The effect of factor XIII on endothelial barrier function was studied in a model of cultured
monolayers of porcine aortic endothelial cells and saline-perfused rat hearts. The thrombin-activated plasma factor XIII (1 U/ml) reduced albumin permeability of endothelial monolayers
within 20 min by 30 ± 7% (basal value of 5.9 ± 0.4 × 10
6 cm/s), whereas the nonactivated
plasma factor XIII had no effect. Reduction of permeability to the same extent, i.e., by 34 ± 9% could be obtained with the thrombin-activated A subunit of factor XIII (1 U/ml), whereas the
iodoacetamide-inactivated A subunit as well as the B subunit had no effect on permeability.
Endothelial monolayers exposed to the activated factor XIII A exhibited immunoreactive deposition of itself at interfaces of adjacent cells; however, these were not found on exposure to
nonactivated factor XIII A or factor XIII B. Hyperpermeability induced by metabolic inhibition (1 mM potassium cyanide plus 1 mM 2-deoxy-D-glucose) was prevented in the presence
of the activated factor XIII A. Likewise, the increase in myocardial water content in ischemic-reperfused rat hearts was prevented in its presence. This study shows that activated factor XIII
reduces endothelial permeability. It can prevent the loss of endothelial barrier function under
conditions of energy depletion. Its effect seems related to a modification of the paracellular passageways in endothelial monolayers.
Key words:
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Introduction |
The endothelium forms a barrier for solutes and macromolecules between the luminal and interstitial space.
Under pathophysiologic conditions, loss of endothelial barrier function is predominantly due to an increase in paracellular permeability leading to enhanced extravasation of
macromolecules and fluid. The resulting extracellular edema
can compromise the function or may even jeopardize survival of the affected organ.
Factor XIII is a transglutaminase (endo-
-glutamine:
-lysine transferase, EC 2.3.2.13) that catalyzes the formation of
-glutamyl-
-lysyl cross-links between adjacent
polypeptide chains. It plays an important role in the course
of coagulation and fibrinolysis (for reviews, see references
1 and 2). The plasma proenzyme is a heterotetramer consisting of two types of subunits (A and B, with molecular
masses of ~83 and 77 kD, respectively) which are noncovalently associated. The plasma factor XIII is activated by
thrombin-mediated cleavage of an NH2-terminal peptide
from the A subunits which then become the active transglutaminases. The function of the B subunits is not fully
understood at present. It seems to protect the A subunits from spontaneous nonproteolytic activation (3) or the activated A subunits from deactivation (4). The well-known
main function of factor XIII in blood consists in the stabilization of a formed thrombus by cross-linking of fibrin
chains. Factor XIII also appears to be involved in cell adhesion and migration (5), assembly of extracellular matrix
(8, 9), and tissue repair and wound healing (10, 11). The latter effects have been attributed to the ability of factor XIII to
cross-link a variety of proteins of the extracellular matrix, e.g.,
fibronectin, collagen, and vitronectin (12).
During the last decade, several clinical observations showed
that systemically applied factor XIII can reduce capillary
hyperpermeability and may thus confer an antiedematous
effect (for reviews, see references 15 and 16). It was found
that the enhanced capillary permeability in patients with
connective tissue disease is attenuated to almost normal levels under therapy with factor XIII (16, 17). It was also reported that factor XIII therapy reduces mucosal edema in inflammatory bowel disease (18, 19) and Henoch-Schönlein purpura (17, 20, 21). In an animal study, Hirahara et al. (22)
have shown that factor XIII can suppress the enhanced vascular permeability of guinea pig skin provoked by an inflammatory response upon injections of an antiendothelial cell
antiserum. The underlying mechanism of these various antiedematous effects of factor XIII has remained unknown.
In this study, the question was addressed whether factor
XIII can directly influence endothelial barrier function.
Cultures of endothelial cells and the coronary system of an
isolated heart were used as experimental models. In monolayers of cultured endothelial cells from porcine aorta, the
paraendothelial passage of albumin was monitored as a parameter of endothelial barrier function (23, 24). Variations
of macromolecule permeability in this model are attributable to changes in paracellular permeability (25). In the isolated rat heart, changes in tissue water content were determined as indication of vascular permeability (26). We found that the activated factor XIII reduces permeability of endothelial monolayers. Specifically, it prevents hyperpermeability provoked by energy depletion in endothelial monolayers and in ischemic-reperfused hearts.
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Materials and Methods |
Cell Cultures.
Porcine aortic endothelial cells were isolated as
described previously (27) by gentle mechanical scraping of the intima of the descending part of porcine aorta. Harvests of endothelial cells were plated at a density of 106 cells per 100-mm plastic
dish. The cells were cultured at 37°C in a humidified atmosphere
of 5% CO2 in air. The "basal culture medium" consisted of medium 199 with Earle's salt, supplemented with 100 IU/ml penicillin G, 100 µg/ml streptomycin, and 20% (vol/vol) newborn calf
serum (NCS).1 The medium was renewed every other day. After
4 d, when the cells had grown to confluence, they were trypsinized
in PBS (composed of [mM]: 137 NaCl, 2.7 KCl, 1.5 KH2PO4,
and 8.0 Na2HPO4, at pH 7.4, supplemented with 0.05% [wt/vol]
trypsin and 0.02% [wt/vol] EDTA). Endothelial cells were seeded
at a density of 7 × 104 cells/cm2 on either 24-mm round polycarbonate filters (pore size 0.4 µm) or 20-mm round glass coverslips
for determination of albumin flux and immunostaining, respectively, and were cultured in basal culture medium (for compositions, see above). Experiments were performed with confluent
monolayers, 4 d after seeding. The purity of these cultures was
>99% endothelial cells as determined by uptake of DiI-ac-LDL,
contrasted with <1% cells positive for 
smooth muscle actin.
Macromolecule Permeability of Endothelial Monolayers.
The permeability of the endothelial cell monolayer was studied in a two-compartment system separated by a filter membrane (24, 28). Both
compartments contained as basal medium modified Tyrode's solution (composition in mM: 150 NaCl, 2.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.0 CaCl2, and 30.0 N-2-hydroxyethylpiperazine-
N'-2-ethanesulfonic acid; pH 7.4, 37°C) supplemented with 2%
(vol/vol) NCS. There was no hydrostatic pressure gradient between
the two compartments. The "luminal" compartment containing
the monolayer had a volume of 2.5 ml, and the "abluminal" had a
volume of 6.5 ml. The fluid in the abluminal compartment was
constantly stirred. Trypan blue-labeled albumin (60 µM) was
added to the luminal compartment. The appearance of the labeled albumin in the abluminal compartment was continuously monitored by pumping the liquid through a spectrophotometer
(Specord 10; Carl Zeiss). Increases of the concentration of labeled
albumin were detected with a time delay of <15 s. The concentration of labeled albumin in the luminal compartment was determined every 10 min of incubation. It did not change significantly
in the time frame of the experiments.
The albumin flux (F, expressed as mol/[s × cm2]) across the
monolayer with the surface (S) was determined from the rise of albumin concentration (d[A]2) during the time interval (dt) in the
abluminal compartment (volume V):
To facilitate the comparison of data obtained in this study with
those of other studies, the permeability coefficient (P, expressed as
cm/s) of the combined system of monolayer and filter support was
calculated from F according to Fick's law of diffusion as follows:
where [A]1 and [A]2 denote tracer concentrations in the luminal
and abluminal compartments, respectively. Because the driving force ([A]1
[A]2) remained virtually unchanged in the course of
the described experiments, the relative changes in F correspond to similar changes in the permeability coefficient.
Experimental Conditions.
The basal medium used in incubations was modified Tyrode's solution (see above). Macromolecule permeability of the endothelial monolayer, transferred to the
incubation chamber, was determined after an initial equilibration
period of 20 min. The basal albumin permeability of each monolayer filter system was then determined for another 20 min of incubation. Agents were added as indicated, and the response of the
albumin permeability was recorded for an additional 80 min.
In a set of experiments, endothelial monolayers were preincubated in basal medium (for composition, see above) supplemented with thrombin-activated factor XIII A (1 U/ml) at 37°C
in a cell culture incubator for 2, 4, and 6 h. The endothelial
monolayers were then transferred to the incubation chamber, and
albumin permeability of these pretreated monolayers was determined after an initial equilibration period of 20 min.
Myocardial Water Content.
Hearts from 250-g male Wistar rats
were mounted immediately after isolation on a Langendorff perfusion system in a temperature-controlled chamber (37°C), as described previously (29). During normoxic perfusion, the chamber
was flushed with humidified air, and during anoxic perfusion, with
a 95% N2 (vol/vol)/5% CO2 (vol/vol) mixture. Under normoxic
conditions, the hearts were perfused at a constant flow of 10 ml/
min with an oxygenated saline medium (composition in mM: 140.0 NaCl, 24.0 NaHCO3, 2.7 KCl, 0.4 KH2PO4, 1 MgSO4, 1.8 CaCl2,
5 glucose, pH 7.4; gassed with 95% O2 [vol/vol]/5% CO2 [vol/
vol]). For low-flow ischemia, this normoxic period was followed by
40 min anoxic perfusion at 0.5 ml/min (composition of the perfusion medium as above; pH 7.4; gassed with 95% N2 [vol/vol]/5% CO2 [vol/vol]). After low-flow ischemia, hearts were again resupplied with oxygen by returning to the initial perfusion conditions. Factor XIII A was added to the perfusion medium 5 min before the onset of low-flow ischemia. It remained in the perfusion medium during the entire period of low-flow ischemia and reperfusion.
Activation of Factor XIII.
Activation of the plasma factor XIII
and factor XIII A was performed by incubations of known
amounts of factor XIII in the presence of sepharose-coupled
thrombin at 37°C in Tris buffer (200 mM, pH 7.4) for 20 min.
The activated factor XIII was then separated from thrombin-sepharose by centrifugation. The contamination with thrombin
of these supernatants was below detection limits. Factor XIII activity was determined by using the assay described by Fickenscher
et al. (30) without thrombin in the assay.
Inactivation of Factor XIII A.
Factor XIII A was inactivated using the alkylating agent iodoacetamide as described by Curtis et al.
(31). To inactivate factor XIII, aliquots of the thrombin-activated
factor XIII A containing ~12 µM (corresponding to 1 mg protein/ml) were incubated in the presence of 24 µM iodoacetamide at 37°C for 10 min. 48 µM glutathione was then added to
react with the residual amounts of iodoacetamide, and incubations
were continued for 5 min at room temperature. After this procedure, the activity of factor XIII A was below detection limits. Aliquots of the inactivated factor XIII A (~10 µg protein equivalent
to 1 U factor XIII A) were added to the cells. The final concentrations of iodoacetamide and glutathione were 0.24 and 0.48 µM,
respectively. At those concentrations, neither substance affected
basal permeability of the endothelial monolayers.
Immunofluorescence Microscopy.
Confluent endothelial monolayers were washed three times with PBS, then fixed with 5%
paraformaldehyde for 10 min at 20°C, and washed again three
times with PBS. The cells were covered with 100 µl polyclonal
rabbit anti-factor XIII A or anti-factor XIII B antibodies (diluted
1:200 in PBS), and incubated for 6 h at 37°C. The coverslips
were then washed three times with PBS, covered with 100 µl of
mouse anti-rabbit IgG coupled to FITC (diluted 1:100 in PBS),
and incubated for 6 h at 37°C. The coverslips were finally embedded in a 40% glycerol/PBS solution (pH 8.5) on glass slides.
Cell monolayers were visualized using an inverse fluorescence microscope (model IX 70; Olympus).
Electron Microscopy.
After permeability experiments, confluent
endothelial monolayers on filter membranes were washed three
times with PBS, and fixed with 5% paraformaldehyde for 10 min
at 20°C as described for immunofluorescence microscopy. The
cells were covered with 100 µl polyclonal rabbit anti-factor XIII
A or anti-factor XIII B antibodies (diluted 1:200 in PBS), and incubated overnight at room temperature. The filters were then
washed three times with PBS, covered by 100 µl of donkey anti-
rabbit IgG coupled to peroxidase (diluted 1:150 in PBS), and incubated at room temperature for 1 h. The filters were washed
twice with PBS and twice with Tris-HCl (10 mM, pH 7.4) and
then incubated with 3,3'-diaminobenzidine (DAB) and hydrogen peroxide as substrates for the peroxidase reaction in the presence of nickel ammonium sulfite for 45 min. The filters were
then washed again three times with Tris-HCl and exposed to a
1% solution of OsO4 at 4°C for 1 h. After washing twice with Tris-HCl and twice with maleate buffer (pH 5.2), the specimens were incubated in a 1% uranyl acetate solution in maleate buffer in the dark at room temperature for 1 h. Subsequently, the specimens were washed again three times with maleate buffer, dehydrated in 70% ethanol, and transferred to 2,2'-dimethoxypropan,
followed by embedding in spurr resin. Polymerization of the embedded specimens was performed at 60-70°C overnight. Ultrathin cross-sections of the monolayers were cut, stained with lead citrate, and viewed with a transmission electron microscope (model
EM 902; Carl Zeiss).
Statistical Analysis.
Data are given as means ± SD of n = 6 experiments using independent cell preparations. Statistical analysis of data was performed according to Student's unpaired t test.
Probability (P) values <0.05 were considered significant.
Materials.
Donkey anti-rabbit IgG coupled to peroxidase was
from Amersham Buchler; Falcon plastic tissue culture dishes were
from Becton Dickinson; polyclonal anti-factor XIII A antibody, and
polyclonal anti-factor XIII B antibody DADE were from Behring
Diagnostics; glutathione was from Boehringer Mannheim; plasma
factor XIII and isolated factor XIII B subunit purified from Fibrogammin HSTM, factor XIII A subunit (recombinant human factor
XIII expressed in yeast and purified to homogeneity [impurities
<100 ppm]), and human thrombin were from Centeon Pharma
GmbH; Transwell® polycarbonate filter inserts (24-mm diameter,
0.4-µm pore size) were from Costar; NCS), medium 199, penicillin-streptomycin, and trypsin-EDTA were from GIBCO Life
Technologies; DAB (ISOPACTM) and DiI-ac-LDL (acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl-1-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate) were from Paesel & Lorei;
spurr resin was from Serva; anti-rabbit IgG coupled to peroxidase
or FITC, and iodoacetamide were from Sigma. All other chemicals
were of the best available quality, usually analytical grade.
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Results |
Effect of Factor XIII on Monolayer Permeability.
It was tested
initially whether the activity of factor XIII added to endothelial monolayers is changed throughout the time course
of a permeability experiment. The following additions to the luminal compartment of the incubation chambers were
made: thrombin-activated or nonactivated plasma factor
XIII, and thrombin-activated or nonactivated factor XIII A
subunit. As shown in Fig. 1, the measured activities remained
stable during the entire experimental period.

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Fig. 1.
Factor XIII activity in the luminal compartment of the experimental two-compartment system. Thrombin-activated plasma factor
XIII ( , 0.8 U/ml), thrombin-activated factor XIII A subunit ( , 1.2 U/ml), nonactivated plasma factor XIII ( , 10 µg/ml), or nonactivated
factor XIII A subunit ( , 10 µg/ml) was added to the luminal compartment containing the endothelial monolayer at time point 0.
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Macromolecule permeability of endothelial monolayers
was continuously monitored by determining the flux of albumin across the monolayers. Under control conditions, mean
permeability was 5.9 ± 0.6 × 10
6 cm/s (Fig. 2). It remained
constant during the entire period of observation. Addition of
the thrombin-activated plasma factor XIII (1 U/ml) caused a
rapid decrease of albumin permeability, which was reduced
by 30% after 20 min. In contrast to the activated plasma
factor XIII, addition of the nonactivated plasma factor XIII
had no effect on permeability.

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Fig. 2.
Effect of the plasma factor XIII on albumin permeability of
porcine aortic endothelial monolayers. At time 0, the following additions
were made: none ( , Control); nonactivated plasma factor XIII ( , 20 µg
protein/ml = 1 U/ml); activated factor XIII ( , 1 U/ml). Data are
means ± SD of n = 5 separate experiments of independent cell preparations. *P < 0.05 vs. control.
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Exposure of endothelial monolayers to the thrombin-
activated factor XIII A subunit (10 µg/ml, equivalent to
~1 U/ml) also led to a rapid reduction of permeability, by
34% within 20 min (Fig. 3). The nonactivated factor XIII A
(10 µg/ml) as well as additions of the iodoacetamide-inactivated factor XIII A (10 µg/ml) had no effect on permeability. Likewise, the isolated factor XIII B subunit (10 µg/ml)
did not affect the albumin permeability of the endothelial
monolayers (Fig. 4). In a set of experiments, it was tested
whether the activated factor XIII A can affect albumin permeability of endothelial monolayers for a prolonged period of time. For that reason, endothelial monolayers were preincubated with thrombin-activated factor XIII A for 2, 4, and
6 h. Albumin permeability was then determined. As shown
in Table I, the reduction of albumin permeability induced
by the activated factor XIII A persists for 6 h.

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Fig. 3.
Effect of the factor XIII A subunit on albumin permeability
of endothelial monolayers. At time 0, the following additions were made:
none ( , Control); nonactivated factor XIII A ( , 10 µg/ml = 1 U/ml);
iodoacetamide-inactivated factor XIII A ( , 10 µg/ml = 1 U/ml);
thrombin-activated factor XIII A ( , 10 µg/ml = 1 U/ml). Data are
means ± SD of n = 5 separate experiments of independent cell preparations. *P < 0.05 vs. control.
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Fig. 4.
Effect of the factor XIII B subunit on albumin permeability
of endothelial monolayers. At time 0, the following additions were made:
none ( , Control); factor XIII B subunit ( , 10 µg protein/ml) not
treated with thrombin; factor XIII B subunit ( , 10 µg protein/ml)
treated with thrombin. Data are means ± SD of n = 5 separate experiments of independent cell preparations. *P < 0.05 vs. control.
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Table I
Effect of Activated Factor XIII A on Albumin
Permeability of Endothelial Monolayers after Various
Times of Incubation
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The activated factor XIII A reduced albumin permeability with increasing activity (Fig. 5), with half-maximal effect at 0.9 U/ml. In contrast, the nonactivated factor XIII
A as well as factor XIII B had no significant effect on albumin permeability when applied in the same range of protein concentration.

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Fig. 5.
Dose-dependent effect of factor XIII on albumin permeability. Permeability was determined 20 min after the following additions:
none ( , control [C]); nonactivated factor XIII A subunit ( ); thrombin-activated factor XIII A subunit ( , 10 µg protein/ml = 1 U/ml);
factor XIII B subunit ( ). Data are means ± SD of n = 5 separate experiments of independent cell preparations. *P < 0.05 vs. control.
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Immunostaining of Endothelial Monolayers.
For immunostaining, a polyclonal rabbit anti-factor XIII A antibody was
used which recognizes the activated as well as the nonactivated factor XIII A (32). Immunostaining of endothelial monolayers incubated for 20 min in the presence of thrombin-activated factor XIII A (1 U/ml) revealed factor XIII
A-positive staining along the interface of adjacent endothelial cells (Fig. 6 A). In monolayers that were exposed to
nonactivated factor XIII A at equivalent protein concentration (10 µg protein/ml), immunostaining for factor XIII A
remained absent (Fig. 6 B). As control, endothelial monolayers that had not been incubated in the presence of factor XIII A were exposed to either the first anti-factor XIII A
and second antibody (FITC-coupled anti-rabbit IgG; Fig.
6 C) or the second antibody alone (Fig. 6 D). No specific
staining was observed with these protocols.

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Fig. 6.
Immunostaining of factor XIII
A in endothelial monolayers. (A) Endothelial cells were incubated for 20 min in the
presence of activated factor XIII A (1 U/ml).
Factor XIII A-positive staining is seen along
the interfaces of adjacent endothelial cells.
(B) Endothelial cells were exposed to nonactivated factor XIII A (10 µg/ml). No positive staining for factor XIII A is observed.
(C) Endothelial cells not preincubated with
factor XIII A were exposed to anti-factor
XIII A and FITC-coupled anti-rabbit IgG
antibody (first and second antibody control).
Only background fluorescence is seen. (D)
Endothelial cells not preincubated with factor XIII A were exposed only to the FITC-coupled anti-rabbit IgG antibody (second
antibody control). Again, only background
fluorescence is apparent. Bar, 10 µm.
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In a second set of experiments, endothelial monolayers
were incubated in the presence of factor XIII B (10 µg
protein/ml) which had been preexposed or not to thrombin. For immunohistochemistry, a specific polyclonal antibody raised against factor XIII B (32) was used, which we
confirmed to stain isolated factor XIII B (not shown). No
specific staining for factor XIII B was detected in the
monolayers (Fig. 7).

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Fig. 7.
Immunostaining of factor XIII B in endothelial monolayers.
(A) Endothelial cells were incubated for 20 min in the presence of isolated
factor XIII B subunit (10 µg/ml) which had been pretreated with thrombin. Only background fluorescence is observed. (B) Endothelial cells were
exposed to factor XIII B subunit (10 µg/ml) which was not pretreated.
Only background fluorescence is present. (C) Endothelial cells not preincubated with factor XIII B subunit were exposed to anti-factor XIII B
and FITC-coupled anti-rabbit IgG antibody (first and second antibody
control). There is only background fluorescence. Bar, 50 µM.
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To analyze the localization of factor XIII A in cross-sections of endothelial monolayers in greater detail, these were
incubated for 20 min in the presence or absence of thrombin-activated or nonactivated factor XIII A. The endothelial monolayers were then processed for transmission electron microscopy. When activated factor XIII A had been
applied, factor XIII A immunoreactivity was identified by
the accumulation of an electron-dense DAB reaction product at the intercellular cleft and of the basal endothelial surface along the margin of the cells (Fig. 8 C). In contrast, no
DAB reaction product was observed in intercellular clefts
of control monolayers (Fig. 8 A) or in endothelial monolayers exposed to nonactivated factor XIII A (Fig. 8 B).

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Fig. 8.
Electron microscopic localization of factor XIII A immunoreactivity in cross-sections of endothelial monolayers. Cross-sections of
the interface of two adjacent endothelial cells are shown. (A) Control
conditions in the absence of factor XIII A. (B) After incubation for 20 min in the presence of nonactivated factor XIII A (10 µg/ml) or (C) of
activated factor XIII A (1 U/ml). Factor XIII A immunoreactivity was
identified by accumulation of electron-dense DAB reaction product in
the intercellular clefts (arrowheads) only in those monolayers exposed to
the activated factor XIII A (C). Reaction product in C is also found at the
basal endothelial surface between the cells and the filter (double arrowheads), and the inner surface of the filter pores (arrow). Bars, 1 µm.
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Effect of Factor XIII A on Hyperpermeability Induced by Endothelial Energy Depletion.
As shown in previous studies
from our laboratory (28, 33), metabolic inhibition (MI) of
mitochondrial and glycolytic energy production causes a
rapid rise in macromolecule permeability. In the present
study, it was tested whether the activated factor XIII A can
attenuate the hyperpermeability in energy-depleted endothelial monolayers. Addition of 1 mM KCN (inhibitor of
mitochondrial respiration) plus 1 mM 2-deoxy-D-glucose
(2-DG, inhibitor of glycolytic ATP production) caused an
increase in permeability by 23% within 10 min (Fig. 9).
Exposure of endothelial monolayers to 1 U/ml of activated
factor XIII A led to a 30% reduction of permeability. In the
presence of activated factor XIII A, addition of the metabolic inhibitors no longer caused an increase in permeability.
The level of permeability remained even as low as that obtained by addition of the activated factor XIII A before MI.

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Fig. 9.
Effect of activated factor XIII A (1 U/ml) on monolayer permeability of endothelial cells under MI (1 mM KCN plus 1 mM 2-DG).
Conditions were as follows: no MI and no factor XIII A ( , Control);
MI and no factor XIII A ( ); addition of factor XIII A and no MI ( );
addition of factor XIII A and MI ( ). Data are means ± SD of n = 5 separate experiments of independent cell preparations. *P < 0.05 vs. control;
#P > 0.05, not significant vs. factor XIII A.
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In immunomicroscopy, the staining of factor XIII A at
cell-cell interfaces was enhanced when the monolayers
were exposed to metabolic inhibitors (Fig. 10). As can be
seen by comparison of immunostaining and phase-contrast
images of the same section, the enlarged zones of factor
XIII A-positive staining correspond to gaps opening between adjacent cells.

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Fig. 10.
Immunostaining of factor XIII A in endothelial monolayers
under MI. (A) Endothelial cells were first incubated in the presence of activated factor XIII A (1 U/ml), then the monolayer was exposed to MI
for 60 min (see Fig. 9). Broad bands of factor XIII A-positive staining are
seen at cell-cell interfaces. (B) Phase-contrast image corresponding to A. Enlarged zones of factor XIII A-positive staining (arrowheads) in A correspond to gaps (arrowheads) in B between adjacent cells. Bar, 10 µm.
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Effect of Factor XIII A on Myocardial Water Content.
To
analyze whether the activated factor XIII A can also affect
endothelial barrier function in the coronary system, the isolated perfused heart was used and changes of myocardial
water content were determined. Under control conditions,
the myocardial water content of the normoxic perfused rat
heart was, on average, 430 ml/100 g dry wt over a period
of 160 min of observation (Fig. 11). To provoke an increase in vascular permeability, hearts were exposed to a
40-min period of low-flow ischemia followed by a period
of 60 min of normoxic reperfusion. Ischemia-reperfusion experiments were performed with addition of either the
nonactivated or the activated factor XIII A 5 min before
onset of anoxic low-flow perfusion. With the nonactivated
factor XIII A, the water content of reperfused hearts rose to
530 ml/100 g dry wt. In the presence of the activated factor XIII A (5 U/ml), myocardial water content remained as
it was before reperfusion.

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Fig. 11.
Effect of factor XIII on changes of myocardial water content under low-flow ischemia and reperfusion. Rat hearts in Langendorff
mode were perfused for 60 min with normoxic perfusate at 10 ml/min,
followed by 40 min low-flow perfusion at 0.5 ml/min, and 60 min reperfusion with normoxic perfusate at 10 ml/min. Control hearts were perfused up to 160 min with normoxic media at 10 ml/min ( ). Perfusate of
low-flow ischemia and reperfusion was supplemented with either nonactivated factor XIII A ( , 50 µg/ml) or thrombin-activated factor XIII A
( , 50 µg/ml = to 5 U/ml). Data are means ± SD of n = 4 separate experiments of independent heart preparations. *P < 0.05 vs. 160 min normoxia; #P < 0.05 vs. activated factor XIII A.
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Discussion |
The central question of this study was whether factor
XIII can directly influence endothelial barrier function. In
the model of cultured endothelial monolayers, we found
that activated factor XIII not only lowers the basal permeability for macromolecules but also prevents the increase in
permeability provoked by an inhibition of endothelial energy production. In the isolated whole heart, activated factor XIII was able to prevent edema formation caused by ischemia-reperfusion. The endothelial effects of factor XIII are
exerted only by the activated form of the A subunit.
Confluent monolayers of cultured porcine aortic cells
were used as a model (24, 28, 33). To characterize the barrier of these monolayers towards macromolecules, the passage of albumin across the monolayers was studied. Changes
in macromolecule permeability in this model are attributed
to changes in paracellular permeability (25). The basal level
of permeability in this model is not the lowest possible, and
can therefore be used to investigate factors improving endothelial barrier function without prior stimulation (23, 34).
The nonactivated plasma factor XIII did not affect permeability of the monolayers. However, when activated by
exposure to sepharose-coupled thrombin, plasma factor
XIII markedly lowered the permeability. To analyze which
part of the heterodimeric complex is responsible for this effect, a recombinant A subunit and a purified B subunit of
factor XIII were applied in the permeability experiments.
The A subunit was equipotent to plasma factor XIII when
activated by exposure to thrombin. The lowering effect on permeability of the factor XIII A was dependent on its enzymatic activity. If factor XIII A was inactivated by the alkylating agent iodoacetamide, it no longer reduced permeability.
The B subunit had no effect. The results thus show that the
activated A subunit of factor XIII represents the active principle of the permeability-lowering effect.
Active factor XIII is a transglutaminase capable of cross-linking various types of proteins (2) and is entrapped in the stable protein meshwork formed. With immunomicroscopy, we found factor XIII deposited at the endothelial
monolayer under exactly those conditions where factor
XIII reduced monolayer permeability, i.e., when the activated A subunit was present. Immunoreactivity of factor
XIII A was localized under these circumstances along the
interfaces of adjacent endothelial cells. Electron microscopy revealed that it was concentrated in the narrow gaps between adjacent cells and at the basal endothelial surface between the cells and the filter support. Mass deposition of
factor XIII A was not found at any other site within the endothelial monolayers. The B subunit did not form depositions on the monolayer when applied. There are a variety
of proteins like fibronectin and vitronectin residing in the
intercellular clefts and the subendothelial matrix which are
involved in cell-to-cell and cell-to-matrix adhesion of endothelial cells and which represent substrates for factor XIII
cross-linking reactions (12, 14). Interestingly, the small intercellular clefts represent the principle paracellular pathway for passage of macromolecules in these monolayers.
Therefore, the microscopic observations suggest that active
factor XIII A reduces monolayer permeability because it reacts with extracellular matrix proteins at these strategic sites
of the endothelial barrier. In doing so it may itself become
entrapped, as in fibrin clots.
We showed previously, using the same experimental
model, that energy depletion of endothelial cells causes a
rapid rise in monolayer permeability (28, 33). This rise in
permeability is associated with a widening of intercellular
gaps. We find now that in the presence of active factor XIII
A, the rise in permeability is abolished even though the energy-depleted cells in the monolayer remain retracted from
each other. The latter observation indicates that factor XIII
does not prevent the immediate structural consequences of
energy loss within endothelial monolayers. The explanation for the protective effect of factor XIII seems to lie in
another finding, that the intercellular gaps contain massive
depositions of factor XIII immunoreactivity. This finding is
consistent with the above hypothesis that factor XIII reduces monolayer permeability by cross-linking of proteins
at the paracellular passageways.
To study whether the activated factor XIII A can affect
endothelial barrier function in an intact coronary system,
saline-perfused rat hearts were used as a model. Low-flow
ischemia and subsequent reperfusion caused a marked increase in myocardial water content, as also reported by others (26). When the perfusion medium was supplemented
with the activated factor XIII A before onset of low-flow
ischemia, an increase in myocardial water content did not
occur. These data show that the activated factor XIII A can prevent development of hyperpermeability in this perfused
heart model.
This study has revealed a new function of factor XIII,
i.e., stabilization of endothelial barrier function. It shows
that this function is due to a direct effect on the endothelial
monolayer. The observations in microscopy indicate that
factor XIII can reduce the permeability through an endothelial monolayer by interactions with proteins of the extracellular matrix between cells. As the permeability-lowering
effect is restricted to the active form of factor XIII, which
acts as an enzymatic cross-linker of proteins, this effect
seems to be due to narrowing of the sieving meshwork in
the paracellular transendothelial passageways. The experiments on energy-depleted monolayers and ischemic-reperfused hearts indicate that the active factor XIII can be used
to prevent edema formation caused by endothelial metabolic disturbances.
Address correspondence to Thomas Noll, Physiologisches Institut, Justus-Liebig-Universität, Aulweg 129, D-35392 Giessen, Germany. Phone: 49-641-99-47243; Fax: 49-641-99-47239; E-mail: thomas.noll{at}physiologie.med.uni-giessen.de
This work was supported by the Deutsche Forschungsgemeinschaft, grants A3 and A4 of SFB 547.
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