Interaction of fibrin with VE-cadherin and anti-inflammatory effect of fibrin-derived fragments

Summary

Background

The interaction of fibrin βN-domain with VE-cadherin on endothelial cells was implicated in transendothelial migration of leukocytes and theβ15-42 fragment representing part of this domain was shown to inhibit this process. However, our previous study revealed that only a dimeric (β15-66)₂ fragment corresponding to the full-length βN-domain and mimicking its dimeric arrangement in fibrin bound to VE-cadherin.

Objective

To test our hypothesis that dimerization of β15-42-containing fragments increases their affinity to VE-cadherin and ability to inhibit transendothelial migration of leukocytes.

Methods

Interaction of β15-42-containing fragments with VE-cadherin was characterized by ELISA and surface plasmon resonance. Inhibitory effect of such fragments was tested in vitro using leukocyte transendothelial migration assay and in vivo using mouse models of peritonitis and myocardial ischemia-reperfusion injury.

Results

First, we prepared the monomeric β15-42 and β15-64 fragments and their dimeric forms, (β15-44)₂ and (β15-66)₂, and studied their interaction with fibrin-binding domain of VE-cadherin, VE-cad(3). The experiments revealed that both dimeric fragments bound to VE-cad(3) with high affinity while the affinities of monomeric β15-42 and β15-64 were significantly lower. Next, we tested the ability of these fragments to inhibit leukocyte transmigration in vitro and infiltration into the inflamed peritoneum in vivo and found that the inhibitory effects of the dimers on these processes were also superior. Furthermore, dimeric (β15-44)₂ significantly reduced myocardial injury induced by ischemia-reperfusion.

Conclusion

The results confirm our hypotheses and indicate that dimeric (β15-66)₂ and (β15-44)₂, which exhibited much higher affinity to VE-cadherin, are highly effective in suppressing inflammation by inhibiting leukocyte transmigration.

Introduction

Fibrinogen is a haemostatic plasma protein whose major function is to form fibrin clots that prevent the loss of blood upon vascular injury. In addition, fibrin(ogen) participates in a number of other physiological and pathological processes through the interaction with various proteins and cell types. Among these processes are inflammation and angiogenesis which take place during normal wound healing or in pathological states. It was shown that interaction of fibrin with endothelial cells promotes the formation of capillary tubes, i.e. angiogenesis [1,2]. Numerous studies also implicated interaction of fibrin(ogen) with endothelial cells and leukocytes in promoting transendothelial migration of leukocytes and thereby inflammation [3–6].

Fibrinogen is a chemical dimer consisting of two identical disulfide-linked subunits each formed by three non-identical polypeptide chains, Aα, Bβ, and γ [7]. These chains are folded into a number of domains that contain numerous binding sites designed for interaction with various proteins and cellular receptors. The central region of fibrinogen molecule is formed by N-terminal portions of all six chains linked together by 11 disulfide bonds and is often called N-terminal disulfide knot, NDSK [8]. Digestion of fibrinogen with CNBr results in NDSK fragment corresponding to this region. This fragment preserves some binding sites and, therefore, is often used in functional studies. Namely, it contains two pairs of fibrin polymerization sites [7] and sites involved in the interaction with endothelial cell receptor VE-cadherin [2,9,10], cell proteoglycans [11,12], and integrin receptor αXβ2 [13]. The VE-cadherin and proteoglycan binding sites are located in the N-terminal portions of two fibrin β chains each forming βN-domain [10–12] whileαXβ2 binding sites are in the N-terminal portions of fibrin α chain [13]. These sites are cryptic in fibrinogen and become exposed in fibrin after thrombin-mediated removal of fibrinopeptides A and B from the N-termini of the Aα and Bβ chains, respectively.

The ability of fibrin(ogen) to interact with different cellular receptors together with its dimeric nature make this protein well suited for bridging different cell types. It was suggested more than a decade ago that fibrin(ogen) binding to a variety of vascular cell receptors mediates a specific pathway of cell-to-cell adhesion by bridging together leukocytes and endothelial cells [3]. Further, it was proposed that such bridging may occur through the interaction of fibrin(ogen) with the leukocyte receptor Mac-1 and endothelial cell receptor ICAM-1, and may contribute to transendothelial migration of leukocytes to injured tissues [3,4]. More recent studies revealed that fibrin-derived NDSK fragment devoid of fibrinopeptides A and B (NDSK-II) promotes transendothelial migration of leukocytes [5]. It was suggested that such NDSK-II induces leukocyte transmigration by a similar mechanism, namely, by bridging inflammatory cells to the endothelium through its interaction with leukocyte integrin αXβ2 and endothelial cell VE-cadherin [5,6]. Furthermore, it was shown that NDSK-II-induced leukocyte transmigration can be inhibited by aβ15-42 peptide [5], which represents part of the fibrin βN-domain and can be easily prepared by plasmin digestion of fibrin or synthesized. The same study [5] also demonstrated that in rat and mouse models of myocardial ischemia-reperfusion injury the β15-42 peptide, by inhibiting leukocyte transmigration, substantially reduced myocardial inflammation and infarct size. This peptide was proposed to be a potential drug candidate for myocardial reperfusion therapy in humans [5,6].

Our studies on the interaction of fibrin with VE-cadherin resulted in identification in the fibrin β chain of amino acid residues critical for this interaction and localization of the fibrin-binding site in the third domain of VE-cadherin [10,14]. They also revealed that a recombinant dimeric (β15-66)₂ fragment, which corresponds to the full-length βN-domain and mimics its dimeric arrangement in fibrin, bound to VE-cadherin with high affinity while the recombinant monomericβ15-64 fragment and the proteolytically prepared β15-42 peptide failed to bind in the studied concentration range [10]. Based on these findings, we hypothesized that dimerization is important for the high affinity interaction of β15-42-containing fragments with VE-cadherin and dimeric β15-42-containing fragments should be more potent inhibitors of leukocyte transmigration and thereby inflammation than monomeric β15-42. The major goal of the present study was to test this hypothesis.

Materials and methods

Reagents

Human fibrinogen (plasminogen, von Willebrand factor, and fibronectin depleted) was purchased from Enzyme Research Laboratories (South Bend, IN). Phorbol 12-myristate 13-acetate (PMA) was from Promega (Madison, WI), N-formyl-Met-Leu-Phe (fMLP), extravidin-alkaline phosphatase, DMSO, and triphenyltetrazolium were from Sigma (St. Louis, MO), Calcein AM fluorescent dye was from BD Biosciences (Franklin Lakes, NJ).

Mice

C57BL/6 mice of 8–12 weeks old (20–22 grams) were from the Jackson Laboratory (Bar Harbor, ME). All mice were housed in a pathogen-free facility and all procedures were performed in accordance with University of Maryland Institutional Animal Care and Use Committee approval.

Preparation of fibrin-binding VE-cadherin fragment

The recombinant fibrin-binding VE-cad(3) fragment (residues Phe²¹⁰-Phe³²⁵) corresponding to the third domain of the extracellular portion of VE-cadherin tagged with six His residues at the C-terminus was expressed in Escherichia coli and purified and refolded as described earlier [14]. VE-cad(3) was biotinylated with EZ-Link Sulfo-NHS-LC-LC-Biotin (Thermo Scientific, Rockford, IL) as recommended by the manufacturer.

Preparation of fibrin(ogen) fragments

NDSK fragment corresponding to the central region of fibrinogen was prepared by digestion of human fibrinogen with CNBr [9]. The recombinant Bβ1-64 fragment corresponding to the fibrinogen βN-domain and its dimeric version, (Bβ1-66)₂, were produced in Escherichia coli and purified as described earlier [10].

Thrombin-treated NDSK fragment lacking fibrinopeptides A and B (NDSK-II) was prepared by incubation of NDSK with thrombin-agarose from the Thrombin CleanCleave Kit (Sigma) for 2 hours at room temperature and subsequent purification by size-exclusion chromatography on Superdex 75. The β15-64 and (β15-66)₂ fragments were also prepared by incubation of Bβ1-64 and (Bβ1-66)₂ with thrombin-agarose, as above; however, the final purification from contaminating fibrinopeptides was performed on Superdex Peptide column (GE Healthcare, Piscataway, NJ).

The β15-42 fragment representing a part of the fibrin βN-domain was synthesized by Bachem (Torrance, CA). Its dimeric version, (β15-44)₂, was made by addition of two more amino acid residues, Cys43 and Gly44, to the C-terminus of β15-42 and dimerization of the resulting β15-44 fragment through Cys43 by incubation in 0.05 M ammonium acetate buffer, pH 8.0. The dimeric (β15-44)₂ fragment with the scrambled β15-42 sequence and Cys43-Gly44 at the C-terminus [scrambled (β15-44)₂] was prepared similarly. All fragments used in the inhibition studies were additionally purified on Detoxi-Gel column (Thermo Scientific) to remove endotoxin contamination.

Solid-phase binding assay

Wells of Immulon 2HB microtiter plates were coated overnight at 4 °C with 2 μg/mL (β15-66)₂, (β15-44)₂, β15-64, or β15-42 in 0.1 M Na₂CO₃, pH 9.5 (coating buffer). The wells were then blocked with SuperBlock blocking buffer in TBS (20 mM tris, pH 7.4, 150 mM NaCl) (Thermo Scientific) for 1 hour at room temperature. Following washing of the wells with TBS containing 0.05% Tween 20 and 1 mM CaCl₂ (binding buffer), the indicated concentrations of biotinylated VE-cad(3) in the binding buffer were added to the wells and also to control wells coated with just SuperBlock and incubated for 1 hour at 37 °C. Bound VE-cad(3) was detected with extravidin conjugated to alkaline phosphatase. The alkaline phosphatase substrate, 1-Step p-nitrophenyl phosphate (Thermo Scientific), was added to the wells and the amount of bound ligand was measured spectrophotometrically at 405 nm. Data were analyzed by nonlinear regression analysis using the equation:

$$A=A_{\mathit{max}}/(1+K_{\text{d}}/[\text{L}])$$ (1)

where A represents absorbance of the oxidized substrate, which is assumed to be proportional to the amount of ligand bound, A_max is the absorption at saturation, [L] is a molar concentration of ligand, and K_d is the dissociation constant.

Surface plasmon resonance analysis

The interaction of β15-42-containing fragments with immobilized VE-cad(3) was studied by surface plasmon resonance (SPR) using the BIAcore 3000 biosensor (BIAcore AB, Uppsala, Sweden), essentially as described earlier [14]. Briefly, immobilization of VE-cad(3) to the surface of CM5 sensor chip was performed using the amine couplingkit. Binding experiments were performed at 20 μl/min flow rate in binding buffer HBS-P (GE Healthcare) containing 1 mM CaCl₂. Fibrin fragments were injected at increasing concentrations, and the association/dissociation between them and immobilized VE-cad(3) were monitored by the change in the SPR response. Experimental data were analyzed using BIAevaluation 4.1 software. The dissociation equilibrium constant, K_d, was calculated as K_d = k_diss/k_ass, where k_ass and k_diss represent kinetic constants that were estimated by global analysis of the association/dissociation data using the 1:1 Langmurian interaction model (kinetic analysis). To confirm the kinetic analysis, K_d was also estimated by analysis of the association data using the steady-state affinity model (equilibrium analysis).

Cell culture and treatments

Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (Walkersville, MD), grown in EGM-2 complete media and used at passage 4 to 6. The HL-60 promyelocytic cell line (American Type Culture Collection, Manassas, VA) was cultured in IMDM (Iscove’s modified Dulbecco’s medium, Invitrogen) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and antibiotics. Differentiation of HL-60 to a neutrophil-like lineage was achieved by adding DMSO to final concentration of 1.3% and incubating for 5 days [15]. To assess the morphology of DMSO-induced HL-60 cells, samples containing 100 μL of non-induced or induced cells with concentration of 0.5 × 10⁶ cells/mL were span at 500 rpm for 5 min on cytospin slide; the slide with cells was dried and stained with Mayer’s hematoxylin solution (Sigma). Differentiated HL-60 cells were labeled with Calcein AM fluorescent dye (BD Biosciences) in serum-free IMDM medium, as recommended by the manufacturer. All cell lines were cultured or incubated at 37°C and 5% CO₂.

Leukocyte transendothelial migration assay

Transendothelial migration experiments were performed using 24-well plates containing 8-μm pore size PET membrane inserts (BD Biosciences). 1×10⁵ HUVEC in 300 μL were seeded onto the insert membrane pre-coated with 0.1 % gelatin (Millipore) and grown to confluence for 3 days without medium in lower chamber. Integrity of confluent endothelial monolayers was confirmed by staining the membrane with fixed cells with coomassie dye. HUVEC were washed twice with IMDM and serum-starved for 2 hours before experiments. Calcein AM-labeled differentiated HL-60 cells were stimulated with 100 nM PMA for 30 min, washed twice with IMDM, and 5×10⁵ stimulated cells in 300 μL IMDM containing 1.5 μM NDSK-II without or with 10 μM β15-42, (β15-44)₂, or 10 μM (β15-66)₂ were added on top of HUVEC monolayer. The inserts were placed into the wells each containing 700 μL 100 nM chemoattractant fMLP in IMDM. Transmigration proceeded for 4 hours and was stopped by removing the inserts from the wells. Migrated HL-60 cells were recovered from the bottom of the wells and counted using fluorescence plate reader at 480 nm/530 nm.

Mouse model of peritonitis

Mice (5 per group) were injected intraperitoneally (i.p.) with 3.85% Bacto Fluid thioglycollate (1 mL per mouse) to induce leukocyte infiltration into the peritoneum. To compare the effect of the β15-42, (β15-44)₂, and (β15-66)₂ fragments on leukocyte infiltration, mice received an intravenous (i.v.) injection (via the tail vein) of the fragments, all at the indicated amounts in 200 μL PBS (Phosphate Buffered Saline, Lonza), prior to i.p. injections of thioglycollate. Mice in control groups received an i.v. injection of the same volume of PBS or the scrambled peptide in PBS. Four hours after the injections, each group of mice was euthanized by CO₂ inhalation followed by cervical dislocation, injected i.p. with 3 mL ice-cold PBS, and total lavage fluid was withdrawn after massaging their abdomens. Total cell number in lavage fluid was determined using a hemocytometer and the percentage of neutrophils (~90%) was determined by cytospin, as previously described [16].

Mouse model of myocardial ischemia-reperfusion injury

C57BL/6J mice (8 to 12 weeks of age) were anesthetized initially with 4.5% Isoflurane and then maintained via face mask at 2 % Isoflurane. An ocular lubricant paralube was applied to the animal eyes to prevent corneal desiccation. A 1 cm incision on the ventral surface of the neck over the trachea was made to expose trachea for visualization during orotracheal intubation with a 20G catheter (0.9-mm outside diameter). Mouse was connected to Harvard Rodent Ventilator, which is supplied with room air supplemented with oxygen at a rate of 105 breaths/min and with a tidal volume of 10–15 mL/kg body weight. The left jugular vein was isolated and ligated with 6–0 silk suture. A saline filled PE 10 tube was cannulated into the vein, and positioned to the superior vena cava. A midline thoracotomy was then made between the 3^rd and 4^th rib. The left coronary artery near the atrial appendage was ligated with a 8–0 silk suture, and ischemia was maintained for 20 min after which the suture was removed to initiate blood flow into the ischemic myocardium. The (β15-44)₂ fragment at 320 μM in 50 μL PBS (total dose – 16 nmol) or PBS (50 μL) was bolus injected via the jugular vein catheter into the mice twice, 1 min prior to reperfusion and 30 min after reperfusion. After 2 hours reperfusion, mice were euthanized and the hearts were retrieved. To identify infarcted areas, the hearts were perfused with 1% triphenyltetrazolium, cut into 2 mm-thick sections using a standard heart Matrix (Roboz, Gaithersburg, MD), and the size of infarcted areas, which appear in pale color, was estimated using ImageJ program (NIH).

Statistical analysis

Statistical analysis was done using Student’s t-test with a P value of less than 0.05 being considered significant. All statistical analyses were performed in SigmaPlot 8.0 software (Systat Software, San Jose, CA).

Results

Interaction of β15-42-containing fibrin fragments with VE-cadherin

The previous finding that the dimeric (β15-66)₂ fragment has a high affinity for VE-cadherin (K_d = 80 nM) while monomeric β15-64 and its truncated variant, β15-42, fail to bind even at high concentrations (up to 400 nM) [10] rises two questions. The first question concerns the effect of dimerization of the βN-domain (amino acid residues β15-64) on its interaction with VE-cadherin, while the second question concerns the contribution of the β43-64 region of this domain, which is missing in the β15-42 fragment, to this interaction. To address these questions, we prepared recombinant monomeric β15-64 fragment and its dimeric version, (β15-66)₂, as well as synthetic β15-42 and its dimeric version, (β15-44)₂, and measured their affinity for VE-cad(3), a recombinant fragment corresponding to the fibrin binding domain of VE-cadherin [14].

To measure the affinity of this interaction by ELISA, microtiter wells were coated with the above mentioned fibrin fragments and then incubated with increasing concentrations of VE-cad(3) (up to 500 nM). VE-cad(3) exhibited a prominent binding to both dimeric fragments, (β15-66)₂ and (β15-44)₂, while no binding was observed to monomeric β15-64 and β15-42 in the tested concentration range (Fig. 1). The K_d value of 18 nM determined for the interaction of VE-cad(3) with (β15-66)₂ was comparable with that determined earlier using the same method [14], while the interaction of VE-cad(3) with (β15-44)₂ was weaker, with a K_d value of 146 nM (Table 1). We were not able to test whether the monomeric fragments exhibit any binding at higher concentrations of VE-cad(3) since further increasing of its concentration resulted in formation of aggregates that prevented reliable measurements. To overcome this problem, we turned to SPR measurements.

Fig. 1.

Analysis of the interaction between β15-42-containing fibrin fragments and VE-cadherin by ELISA. Increasing concentrations of the VE-cad(3) fragment representing the fibrin-binding domain of VE-cadherin were added to the immobilized (β15-66)₂, (β15-44)₂, β15-64, or β15-42 fibrin fragments (filled circles, empty circles, filled triangles, and empty triangles, respectively) and bound VE-cad(3) was detected as described in Materials and methods. The curves represent the best fit of the data to eq. 1. All results are means ± the standard deviation of triplicate determination.

Table 1.

Equilibrium dissociation constants for the interaction between the β15-42-containing fibrin fragments and the VE-cad(3) fragment determined by ELISA and surface plasmon resonance (SPR).

Fragments	Kd (ELISA)a	Kd (SPR)b
(β15-66)2	18 ± 6 nM	93 ± 16 nM
(β15-44)2	146 ± 37 nM	715 ± 61 nM
β15-64	n.b.c	33 ± 5 μM
β15-42	n.b. c	267 ± 42 μM

^aValues are means ± SD of three independent experiments.

^bValues are means ± SD of at least three independent experiments.

^cNo binding observed up to 500 nM.

In SPR experiments, VE-cad(3) was immobilized on the surface of a sensor chip and the β15-42-containing fragments were added at increasing concentrations. The dimeric (β15-66)₂ fragment exhibited a prominent binding (not shown) with K_d = 93 nM (Table 1), essentially the same value that was obtained by this method earlier [10,14]. The binding of dimeric (β15-44)₂ was observed at higher fragment concentrations, up to 1 μM (Fig. 2A). The K_d value for this binding was found to be 715 nM (Table 1), i.e. almost one order of magnitude higher than that for (β15-66)₂. The binding of the monomeric fragments, β15-42 and β15-64, was observed only at much higher concentrations, up to 100 μM (Fig. 2B and C), and the determined K_d values were 267 and 33 μM, respectively (Table 1). Again, as in the case of the dimeric fragments, the K_d value for the truncated fragment, β15-42, was almost one order of magnitude higher than that for β15-64. These results suggest that the β43-64 portion of the full-length βN-domain contributes to its interaction with VE-cadherin. At the same time dimerization of this domain seems to make the major contribution to this interaction since the K_d values for the dimeric fragments, (β15-66)₂ and (β15-44)₂, were much lower than those for monomeric β15-42 and β15-64.

Fig. 2.

Analysis of the interaction between β15-42-containing fibrin fragments and VE-cadherin by surface plasmon resonance. The (β15-44)₂ (panel A), β15-42 (panel B), or β15-64 (panel C) fragments, each at increasing concentrations [(β15-44)₂ at 10, 25, 50, 100, 250, 500, and 1000 nM; β15-42 at 5, 10, 25, 50, and 100 μM; β15-64 at 0.25, 0.5, 1, 2.5, 5, 10, and 25 μM] was added to the immobilized VE-cad(3) fragment, and its association and dissociation represented by solid curves were monitored in real time; the dotted curves represent the best fit of the binding data using global fitting analysis the results of which are presented in Table 1.

In vitro study of the inhibitory effect of β15-42-containing fragments

The fact that the dimeric (β15-66)₂ and (β15-44)₂ fragments have much higher affinities to VE-cadherin than their monomeric analogs suggests that both these dimers should be more potent inhibitors of leukocyte transmigration than monomeric β15-42 used in the previous studies [5,17,18]. To test this suggestion, we first compared the inhibitory effect of (β15-66)₂, (β15-44)₂, and β15-42 using an in vitro transendothelial leukocyte migration assay. For this assay, HL-60 cells were induced with DMSO to differentiate them into neutrophil-like cells [19] (Fig. 3A–B), which were labeled with Calcein AM and stimulated with PMA. It was shown earlier that such cells are a valid model system for the analysis of human neutrophil migration [19]. The cells were then added to the upper chambers, which was separated from the lower chamber by the insert membrane coated with confluent endothelial cell (HUVEC) monolayer, and their transmigration to the lower chamber was stimulated by fMLP (control) and by fMLP in the presence of NDSK-II or NDSK-II with the β15-42-containing fragments. As shown in Fig. 3C, NDSK-II added to the upper chambers stimulated transendothelial migration of DMSO-differentiated HL-60 cells, as expected based on the previous observation [5]. When NDSK-II was added in the presence of (β15-66)₂, (β15-44)₂, or β15-42, all at 10 μM, the transmigration was reduced indicating that all three fragments inhibited NDSK-II-induced transmigration of these cells. However, the inhibitory effect of the dimeric fragments, (β15-66)₂, (β15-44)₂, was much more pronounced than that of monomeric β15-42. Even at 3-fold higher concentration, 30 μM, the effect of β15-42 monomer was lower and did not exceed that of (β15-44)₂ added at 5 μM, as revealed in subsequent dose-dependence experiments (Fig. 3C, inset).

Fig. 3.

Inhibitory effect of β15-42-containing fibrin fragments on NDSK-II dependent transendothelial migration of neutrophils in vitro. HUVECs were grown to confluency on gelatin-coated cell culture inserts. Calcein AM-labeled HL-60 cells (panel A) were differentiated into neutrophil-like cells (panel B) and added to the upper chambers (inserts) on top of the HUVEC monolayer in the presence of 1.5 μM NDSK-II without or with 10 μM β15-42, (β15-44)₂, or (β15-66)₂ fragments. The cells were allowed to migrate into the lower chambers for 4 h at 37 °C, collected, and measured by fluorescence at 530 nm (panel C). The results in panel C are expressed as % of NDSK-II dependent cell migration that was calculated by subtracting migrated cells in the absence of NDSK-II from total migrated cells. The graph shows combined data obtained in 4 independent experiments; bars denote means ± the standard error (n = 15-20 wells for each condition); ***, P < 0.001. The inset in panel C shows dose-dependence of the inhibitory effect of the β15-42 and (β15-44)₂ fragments (empty and filled circles, respectively) on NDSK-II dependent neutrophil transmigration.

In vivo study of the anti-inflammatory effect of β15-42-containing fragments

To compare the effects of the dimeric (β15-66)₂ and (β15-44)₂ and monomeric β15-42 fragments on leukocyte transmigration in vivo we used a peritonitis mouse model. In this model, leukocyte migration from the circulation into the peritoneum is stimulated by intraperitoneal (i.p.) injection of thioglycollate, and leukocyte (neutrophil) accumulation is evaluated after 4 hours by counting the cell number in the peritoneal lavage. In our experiments, each mouse was injected intravenously (i.v.) with 200 μL of either β15-42, (β15-44)₂, or (β15-66)₂, all at 80 μM (total dose per mouse – 16 nmol) prior to i.p. injection of thioglycollate; control mice were injected i.v. either with the same amount of the scrambled dimeric (β15-44)₂ fragment or PBS. The experiments revealed that the number of neutrophils accumulating in the peritoneum of fragment-treated mice was significantly lower than that in control mice (Fig. 4). No statistically significant difference was observed between accumulation of neutrophils in control mice injected with the scrambled fragment and PBS. This finding indicates that in this model all three fragments inhibited leukocyte transmigration and thereby inflammation. However, the extent of inhibition by the dimeric (β15-66)₂ and (β15-44)₂ fragments in the selected experimental conditions was almost twice as much as that of the monomeric β15-42 fragment.

Fig. 4.

Inhibitory effect of β15-42-containing fibrin fragments on neutrophil infiltration in vivo in a mouse model of peritonitis. The indicated fragments were injected i.v., each in 200 μL PBS at 80μM (total dose per mouse was 16 nmol); control mice were injected i.v. with 200 μL PBS or scrambled (β15-44)₂ fragment at 80 μM in 200 μL PBS. The graph shows combined data obtained in 4 independent experiments and the results are means ± the standard error [the combined number of mice in each group was the following: injected with PBS or the scrambled fragment, n = 25 and 9, respectively; injected with β15-42, (β15-44)₂, or (β15-66)₂, n = 15, 25, and 10, respectively]. **, P < 0.01; ***, P < 0.001.

Next, using the same peritonitis model we performed a systematic study of the anti-inflammatory effect of (β15-66)₂, (β15-44)₂, and β15-42 to identify optimal physiologically active amounts of these fragments for further in vivo experiments. The results of the study, in which 200 μL of each fragment at different concentrations (total dose per mouse - 4, 8, and 16 nmol) were injected i.v., revealed that both dimeric fragments, (β15-66)₂ and (β15-44)₂, exhibited similar inhibitory effects on transendothelial migration of leukocytes and were about twice as potent inhibitors of leukocyte transmigration as monomeric β15-42 (Fig. 5). These results further reinforce the above conclusion that the dimeric fragments are more potent inhibitors of leukocyte transmigration than monomeric β15-42. They also suggest that the maximal inhibitory effect can be achieved at fragment amount of 8 nmol per mouse and above. This was taking into account in our further in vivo experiments.

Fig. 5.

Dose-dependence of the inhibitory effect of β15-42-containing fibrin fragments on neutrophil infiltration in vivo in a mouse model of peritonitis. The β15-42, (β15-44)₂, or (β15-66)₂ fragments (circles, squares, and triangles, respectively) were injected i.v. in the indicated amounts (each in 200 μL at 20, 40 and 80 μM; total dose per mouse was 4, 8 and 16 nmol, respectively). The results are expressed as % of control groups and are means ± the standard error (n = 5).

Testing the cardioprotective effect of the (β15-44)₂ fragment

By inhibiting leukocyte transmigration, the monomeric β15-42 fragment was shown to reduce myocardial inflammation and infarct size in animal models for myocardial ischemia followed by reperfusion [5,17,18]. To evaluate the potential of the dimeric β15-42-containing fragments for myocardial infarction therapy we used a mouse model of myocardial ischemia-reperfusion injury. In this model, ischemia is achieved by ligation of the left coronary artery with a suture; after 20 min the suture is cut to initiate blood flow into the ischemic myocardium (reperfusion) and the size of infarcted area is evaluated after 2 hours of reperfusion. Using this model, we tested the cardioprotective effect of the (β15-44)₂ fragment which, together with (β15-66)₂, demonstrated the highest inhibitory effect on leukocyte transmigration in the experiments described above. This fragment was injected via the jugular vein into the mice twice (16 nmol per mouse in each injection), 1 min prior and 30 min after reperfusion; control mice were injected with PBS. The results presented in Fig. 6 indicate that infarct size in mice treated with (β15-44)₂ was reduced by more than two-fold in comparison with that in control mice. Thus, in this in vivo model the dimeric (β15-44)₂ fragment exhibited significant cardioprotective effect.

Fig. 6.

Cardioprotective effect of the (β15-44)₂ fragment during myocardial reperfusion injury. Representative mouse heart slices after myocardial ischemia-reperfusion in mice treated with PBS (panel A) or the (β15-44)₂ fragment (panel B). The size of infarcted areas, which appear in pale color (contoured by broken lines in A and B), was determined using ImageJ program (NIH) and the results are presented in panel C as % of total area of the slices. The results are means ± the standard error (n = 5). ***, P < 0.001.

Discussion

Previous studies revealed that the interaction of fibrin with VE-cadherin on endothelial cells promotes angiogenesis, this interaction occurs through the βN-domain of fibrin and is inhibited by the β15-42 fragment representing part of this domain [1,2,9]. A subsequent study [5] demonstrated that this interaction also promotes transendothelial migration of leukocytes and thus inflammation. Furthermore, it has been shown that β15-42 inhibits VE-cadherin dependent leukocyte transmigration thereby reducing reperfusion-induced inflammation and infarct size in animal models of ischemia-reperfusion [5]. At the same time, our study [10] revealed that a dimeric fragment (β15-66)₂, which corresponds to the full-length βN-domain and mimics its dimeric arrangement in fibrin, has much higher affinity to VE-cadherin than β15-42 suggesting that this dimer may be more potent inhibitor of leukocyte transmigration. The present study clarified the structural basis for the increased affinity of the (β15-66)₂ fragment to VE-cadherin and confirmed its superior inhibitory activity over β15-42. In addition, this study revealed that a truncated variant of this dimer, (β15-44)₂, also preserves high affinity to VE-cadherin and inhibitory activity towards leukocyte transmigration.

The major difference between the (β15-66)₂ and β15-42 fragment is the dimeric nature of the former and the absence of the β43-64 region in the later. Thus, to check the contribution of this region as well as that of the dimerization to the interaction with VE-cadherin, we prepared monomeric β15-64 and β15-42 fragments and their dimeric variants, (β15-66)₂ and (β15-44)₂, and studied their interaction with VE-cadherin by ELISA and SPR. The recombinant third domain of VE-cadherin, VE-cad(3), was used as a simple model of VE-cadherin since its affinity to fibrin is comparable with that of the extracellular portion of VE-cadherin [10,14]. The K_d values for the interaction of (β15-66)₂ and (β15-44)₂ with VE-cad(3) determined by ELISA were found to be lower than those determined by SPR. A similar difference for the interaction of VE-cad(3) with (β15-66)₂ was also observed in our previous study [14]. The lower apparent K_d values determined by ELISA may be connected with the observed tendency of VE-cad(3) to form aggregates whose binding to immobilized (β15-66)₂ or (β15-44)₂ may increase apparent concentration of bound VE-cad(3) thus decreasing K_d. This possible problem was absent in SPR experiments, in which VE-cad(3) was immobilized on the surface of a sensor chip and fibrin fragments were kept in solution. Thus, the data obtained by SPR should provide more accurate estimate of the K_d values than those obtained by ELISA.

Analysis of the SPR data revealed substantial differences in the affinities to VE-cadherin between the full-length and truncated fibrin fragments, both monomeric and dimeric. Namely, the K_d values for the interaction of (β15-66)₂ and β15-64 with VE-cad(3) were found to be 8-fold lower than those determined for their truncated variants, (β15-44)₂ and β15-42 (Table 1). This indicates that removal of the β43-64 region from both the dimeric and monomeric full-length fragments substantially decreased their affinity to VE-cad(3) suggesting the involvement of this region in the interaction with VE-cadherin. Our previous study localized the VE-cadherin binding sites in the N-terminal portion of the fibrin β chains (βN-domains) and identified two residues, His16 and Arg17, that are critical for the binding [10]. The results of the present study suggest that the VE-cadherin binding site is more complex and may also involve distant residues from the β43-64 region.

Our previous study also revealed that dimerization of the βN-domain is important for the interaction with VE-cadherin since the dimeric (β15-66)₂ fragment bound to VE-cadherin with high affinity while β15-64 monomer, as well as β15-42, failed to bind [10]. However, the highest concentration of β15-64 and β15-42 tested in that study was 400 nM. In the present study, we performed binding experiments at higher concentrations of these fragments and found that they do bind to VE-cadherin; however, their affinities were much lower than those determined for their dimeric variants. Namely, the K_d values determined for the interaction of (β15-66)₂ and (β15-44)₂ dimers with VE-cad(3) were found to be 355- and 373-fold lower than those determined for β15-64 and β15-42 monomers, respectively (Table 1). This difference is much higher than that between the full-length and truncated variants. Thus, this finding indicates that while the β43-64 region contributes to the interaction with VE-cadherin, dimerization of these fragments plays the major role in the high affinity interaction. This finding also raises a question of whether shorter variants of the dimeric (β15-44)₂ fragment will preserve high affinity to VE-cadherin.

Our finding that the dimeric fragments, (β15-66)₂ and (β15-44)_2, have much higher affinity to VE-cadherin than monomeric β15-42 suggested that they both may be more potent inhibitors of transendothelial migration of leukocytes than β15-42. To test this suggestion, we compared the inhibitory effects of all three fragments on leukocyte transmigration in vitro and in vivo. In the in vitro transmigration assay, in which we used HL-60 cell line differentiated into neutrophil-like cells, both dimeric fragments exhibited similar inhibitory effects on NDSK-II-induced migration of these cells through HUVEC monolayer while the effect of β15-42 was lower. The difference was also observed in the in vivo experiments using a peritonitis model, a well established mouse model for leukocyte migration into sites of acute inflammation [16,20-22]. In these experiments, the inhibitory effect of the dimeric fragments exceeded that of β15-42 by more than two-fold (Fig. 4 and 5). Altogether, these experiments confirmed the hypothesized superior anti-inflammatory properties of the dimeric (β15-66)₂ and (β15-44)₂ fragments. It remains to be tested whether shorter variants of the dimeric (β15-44)₂ fragment, which may preserve high affinity to VE-cadherin, also exhibit superior anti-inflammatory effect.

It should be noted that in spite of a very low affinity of β15-42 monomer to VE-cadherin its inhibitory effect on leukocyte transmigration in our in vitro and in vivo experiments was fairly high, about half of that exhibited by (β15-66)₂. Such a discrepancy can be explained by the fact that its affinity to endothelial cell surface (K_d = 0.18 μM), which was determined earlier [2], is much higher than that to VE-cadherin determined in this study (K_d = 267 μM, Table 1). This suggests that β15-42 monomer may interact with other endothelial cell receptor(s). Indeed, since fibrin and its β15-42 containing fragments interact with heparin [11,12] they should also interact with cell surface proteoglycans and this interaction may contribute to their increased affinity to endothelial cell surface. Thus this interaction may represent an additional mechanism contributing to fibrin-dependent leukocyte transmigration; the existence of other mechanisms cannot be excluded.

Since the difference in the anti-inflammatory effects of both dimeric fragments, (β15-66)₂ and (β15-44)_2, was statistically insignificant, we tested the effect of the smaller fragment, (β15-44)_2, on infarct size in a mouse model of myocardial ischemia-reperfusion injury. The advantage of this short dimer is that its monomeric unit contains only 30 amino acid residues and can be easily synthesized and dimerized through its Cys43. The experiments revealed that treatment with this fragment of mice subjected to ischemia-reperfusion reduced myocardial infarct size by more than two-fold indicating that (β15-44)₂ has significant cardioprotective effect in this model. The question of whether this dimeric fragment, as well as (β15-66)₂, is more efficient inhibitor of ischemia-reperfusion induced myocardial inflammation than monomericβ15-42 remains to be addressed.

A number of recent studies with the β15-42 fragment further confirmed its cardioprotective effect in rodent and pig models of myocardial ischemia-reperfusion injury [17,18] and revealed its organ protective effect in pig models of hemorrhagic shock and reperfusion [23,24]. Further, the results of a recent trial on the efficacy of β15-42 in prevention of myocardial reperfusion injury revealed the potential of this fragment to protect during myocardial infarction in humans [25]. At the same time, they also suggest that more potent fibrin-derived inhibitors of inflammation may be required for more efficient treatment of reperfusion injury in human patients. In this connection, the dimeric fragments described in the present study, (β15-66)₂ and (β15-44)₂, which exhibited much higher anti-inflammatory effect than β15-42 in animal models, may also have higher cardioprotective effect in humans.

In summary, the present study revealed that dimerization of β15-42-containing fragments, (β15-66)₂ and (β15-44)₂, plays the major role in the increasing of their affinity to VE-cadherin and that such dimeric fragments are more potent inhibitors of leukocyte transmigration than monomeric β15-42 in the in vitro transendothelial migration assay and in vivo peritonitis model. The study also demonstrated a significant cardioprotective effect of the dimeric (β15-44)₂ fragment in a mouse model of myocardial ischemia-reperfusion injury. Whether dimeric β15-42-containing fragments and their truncated variants are more potent cardioprotective agents in animals and humans remains to be tested.