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analogs on collagen
gel compaction in vitro and interstitial pressure in vivo
1 Department of Physiology,
University of Bergen, N-5009 Bergen, Norway;
2 Department of Medical and
Physiological Chemistry, Acute inflammation in skin is accompanied by
increased negativity of interstitial fluid pressure
(PIF), which will increase capillary fluid filtration and thereby potentiate edema formation. A
series of studies indicates that the connective tissue cells in rat
dermis are involved in the control of
PIF and mediate this response. The
present study describes a novel effect of prostaglandin (PG)
E1 isopropyl ester,
carbaprostacyclin (PGI2 analog),
and latanoprost (PGF2
acute inflammation; edema; loose connective tissue; latanoprost
INCREASED CAPILLARY FLUID filtration and
increased tissue fluid volume content are important elements of acute
inflammatory reactions (2, 23). These changes lead to impaired tissue function that in some conditions may become life threatening, such as
in angioedema (Quincke's edema). During the last few years we have
demonstrated that loose connective tissues can participate actively in
control of interstitial fluid pressure
(PIF) (23). This concept is
based partly on Meyer's (18) observation that loose connective tissue
that is allowed free access to fluid will swell because of its
glycosaminoglycan and hyaluronan content. Furthermore, Meyer (18)
demonstrated that under normal conditions the tendency of the loose
connective tissue to expand is counteracted by collagen and microfibril
networks physically restraining the swelling hyaluronan. During acute
inflammation there is an increased negativity of
PIF. This observation has been
interpreted to mean that the tensile forces exerted by connective
tissue cells on these fiber networks are reduced, allowing tissues to
swell and thereby lower PIF
because no fluid is initially added to the tissue. Increased negativity of PIF can be
seen after a number of inflammatory reactions and after blockade of
The present study was performed to investigate whether prostaglandins
may also induce increased negativity of
PIF in parallel with their known
action on the collagen gel contraction assay. The prostaglandins are
autacoids, synthesized from arachidonic acid (21), with a role both in
normal physiology and in inflammation (11). In epidermis they are
involved in almost every step of the inflammatory process (8).
Prostanoids have a wide range of biological activities in different
cells and tissues, acting as local hormones and functioning through
different G protein-linked receptors. However, their biological
response is not only determined by a particular intracellular effector
or second messengers but also by the distribution and number of
specific receptors on different cells (5). In skin the early responses
to an injurious agent are increase in vascular flow (8) and edema
formation. The prostaglandins contribute to the increased blood flow
and edema formation by modulating the vascular tone and microvascular
permeability (4). Prostacyclin
(PGI2) and
PGE1 are potent vasodilatators (15, 19). PGF2 Based on the effects of PGE1,
PGI2, and
PGF2 Measurements of PIF were performed
on female Wistar-Møller rats (200-250 g) anesthetized by
intraperitoneal injections of pentobarbital sodium (Mebumal, 50 mg/kg
body wt). The rats were not fasting before experiments and were kept at
37.5°C on a servo-controlled heating pad when anesthetized. When
necessary, the right external jugular vein was cannulated with a
polyethylene catheter (PE-50) for intravenous administration.
Anaphylactic reactions were induced by injecting Dextran 70 (60 mg/ml
iv; Pharmacia, Uppsala, Sweden; Ref. 32). In the experiments for
measurement of PIF, circulatory arrest was induced to limit transcapillary fluid flux and edema formation induced by dextran or by the test substances. Circulatory arrest was induced in pentobarbital anesthesia by rapid intravenous or
intracardiac injection of 0.5 ml of saturated potassium chloride 1 min
after the start of dextran infusion. This will allow distribution of
dextran to the peripheral tissues but keeps edema formation at a
minimum. The edema formation will raise the interstitial fluid volume
(IFV) and thereby PIF and
potentially cause an underestimation of an increased negativity of
PIF.
The procedures described in this article have been carried out with the
approval of and in accordance with the recommendations laid down by the
Norwegian State Commission for Laboratory Animals.
Procedures
PIF.
PIF was measured with sharpened
glass capillaries (tip diameter 3-7 µm) filled with 0.5 M NaCl
colored with Evans blue and connected to a servo-controlled
counterpressure system (33). A calibration was performed before each
experiment. PIF was obtained after
punctures through intact skin using a micromanipulator and under
guidance of a stereomicroscope (Wild M5, Heerbrugg, Switzerland). Measurements were performed on the dorsal side of the hind paw at a
depth of 0.3-0.5 mm below the skin surface (dermal layers). The
animal was placed in a supine position during the experiment. To
eliminate movement of the hind paw, the distal part was carefully fixed
to the puncture table with surgical tape. There was no visible compression or retraction of the skin after this procedure. The pressures were accepted when the following conditions were met. 1) Feedback gain of the
servo-controlled counterpressure system could be varied without
changing the recorded pressure. 2)
After fulfillment of criterion
1, communication between the fluid in the pipette and the interstitial fluid was tested by applying suction
to the pipette. This should result in an increased electrical resistance in the pipette because of entrance of fluid of lower tonicity than the 0.5 M NaCl contained in the pipette.
3) Recording of zero pressure in a
cup filled with saline at the level of the puncture site did not change
from the beginning to the end of the recording.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
analog)
on edema formation and PIF in
parallel with their action on the fibroblast-populated collagen gel
contraction assay. The prostaglandins were injected subdermally in
pentobarbital-anesthetized rats.
PIF was measured with a
servo-controlled counterpressure system after circulatory arrest had
been induced with saturated potassium chloride. Circulatory arrest was
induced to limit edema formation that would raise interstitial fluid
volume and thereby attenuate a possible increased negativity of
PIF.
PGE1 (0.91 mM) and
carbaprostacyclin (1.28 mM) lowered
PIF from a control value of
0.8 ± 0.4 mmHg to 3.0 ± 0.4 (P < 0.01) and 3.7 ± 0.9 (P < 0.01) mmHg,
respectively, within 45 min in a dose-dependent manner. Edema formation
was measured in separate experiments. PGE1 and carbaprostacyclin
significantly increased interstitial fluid volume (extravascular
51Cr-EDTA space) at concentrations
as low as 0.1 and 1.1 µM, respectively. Latanoprost had no effect on
PIF or edema formation. However, latanoprost reversed, in a dose-dependent manner, an increased negativity of PIF accompanying the
anaphylactic reaction to dextran. In the gel contraction assay with
human diploid fibroblasts (AG 1518), a corresponding specificity was
observed where PGE1 and carbaprostacyclin effectively inhibited gel contraction although latanoprost had no effect. Thus the present data demonstrate a novel
effect of prostaglandins and provide further evidence for active
modulation of PIF via loose
connective tissue cells.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
1-integrins (25), in particular the collagen/laminin receptor
21
(25, 26). Furthermore, platelet-derived growth factor (PDGF) is able to
counteract increased negativity of
PIF occurring in dextran
anaphylaxis and after block of
21-integrin
in rat paw skin (26). As an analogy to this in vivo response, we have
studied dermal fibroblasts cultured in a three-dimensional collagen gel
that will contract or compact the gel under optimal conditions to 10%
of its original volume in ~24 h (3, 20). This process depends on the
collagen binding 1-integrins
and is stimulated by PDGF likely via the activity of the
1-integrins (10, 12, 28). In
contrast, several inflammatory mediators such as prostaglandin (PG)
E1 and interleukin-1 inhibit contractions of fibroblast-mediated collagen gel contraction (7, 9,
31). So far there has been a concordance between observations made
using the collagen gel contraction assay and effects on
PIF in that substances that slow
the rate of collagen gel contraction induce increased negativity of
PIF.
is a
vasoconstrictor and directly antagonizes PGE-induced edema formation
(6).
in the circulatory system,
the aim of the present study was to investigate the effects of these
prostanoids on edema formation and
PIF in parallel with their action
on the collagen gel contraction assay.
METHODS
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Abstract
Introduction
Methods
Results
Discussion
References
IFV, total tissue water, and transcapillary albumin extravasation. TISSUE SAMPLES. Tissue samples were obtained by removing the skin on the dorsal side of the hind paws with a pair of scissors. The samples were placed in preweighed vials that were bottled immediately and reweighed as soon as possible. The wet weights of the tissue samples were ~0.1-0.2 g. After measurement of radioactivity (see IFV), the samples were dried at 65°C until they reached constant weight (usually 2-3 wk).
TOTAL TISSUE WATER. Total tissue water (TTW) in the tissue samples was estimated as the water content per gram dry tissue weight [(wet wt dry wt)/(dry wt)].
IFV.
IFV was measured as the extravascular distribution space of
51Cr-EDTA after nephrectomy.
Nephrectomy was performed by ligating the renal pedicles bilaterally
via flank incisions. Thereafter, 0.7 MBq of
51Cr-EDTA was injected in a volume
of 0.3 ml through a PE-50 catheter in the external jugular vein. One
hour later test substances (5 µl) were injected bilaterally in the
paws (see below) and 2 min thereafter 0.05 MBq of
125I-labeled human serum albumin
(HSA; Institute for Energy Technique, Kjeller, Norway) was injected
intravenously. After 25 min 0.05 MBq of
131I-labeled HSA (Institute for
Energy Technique) was injected intravenously in a volume of 0.3 ml.
Five minutes thereafter, blood samples were obtained by cardiac
puncture, and the rat was killed by intravenous injection of 0.5 ml of
saturated potassium chloride. Tissue samples were then obtained as
described in TISSUE SAMPLES. IFV was
estimated as the extravascular distribution volume of
51Cr-EDTA, i.e., [counts per
min (cpm) 51Cr per g dry tissue
wt]/(cpm 51Cr per ml plasma) plasma volume (cpm 131I
per g dry tissue wt/cpm 131I per
ml plasma). Radioactivity was determined in a gamma-counting system
(LKB Wallac 1285) with automatic background subtraction and spillover
correction. Five microliters of test substance were injected in the
right paw, and the left paw was given five microliters of saline as
control. Edema formation induced by the test substance was estimated as
the difference in water content and extravascular 51Cr-EDTA space in the skin of the
two paws at 30 min after injection of the test substances.
ALBUMIN EXTRAVASATION.
Albumin extravasation (EAlb) was
also measured in the experiments used to estimate TTW and IFV.
131I-HSA was given intravenously
just before the end of the experiment. The increase in
EAlb caused by the test substance
was calculated as the difference between the distribution volumes of
131I-HSA and
125I-HSA. All calculations were
made per gram dry tissue weight.
CELL CULTURE.
Explant cultures were initiated from the back skin of adult
Sprague-Dawley rats by established procedures (17). Briefly, small
pieces of skin tissue (~1 mm in diameter) were placed in 60-mm tissue
culture dishes and cultured for 2 wk in Dulbecco's modified Eagle's
medium (DMEM; National Veterinary Institute, Sweden) supplemented with
10% fetal bovine serum (FBS; Integro, Zaandam, The
Netherlands). Cells migrating out from the skin pieces were isolated and propagated in the same medium. These cells possess a
fibroblast-like morphology, contract three-dimensional collagen gels,
and can actively produce collagen type I (unpublished observations). The rat skin fibroblasts (RSKF) were used in passages
5-15. Normal human diploid fibroblasts (AG 1518)
were obtained from Genetic Mutant Cell Repository (Camden, NJ) and used
in passages 15-25. Both cell
types were cultured in DMEM supplemented with 10% FBS, antibiotics,
and L-glutamine. The
characteristics of RSKF and AG 1518 fibroblasts with regard to
contraction of three-dimensional collagen lattices have been previously
reported (3, 9, 10).
COLLAGEN GEL CONTRACTION.
Quantification of fibroblast-mediated collagen gel contraction was
performed essentially as described previously (10, 12). Briefly,
96-well microtiter plates were coated with sterile, filtered 2% bovine
serum albumin in phosphate buffered saline (PBS), incubated at
37°C, and then washed three times with sterile PBS. Fibroblasts from confluent cultures were trypsinized, suspended in serum-free MCDB
104 medium (National Veterinary Institute, Sweden) and diluted to
500,000 cells/ml. The cell suspension was mixed on ice with a collagen
solution [5 vols 2× MCDB 104, 1 vol 0.2 M
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 9.0), and 4 vols Vitrogen 100 (3 mg/ml)] at a 1:9
(vol/vol) ratio. One hundred microliters of the
cell-collagen suspension were added per well, and the plate was
incubated at 37°C to allow polymerization. After 1 h, the gels were
floated with 100 µl of MCDB 104 containing the indicated
concentrations of various prostanoids in the presence or absence of 1.7 nM PDGF BB isoform (PDGF-BB). Collagen gel contraction was measured as
decrease in gel area, using an inverted light microscope.
Test Drugs
PGE1 isopropyl ester was obtained from Sigma Chemical. Carbaprostacyclin and latanoprost (13,14-dihydro-17-phenyl-18,19,20-trinor-PGF2-isopropyl ester) were obtained from Pharmacia and Upjohn (Uppsala, Sweden). For
the in vitro experiments the lithium salt of latanoprost acid was used.
PGE1 is an nonselective prostanoid
EP receptor agonist, carbaprostacyclin a prostanoid IP receptor
agonist, and latanoprost a selective prostanoid FP receptor agonist.
Human recombinant PDGF-BB was donated by Dr. Carl-Henrik Heldin (Ludwig
Institute for Cancer Research, Uppsala, Sweden).
Experimental Protocol
All substances were diluted in sterile PBS or isotonic saline.Series I: PIF. The test substances were used in decreasing concentrations until there was no longer a measurable effect on PIF.
PGE1 AND CARBAPROSTACYCLIN. i) Saline controls. Five microliters of PBS were injected subdermally after circulatory arrest and PIF was measured as described in PIF (n = 6). ii) PGE1. Five microliters of PGE1 were injected subdermally at concentrations of 0.91 (n = 6), 0.30 (n = 6), and 0.09 (n = 6) mM after circulatory arrest, and PIF was measured. iii) Carbaprostacyclin. Five microliters of carbaprostacyclin were injected subdermally at concentrations of 1.28 (n = 6), 0.43 (n = 6), and 0.13 (n = 6) mM after circulatory arrest was induced, and PIF was then measured. iv) Carbaprostacyclin with PDGF-BB. A mixture (5 µl) of carbaprostacyclin (1 mM) and PDGF-BB (60 nM) was injected subdermally after circulatory arrest (n = 6), and PIF was then measured. LATANOPROST. In experiments in which dextran anaphylaxis was used to induce increased negativity of PIF, the dextran was allowed to circulate for 1 min before circulatory arrest was induced. PIF was measured during the subsequent 10 min. Five microliters of latanoprost were thereafter injected subdermally as described in PIF, and PIF was followed for the next 90 min. i) Latanoprost alone. Five microliters (1.42 mM) were injected subdermally after circulatory arrest and PIF was measured (n = 6). This group did not receive intravenous dextran. ii) Dextran series. One milliliter of Dextran 70 (60 mg/ml) was injected intravenously over the course of 20 s and allowed to circulate for 1 min before circulatory arrest was induced as described in PIF. PIF was measured during the next 10 min, and one of the following two test substances was injected at 10 min after induction of cardiac arrest and continued for the subsequent 90 min: 5 µl PBS was injected subdermally and PIF was measured (n = 6); or latanoprost (5 µl) was injected at concentrations of 1.42 (n = 6), 0.47 (n = 6) and 0.15 (n = 6) mM.Series II: IFV, TTW, and EAlb. Five microliters of the test substance were injected in the right paw, and the left paw was given 5 µl saline as control.
i) Saline controls. Five microliters of PBS were injected subdermally as test substance and edema formations as well as EAlb were measured as described in IFV, total tissue water, and transcapillary albumin extravasation (n = 8). ii) PGE1. Five microliters were injected subdermally at concentrations of 1.10 mM (n = 8), 0.11 mM (n = 8), 0.01 mM (n = 8), 1.10 µM (n = 8), and 0.11 µM (n = 8), and edema formation and EAlb were measured. iii) PGE1 with PDGF-BB. PGE1 (0.1 mM, 5 µl) was injected subdermally in the left paw as a control. In the right paw a mixture (5 µl) of PGE1 (0.1 mM) and PDGF-BB at concentrations of 60 (n = 8), 7.4 (n = 8) and 0.7 (n = 8) nM was used, and edema formation and EAlb were measured. iv) Carbaprostacyclin. Carbaprostacyclin (5 µl) was injected subdermally at concentrations of 1.08 mM (n = 8), 0.11 mM (n = 8), 0.01 mM (n = 7), and 1.10 µM (n = 7), and edema formation and EAlb were measured. v) Carbaprostacyclin with PDGF-BB. Carbaprostacyclin (0.1 mM, 5 µl) was injected in the left paw as control. In the right paw a mixture (5 µl) of carbaprostacyclin (0.1 mM) and PDGF-BB at concentrations of 60 (n = 8), 7.4 (n = 8) and 0.7 (n = 8) nM was used, and edema formation and EAlb were measured. vi) Latanoprost. Five microliters of latanoprost at concentrations of 1.42 (n = 8) and 0.15 (n = 7) mM were subdermally injected, and edema formation and EAlb were measured.Series III: Collagen gel contraction.
Latanoprost (analog of PGF2),
carbaprostacyclin (analog of
PGI2), and
PGE1 isopropyl ester were compared
with regard to their effect on human foreskin fibroblast (AG 1518)- and
RSKF-mediated collagen gel contraction. All substances were tested in
triplicate at concentrations ranging from 1 nM to 1 mM. The experiments
were repeated three times. The effect of the different prostanoids on
the collagen contraction assay was also tested in combination with
PDGF-BB.
Statistical Methods
Statistical analysis was performed by one-way analysis of variance with repeated measures and subsequent Bonferroni and Student's t-test. A value of P < 0.05 was considered statistically significant. Data are given as means ± SD unless otherwise stated.| |
RESULTS |
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Abstract
Introduction
Methods
Results
Discussion
References
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Series I: PIF
PGE1 and carbaprostacyclin.
Control PIF averaged 0.81 ± 0.25 and 0.79 ± 0.37 mmHg (grand mean;
n = 50) before and after
induction of circulatory arrest, respectively
(P > 0.05). Subdermal injection of 5 µl saline did not change the PIF
compared with control values (Fig. 1).
PIF was lowered significantly by
both PGE1 and carbaprostacyclin at concentrations as low as 0.30 and 0.47 mM, respectively (Fig. 1,
A and
B).
PGE1 (0.91 mM) lowered
PIF significantly from control values of 0.8 ± 0.3 to 3.0 ± 0.4 mmHg within
30-45 min after injection (P < 0.01; Fig. 1A). During the same
time interval, carbaprostacyclin (1.28 mM) injected subdermally lowered
PIF from 0.8 ± 0.3 to
3.7 ± 0.9 mmHg (P < 0.01;
Fig. 1B).
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Latanoprost.
Latanoprost injected subdermally had no effect on
PIF at a concentration of 1.42 mM
(Fig. 1C) but reversed the increased
negativity of PIF induced by
dextran in a dose-dependent manner (Fig.
2). Dextran alone lowered
PIF from control values of
0.9 ± 0.2 to 3.2 ± 1.1 mmHg
(n = 24) 10 min after intravenous
administration (P < 0.01).
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PDGF-BB and prostaglandins.
Both PGE1 and carbaprostacyclin
induced a dose-dependent lowering of
PIF (Fig. 1,
A and
B).
PIF before subdermal injection of
a solution containing a mixture of carbaprostacyclin (1.0 mM) and
PDGF-BB (60 nM) was 1.1 ± 0.3 mmHg.
PIF after subdermal injection of
the mixture containing PDGF-BB and carbaprostacyclin did not differ
from PIF after subdermal injection
of carbaprostacyclin (1.28 mM) alone (data not presented), and
PIF under these conditions was
measured at 1.8 ± 0.9 mmHg after 10 min.
Series II: IFV, TTW, and EAlb
PGE1 and carbaprostacyclin induced edema formation and increased EAlb at concentrations as low as 0.11 and 1.10 µM, respectively (Table 1). Latanoprost did not affect edema formation and transcapillary EAlb at concentrations of 1.47 and 0.15 mM (Table 1). PGE1 (1.10 mM) and carbaprostacyclin (1.08 mM) increased TTW during the first hour to 0.36 and 0.11 ml/g, respectively (Table 1). There was no significant difference in IFV, TTW, and EAlb in the paw receiving prostanoids with PDGF-BB compared with the paws receiving prostanoids only (data not presented).
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Series III: Collagen Gel Contraction
Carbaprostacyclin and PGE1 both effectively inhibited AG 1518-mediated contraction at a concentration of 1.0 µM (Fig. 3, A and C). The inhibition was most pronounced at early time points and could be observed already at a concentration of 10 nM (data not shown). In combination with PDGF-BB, which is a potent stimulator of collagen gel contraction, the effect of PGE1 was reduced to control level (Fig. 3C), whereas the inhibitory effect of carbaprostacyclin was unaffected (Fig. 3A). No inhibitory effects of carbaprostacyclin and PGE1 on contraction were demonstrated when RSKF were cultured in collagen gels (Fig. 3, A and C). Latanoprost showed no consistent effect on AG 1518- or RSKF-mediated contraction (Fig. 3B).
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DISCUSSION |
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Abstract
Introduction
Methods
Results
Discussion
References
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In the present study we have demonstrated a novel in vivo effect of prostanoids on PIF. The effect was specific and dose dependent: PGE1 and the PGI2 analog carbaprostacyclin induced an increased negativity of PIF concomitant with rapid edema formation, whereas no such effect was seen with latanoprost, a specific FP receptor agonist. However, latanoprost reversed the increased negativity of PIF accompanying the anaphylactic reaction to dextran. In the rat, intravenous administration of dextran is followed by rapid edema formation with accumulation of fluid in the paw, around the nose, and in the trachea (13, 32). Previously, it was shown that the inflammatory edema in skin induced by dextran is accompanied by an increased negativity of PIF (24). The increased negativity of PIF is a major driving pressure for initial edema formation under these circumstances.
In parallel with the observation on
PIF, contraction of AG 1518 fibroblast-populated collagen gels was inhibited by carbaprostacyclin and PGE1. Taken together with the
effects on PIF, the two sets of
observations are in agreement with the concept that control of
PIF in vivo is not merely a
function of IFV but also depends on the contraction of a
collagen/microfibril network restraining the swelling of a
hyaluronan/proteoglycan gel. Both carbaprostacyclin and
PGE1 significantly inhibited AG
1518 fibroblast collagen gel contraction in vitro in doses as low as 10 nM (data not shown). The inhibitory effect of carbaprostacyclin was
consistently higher than that of
PGE1. This could be explained by
the fact that PGE1 not only binds
to prostanoid receptors of the EP type but also to the prostacyclin
receptor (IP), to which carbaprostacyclin is an agonist (22). Our
results suggest that either IP receptors and not EP receptors are
expressed on the cells or the IP receptors are more abundant or more
potent in inhibiting fibroblast-mediated collagen gel
contraction. Previously, it was reported that
N6,2'-O-dibutyryl
adenosine 3',5'-cyclic monophosphate (cAMP) inhibits fibroblast-mediated collagen gel contraction (7, 27), and it is likely
that the effects of carbaprostacyclin and
PGE1 depend on the ability of IP
receptors to stimulate adenylate cyclase. RSKF-mediated collagen gel
contraction did not respond to carbaprostacyclin and
PGE1, suggesting that these cells
express fewer prostanoid receptors or a different repertoire of such
receptors. The findings that both carbaprostacyclin and
PGE1 possessed strong activity in
vivo whereas the RSKF, originating from rat skin, were relatively insensitive could be explained by the observation that cells change their expression of receptors when established as in vitro cultures (30). PDGF-BB could not overcome the inhibitory effects of
carbaprostacyclin in collagen gel contraction with AG 1518 cells either
in vitro or in vivo, suggesting that carbaprostacyclin elicits a
response that is not modulated by the growth factor. Previous
experiments have suggested that PDGF-BB exerts its effect on
fibroblast-mediated collagen gel contraction by increasing the apparent
avidity of collagen-binding
1-integrins (10), and a similar
mechanism seems to be operating in the in vivo regulation of
PIF. Taken together, the present
data suggest that the effects of carbaprostacyclin and
PGE1 do not primarily involve
alterations in the functions of collagen-binding
1-integrins but work upstream
of these receptors. A likely candidate is the contractile machinery of
the cell, and it has been shown that cAMP exerts an effect on myosin
light-chain kinase leading to an inhibition of collagen gel contraction
(7). Latanoprost, a selective prostanoid FP receptor agonist (29), did
not affect fibroblast-mediated collagen gel contraction in vitro but
reversed the increased negativity of
PIF induced by inflammation. These
findings suggest that FP receptors are not active in native cells but
are able to overcome relaxation induced by inflammatory mediators. The
nature of the latter is presently unknown.
The experimental model used for PIF measurements includes induction of circulatory arrest. This procedure has earlier been shown not to change PIF for up to 90 min compared with the control situation (33). Theoretically, drainage of fluid from the tissue by the lymphatics after circulatory arrest could potentially lower PIF with time, but no such effect was observed in the control groups. Circulatory arrest was used to arrest increased capillary extravasation after exposure to the prostaglandins. With intact circulation fluid, filtration to the tissues, which is a part of the inflammatory response, will increase IFV and thereby PIF (2). If this took place, the measured PIF would represent an underestimate of the increased negativity of PIF.
The test substances were injected subdermally and the pressure measurements were performed in the area adjacent to the deposited volume, in the dermal layer. Earlier it was shown that pressure measurements during steady state, under different experimental conditions, with wick in needle in subcutis, and with micropipettes in dermis were identical over a wide range of hydration and dehydration (34). The fact that we measure at some distance from the deposited substance most likely explains why there is a 100-fold difference in the concentration of the different prostanoids for the measurable effects seen in vitro versus those seen in vivo experiments. Dilution of the prostanoids in the injected volume by diffusion into the surrounding environment will lower their concentration. Furthermore, diffusion of the injected prostaglandin into a sphere with twice the radius of the injected volume will lower the concentration to one-eighth of the injectate. This is less than the distance between the point of injection and the point of pressure measurement, and consequently the concentration must be even lower and likely in the same range of 1-10% of that in the injectate.
The edema-generating effect of the different prostaglandins was investigated with intact circulation. Subdermal injection of PGE1 and carbaprostacyclin caused rapid edema formation (Table 1). The edema was visible within 10 min after subdermal injection of PGE1 and carbaprostacyclin. Edema formation is, according to the Starling equation, caused by increased capillary filtration coefficient (CFC; "water permeability") and/or by increased net capillary filtration pressure (2). Under normal conditions the net driving pressure for ultrafiltration in peripheral tissue is estimated to be 0.5-1 mmHg (2). This small net outward pressure is responsible for the net capillary filtration and therefore formation of lymph, which turns over the IFV in peripheral tissues in 12-24 h (2). The amount of IFV under normal conditions in rat skin is 0.4 ml/g wet weight or 1 ml/g dry weight (2). The gain in TTW was 0.36 ml/g dry weight 30 min after prostaglandin challenge, corresponding to at least a 10-fold increase in transcapillary fluid transport. Increase in the CFC is commonly regarded as the main cause for edema formation in several acute inflammatory responses (2). The increase in CFC in acute inflammatory reactions in peripheral tissues has been measured to be two to three times above control values (1). This, however, is insufficient to explain the rapid edema formation occurring after prostaglandin administration. Both carbaprostacyclin and PGE1, injected subdermally, resulted in increased negativity of PIF. Carbaprostacyclin at a concentration of 1.28 mM lowered PIF ~3 mmHg from control values within 30-45 min after injection. The lowering of PIF will, according to the Starling equation, increase the driving pressure four to seven times above normal, and this favors edema formation. Prostacyclin and PGE1 are vasodilators in almost every vascular bed investigated (15). Vasodilation will normally increase capillary pressure (PC), which is determined by the ratio of precapillary to postcapillary resistance. Precapillary vasodilation increases capillary filtration and net filtration pressure and thereby IFV. The quantitative importance of PC in controlling transcapillary exchange related to the prostaglandins has not been fully determined.
Under normal conditions, PIF acts
to maintain normal interstitial volume and to counteract edema
formation. Thus increased capillary filtration will raise interstitial
volume and PIF to restrict further
fluid filtration to the tissues and is known as one of the "safety
factors" against edema formation. Contrary to this commonly accepted
role for PIF in normal control of
interstitial volume, our observation of increased negativity of
PIF concomitant with edema
formation shows that the tissues can "actively" enhance capillary
filtration and eventually cause edema formation. The change in
PIF is important in the initial
and rapid edema formation during the acute inflammatory response. We
have demonstrated earlier increased negativity of
PIF after mast cell degranulation
in the rat trachea (14), in skin after burn injury (16), and in dextran anaphylaxis (23). Furthermore, increased negativity of
PIF and edema formation can also
be induced in skin with blockade by a polyclonal antibody toward the
1-integrins (25). Similar
results are obtained using a monoclonal antibody toward
21
(26), whereas an antibody toward the
11-integrin
was without effect on PIF (25).
The increased negativity of PIF
obtained after injecting the
anti-21
antibody subdermally is similar to that obtained from a series of acute
inflammatory reactions both in magnitude and time response. In view of
the novel finding that PGE1 and carbaprostacyclin increase PIF
concomitantly with edema formation and the observation that these
prostanoids inhibit compaction of the fibroblast-populated collagen
gels, it is likely that the same cellular mechanism is involved. Our
current thinking about the phenomenon described above is based on the
analogy with the fibroblast contraction of collagen gels and is as
follows. The tendency of tissues to expand when given free access to
saline is caused by their content of hyaluronan/proteoglycans, because enzymatic treatment resulting in loss of these substances will abolish
the swelling (18). We propose that the connective tissue cells keep the
tissue under tension by connecting to the collagen fibers through
cell-surface receptors, thereby counteracting the tendency of the
tissue to expand. If the interaction between connective tissue cells
and the extracellular matrix proteins are lost, the tissue will expand.
If no fluid is available, the expanding tendency will result in an
increased negativity of PIF until
the more negative PIF is again
balanced by the lowered stress in the extracellular fibers and the
swelling hyaluronan.
The effect of latanoprost on PIF
in this study was unique among the prostaglandins. This FP
receptor-selective PGF2 analog had no effect either on PIF or
interstitial volume. This is in accordance with the results of
Crunkhorn and Willis (6), who demonstrated that
PGF2 up to 1 µg (in 0.1 ml)
had no effect on microvascular permeability when injected intradermally
in rats. In the present study, latanoprost reversed the dextran-induced increased negativity of PIF when
given at a time when the anaphylactic reaction had been established.
Thus latanoprost is able to attenuate the effects on
PIF also after an inflammatory
reaction has been initiated. The effect of reversing an increased
negativity of PIF after
dextran-induced anaphylaxis in skin has also been seen with the
experimental drug -trinositol
(D-myo-inositol-1,2,6-trisphosphate; Ref. 27). The exact molecular mechanism of the action of -trinositol on PIF has not been determined but
seems to involve modulation of the intracellular events regulating
1-integrin function and extracellular matrix components (27). The present study does not
determine whether latanoprost modulates
PIF through the same molecular
action on the 1-integrins and
extracellular matrix as does -trinositol.
In summary, the present study is novel in two aspects. First, it
demonstrates for the first time some specific effects of prostaglandins
on PIF.
PGE1 and carbaprostacyclin (a
PGI2 analog) induce a considerable
and rapidly appearing increased negativity of
PIF in skin that will enhance
edema formation. Latanoprost (a
PGF2 analog) reversed the
increased negativity of PIF induced by dextran anaphylaxis. Second,
PGE1 and carbaprostacyclin are
able to inhibit contraction of fibroblast-populated collagen gels,
which depends on 1-integrin
function. Taken together, our data suggest that these prostaglandins
can modulate IFV and PIF through
effects on the connective tissue cells and extracellular matrix
components.
| |
ACKNOWLEDGEMENTS |
|---|
The authors acknowledge the technical assistance of Eli Gunn Kjørlaug and Gerd Signe Salvesen. We are grateful to the Pharmacia and Upjohn Company and Dr. Bahram Resul for supplying us with prostanoid analogs.
| |
FOOTNOTES |
|---|
The present study was financially supported by the Norwegian Research Council and the Swedish Cancer Society.
Address for reprint requests: A. Berg, Dept. of Physiology, Univ. of Bergen, Årstadveien 19, N-5009 Bergen, Norway (E-mail: Ansgar{at}pki.uib.no).
Received 17 April 1997; accepted in final form 16 October 1997.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
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