thrombin (500 nM) or PAR-1 agonist peptide (10 µM) also induced increases in Ppa and lung wet weight. All effects of thrombin (100 or 500 nM) or PAR-1 agonist peptide (5 or 10 µM) were prevented in PAR-1-null lung preparations. Baseline measures of microvessel permeability and Ppa in the PAR-1-null preparations were indistinguishable from those in normal lungs. Moreover, PAR-1-null preparations gave normal vasoconstrictor response to thromboxane analog, U-46619 (100 nM). The results indicate that the PAR-1 receptor is critical in mediating the permeability-increasing and vasoconstrictor effects of thrombin in pulmonary microvessels. proteinase-activated receptor; cultured endothelial cell monolayers; tethered ligand
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Thrombin has important physiological functions 1) hemostasis and facilitation of wound healing, 2) pro-inflammatory action with stimulation of transvascular leukocyte migration, and 3) regulation of microvascular permeability with formation of interendothelial gaps (7, 18). Thrombin has thus been postulated to be a mediator involved in the pathogenesis of acute lung injury (10). In the intact pulmonary microcirculation, the permeability-enhancing effects of thrombin in vivo have been considered an indirect consequence of agonist-stimulated leukocyte extravasation and activation (15). The question of a direct effect of thrombin on endothelial permeability in vivo remains open (12, 28). Thrombin is known to increase permeability to liquid in cultured endothelial cell monolayers (in the absence of leukocytes) (17, 19). The increase in endothelial permeability to albumin correlates with reduced monolayer transendothelial electrical resistance in these cells, implying that thrombin can act through formation of interendothelial gaps (26). Such gaps, which have been directly observed by immunohistochemical methods, are attributed to thrombin-induced cadherin junction disassembly (14, 22). Thrombin induces myosin light chain (MLC) phosphorylation (9), an event that normally precedes actin-myosin interaction and mechanical contraction of the endothelial cell monolayer.
In the present study, we have developed a perfused mouse lung preparation to examine the direct in vivo effects of thrombin on lung microvascular permeability to protein and fluid. Because endothelial cells express multiple thrombin receptors (21, 23, 24), we specifically tested the role of PAR-1 in the regulation of endothelial permeability using PAR-1 knockout mice. Our results show that PAR-1 is requisite for the pulmonary vasoconstrictor and microvessel permeability-increasing properties of thrombin in the intact pulmonary microcirculation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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RT-PCR analysis.
For confirmation of the genotype, total RNA was made from freshly isolated mouse lung tissue, using Trizol reagent (Life Technologies, Grand Island, NY). Total RNA (2 µg) was reverse-transcribed by using SuperScript Preamplification Kit (GIBCO BRL; Life Technologies). One microliter of the RT product was amplified utilizing the following primers: TR4, a forward primer with the sequence 5'-GGCAGCCTTGGACAATGGGGC-3', (covers the first 7 bp of the mouse PAR-1 coding sequence and 14 bp of the 5' untranslated sequence preceding the initiation codon); and TR8, a reverse primer with the sequence 5'-GGTCAGATATCCGGAGGCGTCC-3', derived from nucleotides 285 to 306 of the mouse PAR-1 coding sequence. The TR4 sequence has been removed from the PAR-1 -/- mouse as a result of targeted deletion (6). The resulting PCR product has an expected size of 320 bp. As a positive control, a pair of PCR primers was used for the amplification of a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene fragment with an expected size of 452 bp. PCR was performed using Taq polymerase in a total volume of 50 µl, with the following conditions: annealing at 55°C for 40 s; extension at 72°C for 60 s, and denaturing at 94°C for 40 s. After 30 cycles, 15 µl of the PCR products for the PAR-1 DNA fragment, as well as 4 µl of the PCR products for the housekeeping gene fragment, were analyzed on a 1.1% agarose gel by electrophoresis. The PCR products were visualized by ethidium bromide staining.
Experimental Procedures
Perfused mouse lung preparation.
According to approved protocol of the University of Illinois Animal Care Committee, male CD-1 (n = 60) and PAR-1 knockout mice (n = 48) weighing 30–35 g were placed in an anesthesia chamber and anesthetized with 3% halothane in room air at a flow rate of 2 l/min. After induction, anesthesia was continued by means of a nose cone. The trachea was cannulated with a polyethylene tube (PE-60; Becton-Dickinson, Parsippany, NJ) for constant positive-pressure ventilation (rate of 186 breaths/min) with the anesthetic gas mixture. Heparin (50 U) was injected into the jugular vein as an anticoagulant. The abdominal cavity was opened to expose the diaphragm, which was ventrally punctured and cut free from the rib cage. Then, a thoracotomy was performed and the two halves of the rib cage were retracted to expose the heart and lungs. To make the pulmonary artery accessible for cannulation, the heart was caudally retracted with a silk suture (6-0; Ethicon, Somerville, NJ) through the apical musculature. An incision was made in the right ventricle at the base of the pulmonary artery for introducing an arterial cannula, and another incision was made in the left atrium for drainage of venous effluent. In some preparations, a left atrial catheter was inserted. A polyethylene cannula (PE-60) was advanced into the pulmonary artery via the pulmonic valve and secured by means of a suture around the pulmonary artery that included the aorta. The lungs were perfused in situ using a peristaltic pump. The anesthetic gas flow was terminated when perfusion was begun, and ventilation was continued with room air. The heart and exsanguinated lungs were rapidly excised and transferred en bloc to a perfusion apparatus, where lung preparations were suspended from a 6-cm Perspex lever arm anchored to the sensor element of a force-displacement transducer (model FT03; Astro-Med, West Warwick, RI). The isolated lungs were ventilated (186 breaths/min) and perfused at constant flow (2 ml/min), temperature (37°C), and venous pressure (0 cmH2O) with a modified Krebs-Henseleit solution (composition in mM: 118 NaCl, 4.7 KCl, 1.0 CaCl2, 0.5 MgCl2, 4.43 HEPES sodium, 5.57 HEPES, 3 NaHCO3, 11 glucose, and 0.025 EDTA, pH 7.4), supplemented with 5 g/100 ml of BSA (fraction V, 99% pure and endotoxin-free; Sigma-Aldrich, St. Louis, MO). Pulmonary arterial pressure was monitored throughout the experiment using a Gould pressure transducer (model P23ID; Gould Instruments, Dayton, OH). Lung wet weight was electronically nulled when the tissue was mounted, and subsequent weight changes due to gain or loss of fluid from the lung were recorded. Lung weight and arterial pressure recordings were displayed on a computer video monitor with the aid of amplifiers (model CP122; Astro-Med), an analog-to-digital converter (DAS 1800ST board; Keithley Metrabyte, Solon, OH), and commercial software for acquisition and logging of data (Notebook Pro for Windows; Labtech, Andover, MA). All lung preparations underwent a 20-min equilibration perfusion. Lungs that were not isogravimetric at the end of the equilibration period were discarded.
Infusion of drugs.
-Thrombin was obtained from Enzyme Research Laboratories (South Bend, IN). PAR-1 agonist peptide (TFLLRNPNDK-NH2) and inactive control peptide (FTLLRNPNDK-NH2) were synthesized as described (5). Each drug was dissolved in albumin-Krebs solution at 10 times its final perfusate concentration and delivered through a side-arm in the pulmonary arterial cannula by infusion (200 µl/min), from a 10-ml syringe, with the aid of a syringe pump (model SP100i; World Precision Instruments, Sarasota, FL). Virtually immediate drug application or removal was effected by turning on or off the pump.
Pulmonary capillary filtration coefficient determination.
Capillary filtration coefficient (Kfc) was measured to determine pulmonary microvascular permeability to liquid, as described (8). Briefly, after the standard 20-min equilibration perfusion, outflow pressure was rapidly elevated by 10 cmH2O for 2 min. The lung wet weight changed in a ramplike fashion, reflecting net fluid extravasation. At the end of each experiment, lungs were dissected free of nonpulmonary tissue, and lung dry weight was determined. Kfc (ml· min-1·cmH2O·g dry wt-1) was calculated from the slope of the recorded weight change normalized to the pressure change and to lung dry weight. Normalization of the weight gain by change in outflow pressure can produce slightly higher estimates of Kfc than normalization by change in capillary pressure, as estimated with double-occlusion method. Our procedure was applied consistently before and after administration of test compounds, permitting reliable detection of drug-induced changes of Kfc. The wet-to-dry weight ratio in 6 freshly isolated (nonperfused) mouse lungs was 6.04 ± 0.4.
Pulmonary microvessel 125I -albumin permeability-surface area product measurement.
BSA was labeled with 125I (New England Nuclear, Boston, MA) using the chloramine T method (1). Free 125I was separated from 125I-labeled BSA with a Sephadex G25 column, and contributed less than 0.3% of total radioactivity as determined by trichloroacetic acid (TCA) precipitation. Albumin-Krebs solution containing 125I-BSA (2 x 106 counts/ml) was infused at 200 µl/min into the pulmonary artery for 3 min. A sample of the venous effluent was saved for later determination of the perfusate tracer concentration (counts/ml). Vascular tracer was removed during a 6-min washout period with unlabeled albumin-Krebs solution, as described (8, 25). Then, the lung tissue and the previously collected venous effluent sample were counted for 125I in a gamma counter (Minaxi Auto Gamma 5000 Series; Packard Instruments, Downers Grove, IL). The lung tissue was dried to constant weight in an oven at 80°C. 125I-BSA permeability-surface area product (PS) was calculated from A/(C x t), where A represents measured tissue counts per gram of dry lung tissue, t is duration of exposure to tracer BSA (in min), and C is concentration of 125I-BSA in the perfusing liquid (13)
Experimental Protocols
Effects of PAR-1 agonist peptide and -thrombin on pulmonary microvascular liquid permeability.
We evaluated the change in Kfc produced by PAR-1 agonist peptide or -thrombin in lung preparations obtained from normal or PAR-1-null mice. In one series of experiments, after the 20-min equilibration period, PAR-1 agonist peptide (perfusate concentration, 5.0 or 10.0 µM) was supplied for a 20-min period followed by a 5-min rinse with albumin-Krebs solution containing no additive. Kfc was measured at various times before, during, and after perfusion with PAR-1 peptide. Some preparations received, in place of agonist peptide, an inactive peptide (5 µM), as a control procedure. Each group contained n = 4 lungs. In a second series of experiments, 0.1 or 0.5 µM -thrombin was provided to the pulmonary circulation for 20 min followed by a 5-min rinse. Kfc was measured at various times during and after administration of -thrombin. Each group contained n = 4 lungs.
Effects of PAR-1 peptide or -thrombin on 125I-albumin PS product.
Isogravimetric lung preparations from normal or PAR-1 knockout mice were treated with selective PAR-1 peptide (5 µM) or -thrombin (0.1 µM) for a period of 20 min. Then, the 125I-albumin PS product was determined. For baseline measurements 125I-albumin PS, lungs received no addition but were treated identically in all other respects to experimental lung preparations. Each group contained n = 4 lungs.
Effects of PAR-1 peptide or -thrombin on MLC phosphorylation.
To determine the role of PAR-1 in mediating thrombin-induced changes in MLC phosphorylation, we investigated the effects of selective PAR-1 peptide and thrombin in normal and PAR-1 knockout mice. After the 20-min equilibration perfusion, isogravimetric preparations derived from normal or PAR-1 (-/-) mice received in perfusing liquid either 5.0 µM PAR-1 agonist peptide or 0.1 µM -thrombin. The lungs were then frozen rapidly using liquid nitrogen, after which they were immersed in acetone containing 10% (wt/vol) TCA and 10 mM dithiothreitol (DTT) (acetone/TCA/DTT) cooled to -80°C with crushed dry ice. Lungs were thawed in acetone/TCA/DTT at room temperature and then washed with acetone/DTT. MLCs were extracted for 60 min in 8 M urea, 20 mM Tris, 22 mM glycine, and 10 mM DTT. Proteins were separated by glycerol-urea polyacrylamide gel electrophoresis and blotted to nitrocellulose membranes. MLCs were specifically labeled with polyclonal rabbit anti-MLC20 antibody followed by horseradish peroxidase anti-rabbit IgG (Amersham Pharmacia Biotech, Piscataway, NJ) for visualization of unphosphorylated and phosphorylated bands of MLCs by chemiluminescence. The protein content of MLC and phosphorylated MLC was quantitated by scanning densitometry with the aid of NIH software (NIH Image).
Statistical analysis.
Data are means ± SE. Statistical analysis was performed using two-way analysis of variance and Newman-Keuls test for multiple comparisons. P < 0.05 was the criterion for significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-Thrombin.-thrombin (0.5 µM) increased Ppa by 57% of baseline value within 10 min from beginning of drug infusion (Fig. 2A). As lung preparations were perfused at constant flow rate (see METHODS), the observed increase in Ppa represented a rise in vascular resistance, due to the vasoconstrictor action of thrombin. -Thrombin, at a lower concentration (0.1 µM), did not significantly affect Ppa. No statistically significant effect on Ppa was associated with -thrombin challenge (0.5 µM) in PAR-1-null lungs (Fig. 2B), indicating that PAR-1 mediated the vasoconstrictor effect of thrombin.
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-Thrombin.
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Pulmonary Capillary Filtration Coefficient
-Thrombin.
At a concentration of 0.1 µM, -thrombin induced a significant increase in Kfc in PAR normal mouse lung preparations. This effect was time dependent, as Kfc was increased by 50% and 100% of baseline value after 7 and 20 min of thrombin infusion, respectively (Fig. 6A). A higher concentration of thrombin (0.5 µM) produced a threefold increase in Kfc above the baseline value, measured at 20 min (Fig. 6A). The increased Kfc was not reversible for either concentration of thrombin employed (Fig. 6A). In PAR-1-null lung preparations, thrombin was ineffective in modifying Kfc at either of the tested concentrations (Fig. 6B).
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-Thrombin.-thrombin (0.1 µM, 20 min) produced marked effects on protein permeability, indicated by a sevenfold rise in 125I-albumin PS product (Fig. 8A). This effect of thrombin was absent in PAR-1-null lung preparations (Fig. 8A), thus demonstrating the essential role of PAR-1 in thrombin-induced protein permeability.
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MLC Phosphorylation
We evaluated the effect of thrombin on extent of MLC phosphorylation in mouse lung tissue pretreated with thrombin or PAR-1 agonist peptide for periods of 7 or 20 min. Western immunoblots show MLC and phosphorylated MLC (Fig. 9A). In lung tissue obtained from normal mice, thrombin (0.1 µM) induced a clear increase in MLC phosphorylation. The PAR-1 agonist peptide (5 µM) mimicked this thrombin effect (Fig. 9A). Induction of MLC phosphorylation by thrombin or agonist peptide did not occur in lung tissue taken from PAR-1-null mice, indicating the essential role of PAR-1 in mediating the thrombin action.
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PAR-1 agonist peptide.
PAR-1 agonist peptide, at a concentration of 5 µM, produced a similar threefold increase of MLC phosphorylation above the basal level within the first 7 min of agonist exposure (Fig. 9C). This increase of MLC phosphorylation persisted when agonist exposure was increased to 20 min. In lung tissue obtained from PAR-1-null mice, the PAR-1 agonist peptide was ineffective in augmenting MLC phosphorylation. Thus PAR-1 agonist peptide induces MLC phosphorylation primarily through activation of PAR-1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In our intact mouse lung preparation, lung edema (i.e., increase in lung wet weight) followed the PAR-1 agonist peptide-induced increases in Kfc and 125I-albumin PS product after a 15–20 min lag. Edema formation was less pronounced with thrombin, despite the fact that thrombin (0.5 µM) and PAR-1 agonist peptide (10 µM) produced equivalent increases in Kfc. Because thrombin can act on other PARs besides PAR-1, it is possible that thrombin may have initiated compensatory responses that limited the edema formation. With sufficiently low concentration of either thrombin or PAR-1 agonist peptide, the increase in wet weight was dissociated from any change in Ppa. Hence, we attribute edema formation following PAR-1 activation to a rise in vascular permeability. At relatively high concentrations of PAR-1 agonist, a clearcut pulmonary vasoconstriction was observed both prior to and during the development of edema. The latter finding is strikingly similar to the effects of higher thrombin concentration in perfused rat lung preparations (28), in which increased postcapillary vascular resistance preceded the rise in lung wet weight.
We used PAR-1-deficient mice to explore the in vivo function of PAR-1 in mediating the thrombin-induced increase in pulmonary microvessel endothelial permeability. As shown in Figs. 6–8, basal measures of both liquid and protein permeability did not change significantly from baseline in PAR-1-null lung preparations. The stability of the isogravimetric state in PAR-1-null mouse lung preparations was also similar to the normal preparations. Moreover, baseline values for the perfusion pressure were identical in lung preparations from normal and PAR-1 knockout mice. However, normal responses to thrombin (i.e., increases in lung weight, albumin PS, Kfc, and vascular resistance) were not evident in the PAR-1-deficient lung preparations. In contrast, these preparations gave typical vasoconstrictor responses to the thromboxane analog U-46619, indicating that PAR-1 knockout mice retained normal pulmonary vasoreactivity to other vasoactive mediators. In other experiments, the overexpression of PAR-1 in mice resulted in exaggerated increases in lung wet weight and Kfc in response to thrombin or PAR-1 agonist peptide (Gao X, Stevens M, Vogel SM, and Malik AB, unpublished observations). Taken together, these studies indicate that PAR-1 is the predominant receptor mediating the effects of thrombin in increasing pulmonary microvessel permeability and vasomotor tone. These studies demonstrating the critical role of PAR-1 in mediating the increase in pulmonary microvascular permeability in vivo are consistent with the effects of selective anti-PAR-1 antibody in preventing the thrombin-increase in permeability in cultured endothelial monolayers (20).
We obtained further evidence supporting an important role of PAR-1 by means of a selective PAR-1-activating peptide (TFLLRNPNDK-NH2) (5), which in normal mouse lungs gave similar responses to thrombin. The PAR-1 agonist peptide caused increases in liquid and protein permeability, much like thrombin itself. Interestingly, thrombin and this peptide differed with regard to the reversibility of PAR-1 receptor activation upon removal of the agonist from the perfusate. An explanation lies in the fact thrombin is a proteolytic activator of PAR-1 and irreversibly activates the receptor, and thus the response is inherently irreversible. In contrast, the PAR-1 peptide is an agonist activator that reversibly binds to PAR-1, thus mimicking the "tethered ligand" that thrombin makes available through proteolytic cleavage of substrate. A "control" peptide, identical to the agonist peptide except for the transposition of the first two carboxy-terminal residues, was inactive in all respects, thus ruling out nonspecific peptide effects.
The mechanism by which thrombin induces an increase in liquid and protein permeability could involve endothelial cell contraction with disruption of interendothelial VE-cadherin junctions (3, 11). In previous in vitro studies, activation of the endothelial contractile apparatus was inferred from thrombin-induced increases in intracellular calcium and in MLC phosphorylation by the calcium-sensitive MLC kinase (9, 11). These changes preceded contraction of endothelial cells, resulting from interaction of the phosphorylated form of MLC with actin. In the intact lung, MLC phosphorylation could also play a role in endothelial and vascular smooth muscle contraction that underlies the observed vasoconstrictor and permeability-increasing effects of thrombin. In the present study, we showed that activators of PAR-1 induced the phosphorylation of MLC in normal but not in PAR-1-deficient lung preparations. Thus the results implicate PAR-1 activation in signaling MLC phosphorylation in vivo and in the mechanism of thrombin-induced increases in pulmonary microvessel permeability and vasomotor tone.
In summary, we have used the perfused mouse lung preparation to address the in vivo effects of thrombin on lung microvascular permeability to protein and fluid and vascular resistance. We specifically tested the role of PAR-1 in the regulation of endothelial permeability using PAR-1 knockout mice. The results showed that PAR-1 is requisite for the pulmonary vasoconstrictor and microvessel permeability-increasing properties of thrombin in the intact pulmonary microcirculation. Thus PAR-1 functions as the principle regulator of the pulmonary microvascular actions of thrombin in vivo.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health Grants HL-45638, GM-58531, P01-HL-60678, and T32-HL-07829.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. M. Vogel, Dept. of Pharmacology, Univ. of Illinois College of Medicine, 835 South Wolcott Ave. (M/C 868), Chicago, IL 60612 (E-mail: vogel{at}uic.edu).
* S. M. Vogel and X. Gao contributed equally to this work 
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REFERENCES |
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V. |
) or PAR-1-null (KO; 
, 
) mouse lung preparations. B: effect of PAR-1 agonist peptide (TFLLRNPNDK-NH2) at two concentrations (5 or 10 µM) in normal (solid symbols) or PAR-1-null (open symbols) lung preparations. Note pronounced increase in lung wet weight induced by PAR-1 agonist peptide, 
